U.S. patent application number 10/792201 was filed with the patent office on 2004-09-02 for compositions for inhalation.
This patent application is currently assigned to AstraZeneca AB, a Swedish corporation. Invention is credited to Backstrom, Kjell Goran Erik, Dahlback, Carl Magnus Olof, Edman, Peter, Johansson, Ann Charlotte Birgit.
Application Number | 20040171550 10/792201 |
Document ID | / |
Family ID | 32995675 |
Filed Date | 2004-09-02 |
United States Patent
Application |
20040171550 |
Kind Code |
A1 |
Backstrom, Kjell Goran Erik ;
et al. |
September 2, 2004 |
Compositions for inhalation
Abstract
A pharmaceutical composition including a mixture of active
compounds (A) a pharmaceutically active polypeptide, and (B) an
enhancer compound which enhances the systemic absorption of the
polypeptide in the lower respiratory tract of a patient, the
mixture being in the form of a dry powder for inhalation in which
at least 50% of the total mass of the active compounds consists of
primary particles having a diameter less than or equal to about 10
microns, the primary particles optionally being formed into
agglomerates.
Inventors: |
Backstrom, Kjell Goran Erik;
(Lund, SE) ; Dahlback, Carl Magnus Olof; (Lund,
SE) ; Edman, Peter; (Bjarred, SE) ; Johansson,
Ann Charlotte Birgit; (Lund, SE) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Assignee: |
AstraZeneca AB, a Swedish
corporation
|
Family ID: |
32995675 |
Appl. No.: |
10/792201 |
Filed: |
March 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10792201 |
Mar 3, 2004 |
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08736267 |
Oct 24, 1996 |
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08736267 |
Oct 24, 1996 |
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08265237 |
Jun 23, 1994 |
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Current U.S.
Class: |
424/85.2 ;
514/10.3; 514/10.8; 514/10.9; 514/11.2; 514/11.4; 514/11.5;
514/11.6; 514/11.8; 514/11.9; 514/12.4; 514/9.9 |
Current CPC
Class: |
A61K 9/145 20130101;
A61K 9/0075 20130101; A61K 9/008 20130101; A61K 38/27 20130101;
A61K 47/12 20130101; A61K 38/28 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/16 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 4, 1994 |
SE |
9400371-2 |
Jun 24, 1993 |
SE |
9302198-8 |
Claims
What is claimed is:
1. A pharmaceutical composition, comprising a mixture of active
compounds (A) a pharmaceutically active polypeptide, and (B) an
enhancer compound which enhances the systemic absorption of said
polypeptide in the lower respiratory tract of a patient, said
mixture being in the form of a dry powder for inhalation, in which
at least 50% of the total mass of active compounds consists of
primary particles having a diameter less than or equal to about 10
microns, said primary particles optionally being formed into
agglomerates.
2. A pharmaceutical composition as claimed in claim 1, additionally
comprising a pharmaceutically acceptable carrier, which comprises
either (a) particles having a diameter of less than about 10
microns, such that at least 50% of the resultant powder consists of
optionally agglomerated primary particles having a diameter of less
than about 10 microns; or (b) coarse particles, such that an
ordered mixture is formed between the active compounds and the said
carrier.
3. The composition of claim 1, wherein said polypeptide is a
polypeptide hormone.
4. The composition of claim 3, wherein said hormone is vasopressin,
a vasopressin analogue, desmopressin, glucagon, corticotropin
(ACTH), gonadotrophin (luteinizing hormone, or LHRH), calcitonin,
C-peptide of insulin, parathyroid hormone (PTH), human growth
hormone (hGH), growth hormone (HG), growth hormone releasing
hormone (GHRH), oxytocin, corticotropin releasing hormone (CRH),
somatostatin analogs, gonadotropin agonist analogs (GnRHa), atrial
natriuretic peptide (hANP), thyroxine releasing hormone (TRHrh),
follicle stimulating hormone (FSH), or prolactin.
5. The composition of claim 1, wherein said polypeptide is a growth
factor, interleukin, polypeptide vaccine, enzyme, endorphin,
glycoprotein, lipoprotein, or polypeptide involved in the blood
coagulation cascade, that exerts its pharmacological effect
systemically.
6. The composition of claim 1, wherein said polypeptide has a
molecular weight of less than 30 kD.
7. The composition of claim 1, wherein said polypeptide has a
molecular weight of less than 25 kD.
8. The composition of claim 1, wherein said polypeptide has a
molecular weight of less than 20 kD.
9. The composition of claim 1, wherein said polypeptide has a
molecular weight of less than 15 kD.
10. The composition of claim 1, wherein said polypeptide has a
molecular weight of less than 10 kD.
11. The composition of claim 1, wherein said enhancer compound is a
surfactant.
12. The composition of claim 11, wherein said surfactant is a bile
salt, a bile salt derivative, an alkyl gycoside, a cyclodextrin or
derivative thereof, or a phospholipid.
13. The composition of claim 11, wherein said surfactant is a salt
of a fatty acid.
14. The composition of claim 11, wherein said fatty acid has 10-14
carbon atoms.
15. The composition of claim 14, wherein said fatty acid is capric
acid.
16. The composition of claim 11, wherein said surfactant is sodium
caprate.
17. An inhaler device containing the composition of claim 1.
18. The inhaler device of claim 17, wherein said composition is in
the form of said agglomerates, said device being configured to
induce the majority of said agglomerates to break down into
particles having a diameter less than or equal to about 10 microns,
upon inhalation of said agglomerates from said device.
19. The inhaler device of claim 17, which inhaler device is a unit
dose, breath actuated, dry powder inhaler for single use.
20. The inhaler device of claim 17, which inhaler device is a
multi-dose, breath actuated, dry powder inhaler for multiple use.
Description
[0001] This invention relates to methods and compositions for
delivery of medically useful peptides and proteins.
BACKGROUND OF THE INVENTION
[0002] Although the advent of recombinant DNA technology has
resulted in a rapidly expanding list of peptide-based drugs, a
major drawback of peptide-based therapy has acutely hampered
realization of the full potential of this field: in general,
peptide-based drugs cannot be orally administered in effective
doses, since they are rapidly degraded by enzymes in the
gastrointestinal tract before they can reach the bloodstream.
Unless the polypeptide of interest can be altered to make it
relatively resistant to such enzymes, the only practical method of
delivering the drug is likely to be a parenteral route, such as by
intravenous, intramuscular, or subcutaneous injection.
Administration by other parenteral routes (e.g., by absorption
across nasal, buccal or rectal membranes, or via the lung) has met
with limited success.
SUMMARY OF THE INVENTION
[0003] It has been found that when a peptide or protein
(hereinafter collectively referred to as polypeptides) is combined
with an appropriate absorption enhancer and is introduced into the
lung in the form of a powder of appropriate particle size, it
readily enters the pulmonary circulation by absorption through the
layer of epithelial cells in the lower respiratory tract. This is
conveniently accomplished by inhalation of the powder from an
inhaler device which dispenses the correct dose of powdered
polypeptide/enhancer in a particle size which maximizes deposition
in the lower respiratory tract, as opposed to the mouth and throat.
(For ease of reference, the polypeptide and enhancer are
hereinafter collectively referred to as the "active compounds"). To
accomplish this preferential delivery into the lung, as much as
possible of the active compounds should consist of particles having
a diameter less than approximately 10 .mu.m (e.g., between 0.01-10
.mu.m, and ideally between 1-6 .mu.m). In preferred embodiments, at
least 50% (preferably at least 60%, more preferably at least 70%,
still more preferably at least 80%, and most preferably at least
90%) of the total mass of active compounds which exits the inhaler
device consists of particles within the desired diameter range.
[0004] The invention thus includes a pharmaceutical composition
containing a mixture of active compounds (A) a pharmaceutically
active polypeptide and (B) an enhancer compound which enhances the
systemic absorption of the polypeptide in the lower respiratory
system (preferably the lungs) of a patient, the mixture being in
the form of a dry powder suitable for inhalation, in which at least
50% of the total mass of active compounds (A) and (B) consists of
primary particles having a diameter less than or equal to about 10
microns. The primary particles may be packaged as such, or may
optionally be formed into agglomerates, which then are
substantially deagglomerated prior to entry into the respiratory
tract of the patient. The composition may of course contain other
ingredients as needed, including other pharmaceutically active
agents, other enhancers, and pharmacologically acceptable
excipients such as diluents or carriers. Therefore, the therapeutic
preparation of the present invention may contain only the said
active compounds or it may contain other substances, such as a
pharmaceutically acceptable carrier. This carrier may largely
consist of particles having a diameter of less than about 10
microns so that at least 50% of the resultant powder as a whole
consists of optionally agglomerated primary particles having a
diameter of less than about 10 microns; alternatively the carrier
may largely consist of much bigger particles ("coarse particles"),
so that an "ordered mixture" may be formed between the active
compounds and the said carrier. In an ordered mixture,
alternatively known as an interactive or adhesive mixture, fine
drug particles (in this invention, the active compounds) are fairly
evenly distributed over the surface of coarse excipient particles
(in this invention, the pharmaceutically acceptable carrier).
Preferably in such case the active compounds are not in the form of
agglomerates prior to formation of the ordered mixture. The coarse
particles may have a diameter of over 20 microns, such as over 60
microns. Above these lower limits, the diameter of the coarse
particles is not of critical importance so various coarse particle
sizes may be used, if desired according to the practical
requirements of the particular formulation. There is no requirement
for the coarse particles in the ordered mixture to be of the same
size, but the coarse particles may advantageously be of similar
size within the ordered mixture. Preferably, the coarse particles
have a diameter of 60-800 microns.
[0005] The polypeptide may be any medically or diagnostically
useful peptide or protein of small to medium size, i.e. up to about
40 kD molecular weight (MW), for which systemic delivery is
desired. The mechanisms of improved polypeptide absorption
according to the present invention are generally applicable and
should apply to all such polypeptides, although the degree to which
their absorption is improved may vary according to the MW and the
physico-chemical properties of the polypeptide, and the particular
enhancer used. It is expected that polypeptides having a molecular
weight of up to 30 kD will be most useful in the present invention,
such as polypeptides having a molecular weight of up to 25 kD or up
to 20 kD, and especially up to 15 kD or up to 10 kD. Any desired
polypeptide may be easily tested for use in the present invention
with a particular enhancer, by in vivo or in vitro assays, as
described herein.
[0006] The enhancer compound used in the compositions of the
present invention can be any compound which enhances the absorption
of the polypeptide through the epithelium of the lower respiratory
tract, and into the systemic circulation. By "enhances absorption"
is meant that the amount of polypeptide absorbed into the systemic
circulation in the presence of enhancer is higher than in the
absence of enhancer. Preferably the amount of polypeptide absorbed
is significantly higher. (p<0.05) in the presence of enhancer.
The suitability of any potential enhancer for use in the present
invention may be easily assessed, by means of in vivo or in vitro
assays, as described herein.
[0007] The amount of polypeptide absorbed according to the present
invention is preferably at least 150% of the amount absorbed in the
absence of enhancer. In preferred embodiments, absorption of
polypeptide is at least doubled, more preferably tripled, and most
preferably quadrupled in the presence of the enhancer, compared to
in its absence.
[0008] The enhancer is preferably a surfactant such as a salt of a
fatty acid, a bile salt, a bile salt derivative, an alkyl
glycoside, a cyclodextrin, or a phospholipid. The enhancer may be,
for example, a sodium, potassium, or organic amine salt of the
fatty acid, and the fatty acid is preferably capric acid or another
fatty acid of 10-14 carbon atoms. The preferred enhancer is sodium
caprate. The ratio of polypeptide to enhancer will preferably vary
from about 9:1 to about 1:1. Although proportions of enhancer
greater than 1:1 would presumably enhance uptake as well as or
better than lower proportions, it is believed that the amount of
enhancer used should be no higher than necessary to acheive the
desired level of enhancement, since excess enhancer may trigger
unwanted side effects, such as local irritation.
[0009] Also within the invention is a method of administering
systemically a pharmaceutically active polypeptide, by causing a
patient to inhale the pharmaceutical composition of the invention,
wherein at least 50% of the total mass of the active compounds at
the point of entry to the respiratory tract of the patient consists
of particles having a diameter less than or equal to about 10
microns. This is preferably accomplished by the use of an inhaler
device from which the patient inhales the powder. Where the
powdered composition is in the form of agglomerates of primary
particles, the device is preferably configured to induce
substantial deagglomeration of the agglomerates upon inhalation of
the powder from the device by the patient, so that the majority of
the agglomerates break down into particles having a diameter less
than or equal to about 10 microns, prior to entry of the powder
into the respiratory system of the patient. This deagglomeration
would occur inside the device, and is typically induced by the air
turbulence created in the device by the force of inhalation.
Agglomerates are in general preferably not formed in the ordered
mixture. In the case of an ordered mixture, the active compounds
should be released from the large particles preferably upon
inhalation, either by mechanical means in the inhaler device or
simply by the action of inhalation, or by other means, the active
compounds then being deposited in the lower respiratory tract and
the carrier particles in the mouth.
[0010] The inhaler device is preferably a single dose dry powder
inhaler, but may alternatively be a multi dose dry powder
inhaler.
[0011] The invention also includes processes for the manufacture of
a pharmaceutical composition suitable for administration by
inhalation. In one such process, a solution is first provided in
which are dissolved (a) a pharmaceutically active polypeptide and
(b) an enhancer compound which enhances the systemic absorption of
the polypeptide in the lower respiratory tract of a patient. The
solvent is then removed from the solution to yield a dry solid
containing the polypeptide and the enhancer, and the dry solid is
pulverized to produce a powder. A second such process involves dry
mixing (a) a pharmaceutically active polypeptide and (b) an
enhancer compound, and micronizing the obtained mixture. Yet a
third suitable process includes the steps of providing a first
micronized preparation containing a polypeptide and a second
micronized preparation containing an enhancer compound, and mixing
the two micronized preparations together. When a carrier is to be
included other than when an ordered mixture is desired, this may be
added to the solution, or to the dry-mixture of the
pharmaceutically active polypeptide prior to micronization, or
micronised carrier may be dry mixed with the other micronised
components. In producing an ordered mixture, micronised polypeptide
and enhancer are mixed with a suitable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a graph illustrating the effects of different
concentrations of sodium caprate enhancer on the transport of a
marker compound (mannitol) through a monolayer of cultured
epithelial cells.
[0013] FIG. 2 is a graph illustrating the effects of different
concentrations of sodium caprate enhancer on the transport of a
marker compound (mannitol) through a monolayer of cultured
epithelial cells, in the presence of a polypeptide (sodium
caprate:polypeptide 1:3 by weight).
[0014] FIG. 3 is a graph of plasma polypeptide concentration as a
function of time after inhalation of the polypeptide alone, the
polypeptide with sodium caprate in a ratio of 90:10, and the
polypeptide with sodium caprate in a ratio of 75:25.
DETAILED DESCRIPTION
[0015] Some of the preferred embodiments of the invention are
generally described below.
[0016] The Polypeptide
[0017] The polypeptide is preferably a peptide hormone other than
insulin, such as vasopressin, vasopressin analogues, desmopressin,
glucagon, corticotropin (ACTH), gonadotrophin (luteinizing hormone,
or LHRH), calcitonin, C-peptide of insulin, parathyroid hormone
(PTH), human growth hormone (hGH), growth hormone (HG), growth
hormone releasing hormone (GHRH), oxytocin, corticotropin releasing
hormone (CRH), somatostatin analogs, gonadotropin agonist analogs
(GnRHa), atrial natriuretic peptide (hANP), thyroxine releasing
hormone (TRHrh), follicle stimulating hormone (FSH), and
prolactin.
[0018] Other possible polypeptides include growth factors,
interleukins, polypeptide vaccines, enzymes, endorphins,
glycoproteins, lipoproteins, and polypeptides involved in the blood
coagulation cascade, that exert their pharmacological effect
systemically. It is expected that most if not all polypeptides of
small to medium size, relatively high water solubility, and an
isoelectric point between approximately pH 3 and pH 8 can be
effectively delivered by the methods of the invention.
[0019] The Enhancer
[0020] The use of an absorption enhancer is of critical importance,
as the polypeptide alone is poorly absorbed through the lung. The
enhancer used can be any of a number of compounds which act to
enhance absorption through the layer of epithelial cells lining the
lower respiratory tract, and into the adjacent pulmonary
vasculature. The enhancer can accomplish this by any of several
possible mechanisms:
[0021] (1) Enhancement of the paracellular permeability of a
polypeptide by inducing structural changes in the tight junctions
between the epithelial cells.
[0022] (2) Enhancement of the transcellular permeability of a
polypeptide by interacting with or extracting protein or lipid
constituents of the membrane, and thereby perturbing the membrane's
integrity.
[0023] (3) Interaction between enhancer and polypeptide which
increases the solubility of the polypeptide in aqueous solution.
This may occur by preventing formation of insulin aggregates
(dimers, trimers, hexamers), or by solubilizing polypeptide
molecules in enhancer micelles.
[0024] (4) Decreasing the viscosity of, or dissolving, the mucus
barrier lining the alveoli and passages of the lung, thereby
exposing the epithelial surface for direct absorption of the
polypeptide.
[0025] Enhancers may function by only a single mechanism set forth
above, or by two or more. An enhancer which acts by several
mechanisms is more likely to promote efficient absorption of a
polypeptide than one which employs only one or two.
[0026] For example, surfactants are a class of enhancers which are
believed to act by all four mechanisms listed above. Surfactants
are amphiphilic molecules having both a lipophilic and a
hydrophilic moiety, with varying balance between these two
characteristics. If the molecule is very lipophilic, the low
solubility of the substance in water may limit its usefulness. If
the hydrophilic part overwhelmingly dominates, however, the surface
active properties of the molecule may be minimal. To be effective,
therefore, the surfactant must strike an appropriate balance
between sufficient solubility and sufficient surface activity.
[0027] Another surfactant property that may be of importance is the
net charge of the surfactant at the pH value in the lung
(approximately 7.4). At pH 7.4, some polypeptides have a negative
net charge. This will result in an electrostatic repulsion between
molecules, which will in turn prevent aggregation and thereby
increase the solubility. If the surfactant also is negatively
charged, it can interact with the polypeptide by, for example,
hydrophobic interactions, and additional repulsion among the
polypeptide molecules will occur. In such case an anionic
surfactant will possess the additional advantage (compared to those
having neutral or net positive charge at physiological pH) of
enhancing absorption by helping stabilize the polypeptide in the
monomeric state.
[0028] A number of different compounds potentially useful as
enhancers in the methods of the invention were tested in rats, as
described in Example 2 below. Other substances with known
absorption-enhancing properties, or with physical characteristics
which make them likely candidates for use in the method of the
invention, can be readily tested by one of ordinary skill in that
in vivo assay, or alternatively in the in vitro assay described in
Example 1.
[0029] It is possible that a combination of two or more enhancer
substances also gives satisfactory results. The use of such a
combination in the method of the invention is considered to be
within the invention.
[0030] An enhancer useful in the methods of the invention will
combine effective enhancement of polypeptide absorption with (1)
lack of toxicity in the concentrations used and (2) good powder
properties, i.e., lack of a sticky or waxy consistency in the solid
state. Toxicity of a given substance can be tested by standard
means, such as by the MTT assay, for example as described in Int.
J. Pharm., 65 (1990), 249-259. The powder properties of a given
substance may be ascertained from published data on the substance,
or empirically.
[0031] One very promising type of enhancer is the salt of a fatty
acid. It has been found that the sodium salt of saturated fatty
acids of carbon chain length 10 (i.e., sodium caprate), 12 (sodium
laurate) and 14 (sodium myristate) perform well in the method of
the invention. The potassium and lysine salts of capric acid have
also been found to be effective in the method of the invention. If
the carbon chain length is shorter than about 10, the surface
activity of the surfactant may be too low, and if the chain length
is longer than about 14, decreased solubility of the fatty acid
salt in water limits its usefulness.
[0032] Most preferably in the present invention the substance which
enhances the absorption of polypeptide in the lower respiratory
tract is sodium caprate.
[0033] Different counterions may change the solubility of the
saturated fatty acid salt in water, such that an enhancer having a
carbon length other than 10-14 would prove even more advantageous
than the enhancers specifically mentioned hereinabove. Salts of
unsaturated fatty acids may also be useful in the present invention
since they are more water soluble than salts of saturated fatty
acids, and can therefore have a longer chain length than the latter
and still maintain the solubility necessary for a successful
enhancer of polypeptide absorption.
[0034] All of the bile salts and bile salt derivatives tested
(sodium salts of ursodeoxycholate, taurocholate, glycocholate, and
taurodihydrofusidate) effectively enhance polypeptide absorption in
the lung.
[0035] Phospholipids were also tested as enhancers. It was found
that a single-chain phospholipid (lysophospatidylcholine) was an
effective enhancer, while two double-chain phospholipids
(dioctanoylphosphatidylcho- line and didecanoylphosphatidylcholine)
were not. This may be explained by the fact that the double-chain
phospholipids are much less soluble in water than their
single-chain counterparts; however, it is reasonable to expect that
double-chain phospholipids of shorter chain length, having greater
water-solublility than their longer chain counterparts, will be of
use as enhancers in the present invention so that both single- and
double-chain phospholipids may be used.
[0036] One glycoside, octylglucopyranoside, was tested as an
enhancer in the present invention and was found to have some
absorption enhancing properties. Other alkyl glycosides, such as
thioglucopyranosides and maltopyranosides would also be expected to
exhibit absorption enhancing properties in the methods of the
present invention.
[0037] The cyclodextrins and derivatives thereof effectively
enhance nasal absorption, and may function similarly in the lung.
Dimethyl-.beta.-cyclodextrin has been tested and was found to have
an absorption enhancing effect.
[0038] Other potentially useful surfactants are sodium salicylate,
sodium 5-methoxysalicylate, and the naturally occurring surfactants
such as salts of glycyrrhizine acid, saponin glycosides and acyl
carnitines.
[0039] For ionic enhancers (e.g., the anionic surfactants described
above), the nature of the counterion may be important. The
particular counterion selected may influence the powder properties,
solubility, stability, hygroscopicity, and local/systemic toxicity
of the enhancer or of any formulation containing the enhancer. It
may also affect the stability and/or solubility of the polypeptide
with which it is combined. In general, it is expected that
monovalent metallic cations such as sodium, potassium, lithium,
rubidium, and cesium will be useful as counterions for anionic
enhancers. Ammonia and organic amines form another class of cations
that is expected to be appropriate for use with anionic enhancers
having a carboxylic acid moiety. Examples of such organic amines
include ethanolamine, diethanolamine, triethanolamine,
2-amino-2-methylethylamine, betaines, ethylenediamine,
N,N-dibensylethylenetetraamine, arginine, hexamethylenetetraamine,
histidine, N-methylpiperidine, lysine, piperazine, spermidine,
spermine and tris(hydroxymethyl)aminomethane.
[0040] Since effective enhancement of polypeptide absorption in the
lung was observed for a number of the enhancers tested, it is
expected that many more will be found which also function in this
manner. Starch microspheres effectively enhance the bioavailability
of polypeptide delivered via the nasal membranes and were tested as
an enhancer in the methods of the invention. Although they proved
to be of little use for delivery via the pulmonary route in the
animal model utilized herein, it is thought that this was mainly
due to technical difficulties which, if overcome, may lead to
successful delivery via the pulmonary route.
[0041] Chelators are a class of enhancers that are believed to act
by binding calcium ions. Since calcium ions help maintain the
dimensions of the space between cells and additionally reduce the
solubility of a polypeptide, binding of these ions would in theory
both increase the solubility of polypeptides, and increase the
paracellular permeability of polypeptides. Although one chelator
tested, the sodium salt of ethylenediaminetetraacetic acid (EDTA),
was found to be ineffective in enhancing absorption of insulin in
the rat model tested, other calcium ion-binding chelating agents
may prove to be more useful.
[0042] Proportions of Polypeptide and Enhancer
[0043] The relative proportions of polypeptide and enhancer may be
varied as desired. Sufficient enhancer must be present to permit
efficient absorption of the inhaled polypeptide; however, the
amount of enhancer should be kept as low as possible in order to
minimize the risk of adverse effects caused by the enhancer.
Although each particular polypeptide/enhancer combination must be
tested to determine the optimal proportions, it is expected that to
achieve acceptable absorption of the polypeptide, more than 10% of
the polypeptide/enhancer mixture must be enhancer; for most types
of enhancers, the proportion of enhancer should be more than 15% or
more than 20% and will preferably be between 25% and 50%. The
preferred ratio for each polypeptide/enhancer (or
polypeptide/enhancer/diluent) combination can be readily determined
by one of ordinary skill in the art of pharmacology by standard
methods, based on such criteria as efficient, consistent delivery
of the optimal dosage, minimization of side effects, and acceptable
rate of absorption.
[0044] No further ingredients are needed for the action of the
preparation, but may be included if desired. For example, the
amount of powder which constitutes a single dose of a given
polypeptide/surfactant combination could be increased (e.g., for
use in an inhaler apparatus which by design requires a large powder
volume per dose) by diluting the powder with pharmaceutically
acceptable diluents. Other additives may be included to facilitate
processing or to improve the powder properties or stability of the
preparation. A flavouring agent could be added so that the
proportion of the powder which is inevitably deposited in the mouth
and throat would serve to give the patient positive feedback that a
dose had been delivered from the inhaler device. Any such additive
should have the following properties: (a) it is stable and does not
disadvantageously affect the stability of the polypeptide and
enhancer; (b) it does not disadvantageously interfere with
absorption of the polypeptide; (c) it has good powder properties,
as that term is understood in the pharmaceutical arts; (d) it is
not hygroscopic; and (e) it has no adverse effects in the airways
in the concentrations used. Useful types of such additives include
mono-, di-, and polysaccharides, sugar alcohols, and other polyols:
for example, lactose, glucose, raffinose, melezitose, lactitol,
maltitol, trehalose, sucrose, mannitol, and starch. As reducing
sugars such as lactose and glucose have a tendency to form
complexes with proteins, non-reducing sugars such as raffinose,
melezitose, lactitol, maltitol, trehalose, sucrose, mannitol and
starch may be preferred additives for use in the present invention.
Such additives may constitute anywhere from 0% (i.e., no additive)
to nearly 100% of the total preparation.
[0045] In a preferred embodiment, this invention provides a
therapeutic preparation of a pharmaceutically active polypeptide
and a substance which enhances the absorption of said polypeptide
in the lower respiratory tract, which preparation is in the form of
a dry powder preparation suitable for inhalation of which at least
50% by mass consists of (a) particles having a diameter of less
than about 10 microns or (b) agglomerates of said particles; in
another preferred embodiment, the invention provides a therapeutic
preparation comprising a pharmaceutically active polypeptide, a
substance which enhances the absorption of polypeptide in the lower
respiratory tract, and a pharmaceutically acceptable carrier, which
preparation is in the form of a dry powder suitable for inhalation
of which at least 50% by mass consists of (a) particles having a
diameter of less than about 10 microns, or (b) agglomerates of said
particles; and in a further preferred embodiment this invention
provides a therapeutic preparation comprising active compounds (A)
a pharmaceutically active polypeptide and (B) a substance which
enhances the absorption of said polypeptide in the lower
respiratory tract, wherein at least 50% of the total mass of active
compounds (A) and (B) consists of particles having a diameter of
less than about 10 microns, and a pharmaceutically acceptable
carrier, which preparation is in the form of a dry powder
preparation suitable for inhalation in which an ordered mixture may
be formed between the active compounds and the pharmaceutically
acceptable carrier.
[0046] The described powder preparation could be manufactured in
several ways, using conventional techniques. In many cases, the
purified polypeptide can be obtained from commercial sources.
Alternatively, the polypeptide of interest can be purified from a
naturally occurring source using standard biochemical techniques,
or can be obtained by expression of prokaryotic or eukaryotic cells
genetically engineered to contain a nucleotide sequence which
encodes the polypeptide and has appropriate expression control
sequences linked thereto (including a transgenic animal engineered
to manufacture the desired peptide or protein, for example in its
milk). Such methods are standard in the art (e.g., see Sambrook et
al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989). Peptides (i.e.,
polypeptides having 30 or fewer amino acid residues) can be readily
synthesized by known chemical means.
[0047] Absorption enhancers as described above are also generally
available from commercial sources, or can be manufactured using
published methods. For ionic enhancers, the counterion associated
with the enhancer can be replaced with another, if desired, using
standard ion exchange techniques.
[0048] In manufacturing of the described powder preparation it will
in general be necessary to micronize the powder in a suitable mill,
e.g. a jet mill, at some point in the process, in order to produce
primary particles in a size range appropriate for maximal
deposition in the lower respiratory tract (i.e., under 10 .mu.m).
For example, one can dry mix polypeptide and enhancer powders, and
then micronize the substances together; alternatively, the
substances can be micronized separately, and then mixed. Where the
compounds to be mixed have different physical properties such as
hardness and brittleness, resistance to micronisation varies and
they may require different pressures to be broken down to suitable
particle sizes. When micronised together, therefore, the obtained
particle size of one of the components may be unsatisfactory. In
such case it would be advantageous to micronise the different
components separately and then mix them.
[0049] It is also possible first to dissolve the components in a
suitable solvent, e.g. water, to obtain mixing on the molecular
level. This procedure also makes it possible to adjust the pH-value
to a desired level, for instance to improve absorption of the
polypeptide. The pharmaceutically accepted limits of pH 3.0 to 8.5
for inhalation products must be taken into account, since products
with a pH outside these limits may induce irritation and
constriction of the airways. To obtain a powder, the solvent must
be removed by a process which retains the polypeptide's biological
activity. Suitable drying methods include vacuum concentration,
open drying, spray drying, and freeze drying. Temperatures over
40.degree. C. for more than a few minutes should generally be
avoided, as some degradation of certain polypeptides may occur.
Following the drying step, the solid material can, if necessary, be
ground to obtain a coarse powder, then, if necessary,
micronized.
[0050] If desired, the micronized powder can be processed to
improve the flow properties, e.g., by dry granulation to form
spherical agglomerates with superior handling characteristics,
before it is incorporated into the intended inhaler device. In such
a case, the device would be configured to ensure that the
agglomerates are substantially deagglomerated prior to exiting the
device, so that the particles entering the respiratory tract of the
patient are largely within the desired size range. Where an ordered
mixture is desired, the active compound may be processed, for
example by micronisation, in order to obtain, if desired, particles
within a particular size range. The carrier may also be processed,
for example to obtain a desired size and desirable surface
properties, such as a particular surface to weight ratio, or a
certain ruggedness, and to ensure optimal adhesion forces in the
ordered mixture. Such physical requirements of an ordered mixture
are well known, as are the various means of obtaining an ordered
mixture which fulfills the said requirements, and may be determined
easily by the skilled person according to the particular
circumstances.
[0051] A preferred inhalation apparatus would have the following
design characteristics: protection of the powder from moisture and
no risk of occasional large doses; in addition as many as possible
of the following are desired: protection of the powder from light;
high respirable fraction and high lung deposition in a broad flow
rate interval; low deviation of dose and respirable fraction; low
retention of powder in the mouthpiece--this is particularly
important for a multidose inhaler, where polypeptide retained in
the mouthpiece could degrade and then be inhaled together with
subsequent doses; low adsorption to the inhaler surfaces;
flexibility in dose size; and low inhalation resistance. The
inhaler is preferably a single dose inhaler although a multi dose
inhaler, such as a multi dose, breath actuated, dry powder inhaler
for multiple use, may also be employed. Peferably the inhaler used
is a unit dose, breath actuated, dry powder inhaler for single
use.
[0052] A number of dry powder formulations containing a polypeptide
and various enhancers have been prepared and tested in an in vivo
assay, and are described below. Also described is an in vitro assay
useful for testing polypeptide/enhancer combinations.
EXAMPLE 1
In Vitro Method of Determining Usefulness of Particular
Polypeptides for the Present Invention
[0053] A standard in vitro assay utilizing an epithelial cell line,
CaCo-2 (available through the American Type Culture Collection
(ATCC), Rockville, Md., USA), has been developed to assess the
ability of various enhancer compounds to promote transport of
markers across an epithelial cell monolayer, as a model for the
epithelial cell layer which functions in the lung to separate the
alveolus from the pulmonary blood supply.
[0054] In this assay, the enhancer and polypeptide or other marker
are dissolved in aqueous solution at various proportions and/or
concentrations, and applied to the apical side of the cell
monolayer. After 60 min incubation at 37.degree. C. and 95% RH
(relative humidity), the amount of the marker on the basolateral
side of the cells is determined, e.g., by use of a radioactively
labelled marker.
[0055] For the enhancer tested, sodium caprate, the amount of
marker (mannitol, MW 360) which appears on the basolateral side is
dependent upon the concentration of enhancer used, at least up to
16 mM sodium caprate (FIG. 1). This is true even when the
polypeptide insulin is added to the enhancer/mannitol mixture (1:3
sodium caprate:insulin, by weight) (FIG. 2). This concentration of
sodium caprate (16 mM) was also found to promote absorption across
the cell monolayer of two low molecular weight peptides, insulin
(MW 5734) and vasopressin (MW 1208). The amount of insulin -which
passed across the monolayer doubled in the presence of 16 mM sodium
caprate, compared to the amount in the absence of any enhancer; the
amount of vasopressin which was absorbed across the monolayer
increased 10-15 times compared to the amount in the absence of any
enhancer.
[0056] In contrast, no increase in transport rate was observed for
larger proteins such as cytochrome C (MW 12,300), carbonic
anhydrase (MW 30,000) and albumin (MW 69,000) when tested at up to
16 mM sodium caprate. It is expected that at higher concentrations
of sodium caprate, the permeability of the cells will be further
increased, permitting the transport of larger polypeptides;
however, the potential cytotoxicity of sodium caprate may prevent
the use of substantially higher concentrations of this particular
enhancer.
[0057] Other enhancers may permit transportation of larger
polypeptides; these may also be tested in this in vitro model of
epithelial cell permeability, which can be used as a screening tool
for rapidly testing any desired polypeptide/enhancer combination
for usefulness in the methods of the invention.
EXAMPLE 2
Method for Selecting Enhancers Useful for the Present Invention
[0058] Each of the compounds listed in Table I was tested for its
ability to enhance uptake of a polypeptide (insulin) in a rat
model. The results with insulin are taken as indicative of the
enhancer's potential for enhancement of absorption of other
polypeptides.
[0059] Various forms of insulin were employed in the different
trials: recombinant human, semisynthetic human or bovine. Each
formulation was prepared as above, drying and processing the
insulin/enhancer or insulin/enhancer/lactose solution to produce an
inhalable powder. The powder was administered to rats by
inhalation, and the blood glucose levels of the rats were
subsequently monitored as a measure of insulin uptake. These levels
were compared to the corresponding values obtained from rats which
had inhaled insulin formulations without enhancer.
[0060] The same in vivo model system could be used to test any
given peptide or protein for usefulness in the methods of the
invention, by delivering by the same inhalation method a
formulation containing the desired peptide or protein combined with
an enhancer, and assaying for the concentration of the desired
peptide or protein in the systemic circulation of the test animal
(e.g., by standard immunoassays or biochemical assays as
appropriate for the given peptide or protein).
1 TABLE I Enhancer:Insulin: Substance lactose Effect
Octylglucopyranoside 4:4:92 (+) Sodium ursodeoxycholate 4:4:92 +
Sodium taurocholate 4:4:92 + Sodium glycocholate 4:4:92 +
Lysophosphatidylcholine 4:4:92 + Dioctanoylphosphatidylcholine
2:4:94 (+) Didecanoylphospatidylcholine 4:4:94 - Sodium 2:4:94 +
taurodihydrofusidate Sodium caprylate 25:75:0 - Sodium caprate
10:90:0 (+) Sodium caprate 17.5:82.5:0 (+) Sodium caprate 25:75:0 +
Sodium caprate 4:4:92 + Sodium laurate 25:75:0 (+) Potassium oleate
4:4:92 + Potassium caprate 27:73:0 + Lysine caprate 35:65:0 +
Sodium myristate 30:70:0 + Dimethyl-.beta.-cyclodextrin 75:25:0 + +
effect, i.e. enhancer gives a significant decrease in blood glucose
level - no or very small effect (+) effect, not as marked as
"+"
EXAMPLE 3
Therapeutic Preparation According to the Invention
[0061] Human growth hormone (hGH, MW 22 kD, source Humatrope from
Lilly, 3 parts) was mixed with sodium caprate (1 part). The mixture
was milled in a Retsch mechanical mill to a particle size of mass
median diameter 6.7 .mu.m.
[0062] The resultant powder was administered intratraceally in rats
and the uptake of hGH compared with that of a powder, MMD 9.6
.mu.m, comprising hGH and mannitol in the same proportions and
prepared in the same way as above.
[0063] The results indicated an improvement in the uptake of hGH in
the formulation including sodium caprate, compared with the uptake
in the formulation without enhancer.
EXAMPLE 4
Preparation Containing the Polypeptide Insulin
[0064] Insulin is herein used as indicative of other polypeptides
according to the present invention.
[0065] Biosynthetic human insulin (53 g) was micronised in an
Airfilco Jet Mill (Trade Mark, Airfilco Process Plant Limited),
with pressurised nitrogen (feed pressure 7 bar, chamber pressure 5
bar), to a mass median diameter of 2.4 micrometers.
[0066] Sodium caprate (170 g) was micronised in an Airfilco Jet
Mill (TM), with pressurised nitrogen (feed pressure 5 bar, chamber
pressure 3 bar), to a mass median diameter of 1.6 micrometers.
[0067] The micronised biosynthetic human insulin (45 g) and sodium
caprate (14.26 g) were dry mixed according to the following
procedure: Half of the insulin was added to a mixing device
comprising a mixing cylinder of volume 4.4 litres divided, by a
sieve of width 1 mm, into two compartments, with a metal ring in
each compartment to aid mixing and stirring. The sodium caprate and
finally the rest of the insulin, were added. The mixing cylinder
was closed, turned 180 degrees, and mounted in a motorised shaking
apparatus. The motor was turned on and shaking continued for
approximately two minutes, until all the insulin and sodium caprate
had passed through the sieve. The motor was turned off and the
mixing cylinder turned 180 degrees, again mounted on the shaking
apparatus and shaking was again effected until all the powder had
passed through the sieve. This procedure was repeated a further
eight times to give a total mixing time of approximately 20
minutes.
[0068] The preparation so obtained was administered to 5 dogs by
inhalation, at a dosage level of 1 U./kg, and the plasma insulin
level determined at various time points after administration.
[0069] The results obtained were compared with the plasma insulin
levels obtained when biosynthetic insulin, micronised as above to a
mass median diameter of 2.4 micrometers, were administered to five
dogs in the same way and at the same dosage levels, and with the
plasma insulin levels obtained when a therapeutic preparation of
insulin and sodium caprate in a ratio of 90:10 was administered to
five dogs in the same way and at the same dosage levels as above.
In this case the therapeutic preparation was prepared as follows:
Human semisynthetic insulin was gel filtrated to reduce the zinc
content from 0.52% to 0.01% relative to content of insulin. Insulin
(4.5 g) and sodium caprate (0.5 g) were dissolved in water (232
ml). The solution was stirred until clear and the pH adjusted to
7.0. The solution was concentrated by evaporation at 37.degree. C.
over a period of about two days. The obtained solid cake was
crushed, and sieved through a 0.5 mm sieve, and the resultant
powder micronised through a jet mill to particles with a mass
median diameter of 3.1 micrometers.
[0070] The results of these comparisons are presented in FIG. 3
(p=0.0147 for the difference between 75:25 and 100:0). The results
demonstrate some improvement in the bioavailability of insulin with
the 90:10 formulation, and a dramatic improvement in the
bioavailability of insulin with the 75:25 preparation including
sodium caprate, as compared to insulin alone.
* * * * *