Note: Descriptions are shown in the official language in which they were submitted.
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INSULIN CRYSTALS FOR PULMONARY ADMINISTRATION
Cross Reference
This application claims the benefit of U.S.
Provisional Application No. 60/131,170 filed on April 27,
1999, said application of which is entirely incorporated
herein by reference.
Background of the Invention
1. Field of the Invention. The invention is in
the field of human medicine. More particularly, the
invention is in the field of the treatment of diabetes and
hyperglycemia.
2. Description of Related Art. It has long been
s5 a goal of insulin therapy to mimic the pattern of endogenous
insulin secretion in normal individuals. The daily
physiological demand for insulin fluctuates and can be
separated into two phases: (a) the absorptive phase
requiring a pulse of insulin to dispose of the meal-related
2o blood glucose surge, and (b) the post-absorptive phase
requiring a sustained delivery of insulin to regulate
hepatic glucose output for maintaining optimal fasting blood
glucose. Accordingly, effective therapy for people with
diabetes generally involves the combined use of two types of
25 exogenous insulin formulations: a fast-acting meal time
insulin provided by bolus injections and a long-acting, so-
called, basal insulin, administered by injection once or
twice daily to control blood glucose levels between meals.
Conventional insulin therapy involves only two
3o injections per day. More intensive insulin therapy
involving three or more injections of insulin each day
results in reduction of complications, as demonstrated in
the Diabetes Control and Complications Trial (DCCT) study.
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Unfortunately, many diabetics are unwilling to undertake
intensive therapy due to the discomfort associated with the
many injections required to maintain close control of
glucose levels. A non-injectable form of insulin is
desirable for increasing patient compliance with intensive
insulin therapy and lowering their risk of complications.
Many investigators have studied non-injectable
forms of insulin, such as oral, rectal, transdermal, and
nasal routes. So far, these types of administration have
1o not been effective due to poor insulin absorption, low serum
insulin concentration, irritation at the site of delivery,
or lack of significant decrease in serum glucose levels.
Another well-studied route for administering non-
injectable insulin is by the lung. Due to its relatively
small molecular weight (5,800 daltons) insulin seems to be
an ideal candidate for administration through the lungs.
Administration of insulin by inhalation, and its absorption
through the lung was first reported in 1925.
However, after administration by inhalation,
2o small-sized proteins like insulin are absorbed rapidly from
the lung due to the very large surface area and relatively
porous membrane of the lung. The plasma concentration peaks
quickly, but also decreases quickly. These pharmacokinetics
may be suited for controlling blood glucose during the
absorptive phase, but are completely unsuited during the
post-absorptive phase. Therefore, means for administration
of long-acting insulin by inhalation remains a challenge.
The following are reviews of the inhalation of
insulin and other proteins: "Aerosol Insulin- A Brief
3o Review", Patton, J. S. and Platz, R. M., in Respiratory Drug
Delivery IV, P. Byron, Ed., Interpharm Press (1994);
"Delivery of Biotherapeutics by Inhalation Aerosol", Niven,
R., Crit. Rev. in Therapeutic Drug Carrier Systems 12:151-
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231 (1995); and "Drug Delivery via the Respiratory Tract",
Byron, P. R. and Patton, J. S., J. Aerosol Medicine 7:49-75
(1994).
The efficiency of delivery and deposition of
particles onto the surface of the deep lung, i.e. to the
alveoli, is strongly related to the mass median aerodynamic
diameter (MMAD) of the particles. The optimal MMAD is about
2-3 microns. Above and below this range, less material will
be deposited onto the alveolar surface.
1o Phagocytosis of insoluble or slowly dissolving
particles is the most significant clearance mechanism in the
deep lung [Rudt, S. H., et al., J. Controlled Release
25:123-138 (1993); Tabata, Y., et al., J. Biomedical
Material Res. 22:837-842 (1988); Wang, J. A., et al., In:
Respiratory Drug Delivery. Buffalo Grove, IL; Interpharm
1997, vol. VI, pp. 187-192; Edwards, D. A., et al., Science
276:1868-1871 (1997); Gehr, P. M., Microsc. Res. Tech.
26:423-436 (1993)]. Once deposited on the alveolar surface,
particles having actual dimensions in the range of about 1
to about 10 microns may be phagocytosed by lung macrophages.
It is known that very large particles, such as those having
actual diameters above about 10 microns, are not efficiently
phagocytosed by lung macrophages. Small particles with
actual dimensions in the nanometer size range, likely also
escape macrophage ingestion.
Unfortunately, particles having optimal properties
for delivery and deposition often have actual particle
dimensions that fall in the range within which macrophage
attack is expected.
3o Edwards, et a1. describe protracted release of
insulin from large, porous particles administered into the
deep lung by inhalation [J. Appl. Physiol. 85:379-385
(1998); Science 276:1868-1871 (1997)]. The persistence of
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the administered particles was attributed to the large
actual size of the particles (greater than 10 microns),
while their high rate of deposition into the deep lung was
attributed to the low density of the particles which
resulted in relatively small aerodynamic size (approximately
3-4 micrometers MNlAD) despite their large physical size.
These particles comprise insulin encapsulated in a
biodegradable copolymer (poly-lactic-co-glycolic acid).
Slow degradation of the copolymer releases insulin over a
period of at least four days. These publications
demonstrate that a depot effect in the lung is a feasible
mechanism for producing a sustained delivery of insulin.
Crystallization of insulin, such as with NPH-
insulin and the various Lente products, is a well-known
means to provide extended control of blood glucose in people
with diabetes. Insulin crystals have uniformly been
administered by parenteral routes, ususally subcutaneously.
Acceptably high bioavailability of insulin-containing
crystals when delivered to the deep lung by inhalation has
2o not been achieved. This is likely due to the complex
protective mechanisms involving the biology, immunology, and
chemistry of the lung surface relating to clearing of air-
borne particles, especially microbes and aerosols containing
microbes, which have particle qualities that permit their
evasion of upper respiratory entrapment mechanisms.
Hughes, B. L., et a1. [PCT/US98/23040, filed 29
October 1998 described pharmaceutical compositions and
methods of administering fatty acid-acylated insulin and
insulin analogs by inhalation to treat diabetes. The
3o compositions were solutions or powders of amorphous
materials, but not crystals. Hughes, et al. demonstrated
that derivatized insulins, B28-NE-myristoyl-LysB28,ProB29-
human insulin analog and B29-Ne-palmitoyl-human insulin,
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were absorbed in an amount effective to reduce glucose
levels. The pharmacokinetics of the fatty acid-acylated
insulins administered via the lung was protracted compared
with non-acylated insulin, but the length of protraction was
shorter than that of NPH-insulin delivered subcutaneously.
Havelund, S. [W098/42749, published 1 October
1998] describes zinc-free crystal powders of insulin,
insulin analogs, and insulin derivatives that are claimed to
be resistant to aggregation and clumping, and allegedly
suitable for administration by inhalation.
Whittingham, J. L., et a1. [Biochemistry 36:2826-
2831 (1997)] produced very large crystals comrpised of B29-
N~-tetradecanoyl-des(B30)-human insulin analog and zinc for
structural studies by X-ray crystallography. These crystals
were much too large to expect efficient deposition in the
deep lung when administered by inhalation. These crystals
would have to have been milled to produce material of
suitable particle size to achieve efficient deposition when
administered by inhalation.
2o Brader, M. [U. S. Patent Application No. 09/177685,
filed 22 October 1998; PCT/US98/22434; European Patent
Publication No. 0911035, published 28 April 1999, herein
Brader I] and Brader, M. [U.S. Patent Application No.
09/217275, filed 21 December 1998; PCT/US98/27299, herein
Brader II] describe microcrystals comprising divalent metal
cations together with derivatized proteins, including
derivatized insulin and derivatized insulin analogs,
processes for making the crystals, and methods for
administering them to treat diabetes. The crystals are rod-
3o shaped, and have the size of rod-shaped commercial NPH-
insulin crystals, which is about 5 microns in length. Such
crystals are thought to be too large to obtain optimal
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deposition in the deep lung when administered by inhalation
through the mouth.
Brader II lays out a long list of parameters that
were thought to affect crystallization rate and size, and
among them were mentioned temperature and the concentration
of competing compounds, such as citrate. Brader II does not
however, specify the relationship between crystal size and
temperature, and the effect of competing compound on size is
only inferred from its likely effect in slowing down the
rate of crystallization. Furthermore, Brader II does not
mention the chloride anion among the many parameters thought
to influence crystal size of derivatized proteins.
Summary of the Invention
The invention includes a stable population of
crystals comprised of a derivatized insulin or a de~ivatized
insulin analog and a divalent metal cation, characterized in
that the crystals have a mean diameter in the range of 1-3
microns.
More specifically, the invention is crystals
having a uni-modal, symmetric particle distribution,
comprising:
a) derivatized insulin of the formula B29-N~-X-
human insulin, wherein X is selected from
the group consisting of butyryl, pentanoyl,
hexanoyl, heptanoyl, octanoyl, nonanyl, and
decanoyl;
b) zinc;
c) a phenolic preservative; and
d) protamine;
3o characterized in that the volume mean
spherical equivalent diameter is from 1 microns to 3
microns.
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The invention includes a stable population of co-
crystals comprised of an insulin or an insulin analog and a
derivatized insulin or a derivatized insulin analog,
characterized in that the co-crystals have a mean diameter
in the range of 1-3 microns. Thus, more specifically, the
invention also includes co-crystals having a uni-modal,
symmetric particle distribution, comprising insulin and:
a) derivatized insulin of the formula B29-N~-X-
human insulin, wherein X is selected from
1o the group consisting of butyryl, pentanoyl,
hexanoyl, heptanoyl, octanoyl, nonanyl, and
decanoyl;
b) zinc;
c) a phenolic preservative; and
d) protamine;
characterized in that the volume mean spherical
equivalent diameter is from 1 microns to 3 microns, wherein
the wherein the proportion of derivatized insulin relative
to total protein is at least SOo.
2o The invention also includes pharmaceutical
compositions comprising crystals or co-crystals together
with one or more pharmaceutically acceptable excipients
carriers, or with an aqueous solvent in which the crystals
are stable. The pharmaceutical composition may be for
parenteral administration, or more preferably, for
administration by inhalation through the mouth of the
patient for deposition in the deep lung (the alveolae).
For parenteral pharmaceutical compositions, the
crystals are suspended in a pharmaceutically acceptable
aqueous solvent, comprising optionally an isotonicity agent,
a buffer, a preservative, and insulin or an insulin analog.
For pharmaceutical compositions for pulmonary
administration, the crystals may be in the form of a dry
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powder (for delivery by a dry powder inhaler for example),
optionally together with other dry excipients, dry powders
of insulin or an insulin analog, and carrier particles. The
crystals may also be formulated for administration by
inhalation through the mouth of the patient in a liquid form
by suspending them in a pharmaceutically acceptable aqueous
solvent, comprising optionally insulin or an insulin analog.
The invention provides a process for preparing
crystals and co-crystals of a size that increases the
1o efficiency of their deposition in the deep lung when
administered by inhalation through the mouth. The process
involves preparing a suspension having neutral pH, in the
absence of citrate, by carrying out steps a) - f) in any
order, provided that step f) follows step a), and provided
z5 that if step f) precedes step e), then steps b) and c)
precede step f):
a) dissolving a derivatized insulin in an
aqueous solvent at acidic pH;
b) adding a phenolic preservative;
2o c) adding zinc;
d) adding chloride anion to a final
concentration of from about 100 mM to about
150 mM chloride anion above that introduced
by pH adjustment;
25 e) adding protamine;
f) adjusting to a neutral pH;
and then holding the temperature of the neutral pH
suspension between about 25°C and about 37°C for between 12
hours and about 96 hours.
3o The invention also encompasses the use of crystals
in the manufacture of a medicament for the treatment of
diabetes or hyperglycemia by inhalation, which treatment
comprises administering to a patient in need thereof an
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effective amount of the medicament using an inhalation
device, such that the medicament is deposited in the lungs
of the patient.
The invention also encompasses the use of co-
crystals in the manufacture of a medicament for the
treatment of diabetes or hyperglycemia by inhalation, which
treatment comprises administering to a patient in need
thereof an effective amount of the medicament using an
inhalation device, such that the medicament is deposited in
1o the lungs of the patient.
The present invention also provides a method for
administering crystals or co-crystals by inhalation.
The invention also provides crystals and co-
crystals that are advantageously more physically stable and
z5 easier to resuspend than crystals and co-crystals produced
by the methods described by Brader I and Brader II. The
process for making crystals and co-crystals with improved
properties involves preparing a suspension having neutral
pH, in the absence of citrate, by carrying out steps a) - g)
2o in any order, provided that step g) follows step a), and
provided that if step g) precedes step f), then steps b) and
c) precede step g):
a) dissolving a derivatized insulin in an
aqueous solvent at acidic pH;
25 b) adding a phenolic preservative;
c) adding zinc;
d) adding chloride anion to a final
concentration of from about 15 mM to about
150 mM chloride anion above that introduced
30 by pH adjustment;
e) adding citrate to a concentration of from 1
mM to 10 mM;
f) adding protamine;
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g) adjusting to a neutral pH;
and then holding the temperature of the neutral pH
suspension between about 20°C and about 37°C for between 12
hours and about 96 hours. For co-crystals, insulin is also
dissolved in the above process, and the proportion of
derivatized insulin relative to total protein is at least
500. The crystals produced by this process are more
physically stable than crystals produced according to the
teachings of Brader I and Brader II, and they resuspend more
to readily also. These are advantages to the patient.
The present invention is the discovery of the
dramatic and unexpected effect of chloride ions on particle
size of crystals and co-crystals, and on their physical
stability and re-suspendability. The ability to make
z5 crystals and co-crystals having volume mean spherical
equivalent diameters in the range of about 1 micron-to about
3 microns, which are ideally suited for pulmonary deposition
in high efficiency without additional particle
classification or size-reduction, came from the additional
2o discovery of the combined effects of chloride ion, higher
temperature, and low levels or absence of citrate.
The present invention solves two problems
currently not addressed by the art. First, previous
pulmonary methods for delivering insulin do not provide
25 adequate time action to control blood glucose between meals
and overnight. Second, presently known soluble, long-acting
insulins and insulin derivatives are delivered by
subcutaneous injection, which involves the inconvenience of
preparing a sample for injection, and the pain of a needle
30 stick.
According to the present invention, a patient in
need of insulin to control blood glucose levels will benefit
from an advantageous slow uptake and prolonged persistence
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of insulin activity compared to inhalation of un-derivatized
insulin, and also a reduction of inconvenience and pain
compared with subcutaneous delivery. The crystals or co-
crystals can be delivered in a carrier, as a solution or
suspension, or as a dry powder, using any of a variety of
devices suitable for administration by inhalation. The
acylated insulin can be administered using an inhalation
device such as a nebulizer, a metered-dose inhaler, a dry
powder inhaler, a sprayer, and the like.
to The invention also provides a method for
administering a pharmaceutical composition comprising either
crystals or co-crystals and insulin or an insulin analog to
a patient in need thereof by inhalation. Administering such
combinations provides both post-prandial and basal control
of blood glucose levels. Because the method avoids
injections, patient comfort is improved, and patien-~.
compliance increased compared with conventional insulin
delivery methods.
Brief Description of the Drawing
Figure 1 depicts blood glucose levels following
administration of crystals and co-crystals to F344 rats:
intratracheal instillation of 1 mg/kg of 1000 C8-BHI (solid
squares, solid line); intratracheal instillation of 1 mg/kg
of 75o C8-BHI:25% BHI (open squares, solid line);
subcutaneous administration of 1 mg/kg of 75o C8-BHI:25o BHI
(solid circles, dashed line); and subcutaneous
administration of 1 mg/kg of NPH insulin as control (solid
triangles, solid line).
Detailed Description of the Invention
As used herein, the term "crystal" means a
microcrystal comprising derivatized insulin or derivatized
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insulin analog, a divalent metal cation, a complexing
compound, and a hexamer-stabilizing compound. Crystals of
this type were originally described by Brader, M. [U. S.
Patent Application No. 09/177685, filed 22 October 1998;
PCT/US98/22434; European Patent Publication No. 0911035,
published 28 April 1999].
As used herein, the term "co-crystal" means a
microcrystal comprising insulin or an insulin analog,
derivatized insulin or derivatized insulin analog, a
l0 divalent metal cation, a complexing compound, and a hexamer-
stabilizing compound. Co-crystals of this type were
originally described by Brader, M. [U. S. Patent Application
No. 09/217275, filed 21 December 1998; PCT/US98/27299].
The term "microcrystal" means a solid that is
comprised primarily of matter in a crystalline state, and
are of a microscopic size, typically of longest dimension
within the range 1 micron to 100 microns. The term
"microcrystalline" refers to the state of being a
microcrystal.
The term "rod-like" means the distinctive
microcrystal morphology that is also described as pyramidal-
tipped tetragonal rods. The morphology of microcrystals of
the present invention are easily determined by microscopic
examination.
The term "protein" may have its common meaning,
that is, a polymer of amino acids. The term "protein," as
used herein, also has a narrower meaning, that is, a protein
selected from the group consisting of insulin, insulin
analogs, and proinsulins. The term "un-derivatized protein"
also refers to a protein selected from the group consisting
of insulin, insulin analogs, and proinsulins.
As used in the claims, and elsewhere as the
context dictates, the term "total protein" refers to the
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combined amount of protein (insulin, an insulin analog, or a
proinsulin) and derivatized protein (derivatized insulin, a
derivatized insulin analog, or a derivatized proinsulin).
Although protamine and other known complexing compounds are
also proteins in the broadest sense of that term, the term
"total protein" does not include them.
The term "derivatized protein" refers to a protein
selected from the group consisting of derivatized insulin,
and derivatized insulin analogs that is derivatized by a
to functional group such that the derivatized protein is either
less soluble in an aqueous solvent than is the un-
derivatized protein, is more lipophilic than un-derivatized
insulin, or produces a complex with zinc and protamine that
is less soluble than the corresponding complex with the un-
derivatized protein. The determination of either the
solubility or lipophilicity of proteins and derivatzzed
proteins is well-known to the skilled person. The
solubility of derivatized proteins and protein in complexes
with zinc and protamine can be readily determined by well-
known procedures [Graham and Pomeroy, J. Pharm. Pharmacol.
36:427-430 (1983), as modified in DeFelippis, M. R. and
Frank, B., EP 735,048], or the procedure used herein.
Many examples of such derivatized proteins are
known in the art, including benzoyl, p-tolyl-sulfonamide
carbonyl, and indolyl derivatives of insulin and insulin
analogs [Havelund, S., et al., W095/07931, published 23
March 1995); alkyloxycarbonyl derivatives of insulin
[Geiger, R., et al., U.S. Patent No. 3,684,791, issued 15
August 1972; Brandenberg, D., et al., U.S. 3,907,763, issued
23 September 1975); aryloxycarbonyl derivatives of insulin
[Brandenberg, D., et al., U.S. 3,907,763, issued 23
September 1975); alkylcarbamyl derivatives [Smyth, D. G.,
U.S. Patent No. 3,864,325, issued 4 February 1975; Lindsay,
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D. G., et al., U.S. Patent No. 3,950,517, issued 13 April
1976]; carbamyl, 0-acetyl derivatives of insulin [Smyth, D.
G., U.S. Patent No. 3,864,325 issued 4 February 1975];
cross-linked, alkyl dicarboxyl derivatives [Brandenberg, D.,
et al., U.S. Patent No. 3,907,763, issued 23 September
1975]; N-carbamyl, 0-acetylated insulin derivatives [Smyth,
D. G., U.S. Patent No. 3,868,356, issued 25 February 1975];
various O-alkyl esters [Markussen, J., U.S. Patent No.
4,343,898, issued 10 August 1982; Morihara, K., et al., U.S.
to Patent No. 4,400,465, issued 23 August 1983; Morihara, K.,
et al., U.S. Patent No. 4,401,757, issued 30 August 1983;
Markussen, J., U.S. Patent No. 4,489,159, issued 18 December
1984; Obermeier, R., et al., U.S. Patent No. 4,601,852,
issued 22 July 1986; and Andresen, F. H., et al., U.S.
Patent No. 4,601,979, issued 22 July 1986]; alkylamide
derivatives of insulin [Balschmidt, P., et al., U.S. Patent
No. 5,430,016, issued 4 July 1995]; various other
derivatives of insulin [Lindsay, D. G., U.S. Patent No.
3,869,437, issued 4 March 1975]; and the fatty acid-acylated
2o proteins that are described herein.
The term "acylated protein" as used herein refers
to a derivatized protein selected from the group consisting
of insulin and insulin analogs that is acylated with an
organic acid moiety that is bonded to the protein through an
amide bond formed between the acid group of an organic acid
compound and an amino group of the protein. In general, the
amino group may be the oc-amino group of an N-terminal amino
acid of the protein, or may be the ~-amino group of a Lys
residue of the protein. An acylated protein may be acylated
3o at one or more of the three amino groups that are present in
insulin and in most insulin analogs. Mono-acylated proteins
are acylated at a single amino group. Di-acylated proteins
are acylated at two amino groups. Tri-acylated proteins are
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acylated at three amino groups. The organic acid compound
may be, for example, a fatty acid, an aromatic acid, or any
other organic compound having a carboxylic acid group that
will form an amide bond with an amino group of a protein,
and that will lower the aqueous solubility, raise the
lipophilicity, or decrease the solubility of zinc/protamine
complexes of the derivatized protein compared with the un-
derivatized protein.
The term "fatty acid-acylated protein" refers to a
1o an acylated protein selected from the group consisting of
insulin and insulin analogs that is acylated with a fatty
acid that is bonded to the protein through an amide bond
formed between the acid group of the fatty acid and an amino
group of the protein. In general, the amino group may be
the a-amino group of an N-terminal amino acid of the
protein, or may be the ~-amino group of a Lys residue of the
protein. A fatty acid-acylated protein may be acylated at
one or more of the three amino groups that are present in
insulin and in most insulin analogs. Mono-acylated proteins
2o are acylated at a single amino group. Di-acylated proteins
are acylated at two amino groups. Tri-acylated proteins are
acylated at three amino groups. Fatty acid-acylated insulin
is disclosed in a Japanese patent application 1-254,699.
See also, Hashimoto, M., et al., Pharmaceutical Research,
6:171-176 (1989), and Lindsay, D. G., et al., Biochemical J.
121:737-745 (1971). Further disclosure of fatty acid-
acylated insulins and fatty acylated insulin analogs, and of
methods for their synthesis, is found in Baker, J. C., et
al, U.S. 08/342,931, filed 17 November 1994 and issued as
3o U.S. Patent No. 5,693,609, 2 December 1997; Havelund, S., et
al., W095/07931, published 23 March 1995, and a
corresponding U.S. Patent No. 5,750,497, 12 May 1998; and
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Jonassen, I., et al., W096/29342, published 26 September
1996.
The term "fatty acid-acylated protein" includes
pharmaceutically acceptable salts and complexes of fatty
acid-acylated proteins. The term "fatty acid-acylated
protein" also includes preparations of acylated proteins
wherein the population of acylated protein molecules is
homogeneous with respect to the site or sites of acylation.
For example, N~-mono-acylated protein, B1-Na-mono-acylated
1o protein, A1-NOC-mono-acylated protein, A1,B1-NGC-di-acylated
protein, Ne,A1-NO~.,di-acylated protein, N~,B1-NOC,di-acylated
protein, and N~,A1,B1-NOC,tri-acylated protein are all
encompassed within the term "fatty acid-acylated protein"
for the purpose of the present invention. The term also
refers to preparations wherein the population of acylated
protein molecules has heterogeneous acylation. In the
latter case, the term "fatty acid-acylated protein" includes
mixtures of mono-acylated and di-acylated proteins, mixtures
of mono-acylated and tri-acylated proteins, mixtures of di-
2o acylated and tri-acylated proteins, and mixtures of mono-
acylated, di-acylated, and tri-acylated proteins.
The term "insulin" as used herein, refers to human
insulin, whose amino acid sequence and special structure are
well-known. Human insulin is comprised of a twenty-one
amino acid A-chain and a thirty-amino acid B-chain which are
cross-linked by disulfide bonds. A properly cross-linked
insulin contains three disulfide bridges: one between
position 7 of the A-chain and position 7 of the B-chain, a
second between position 20 of the A-chain and position 19 of
the B-chain, and a third between positions 6 and 11 of the
A-chain.
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The term "insulin analog" means proteins that have
an A-chain and a B-chain that have substantially the same
amino acid sequences as the A-chain and B-chain of human
insulin, respectively, but differ from the A-chain and B-
chain of human insulin by having one or more amino acid
deletions, one or more amino acid replacements, and/or one
or more amino acid additions that do not destroy the insulin
activity of the insulin analog.
"Animal insulins" are analogs of human insulin,
l0 and therefore, are insulin analogs, as defined herein. Four
such animal insulins are rabbit, pork, beef, and sheep
insulin. The amino acid substitutions that distinguish
these animal insulins from human insulin are presented below
for the reader's convenience.
Amino Acid Position
A8 A9 A10 B3 0-
human insulin Thr Ser Ile Thr
rabbitinsulin Thr Ser Ile Ser
pork insulin Thr Ser Ile Ala
beef insulin Ala Ser Val Ala
sheep insulin Ala Gly Val Ala
Another type of insulin analog, "monomeric insulin
analog" is well-known in the art. Monomeric insulin analogs
are structurally very similar to human insulin, and have
activity similar or equal to human insulin, but have one or
2o more amino acid deletions, replacements or additions that
tend to disrupt the contacts involved in dimerization and
hexamerization which results in their having less tendency
to associate to higher aggregation states. Monomeric
insulin analogs are rapid-acting analogs of human insulin,
and are disclosed, for example, in Chance, R. E., et al.,
U.S. patent No. 5,514,646, 7 May 1996; Brems, D. N., et a1.
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Protein Engineering, 5:527-533 (1992); Brange, J. J. V., et
al., EPO publication No. 214,826, published 18 March 1987;
Brange, J. J. V., et al., U.S. Patent No. 5,618,913, 8 April
1997; and Brange, J., et al., Current Opinion in Structural
Biology 1:934-940 (1991). An example of monomeric insulin
analogs is described as human insulin wherein Pro at
position B28 is substituted with Asp, Lys, Leu, Val, or Ala,
and wherein Lys at position B29 is Lys or is substituted
with Pro, and also, AlaB26-human insulin, des(B28-B30)-human
insulin, and des(B27)-human insulin. The monomeric insulin
analogs employed as derivatives in the present crystals, or
employed un-derivatized in the solution phase of suspension
formulations, are properly cross-linked at the same
positions as is human insulin.
Another group of insulin analogs for use in the
present invention are those wherein the isoelectric point of
the insulin analog is between about 7.0 and about 8Ø
These analogs are referred to as "pI-shifted insulin
analogs." Examples of such insulin analogs include
ArgB3l,ArgB32-human insulin, GlyA2l,ArgB3l,ArgB32-human
insulin, ArgAO,ArgB3l,ArgB32-human insulin, and
ArgAO,GlyA2l,ArgB3l,ArgB32-human insulin.
Another group of insulin analogs consists of
insulin analogs that have one or more amino acid deletions
that do not significantly disrupt the activity of the
molecule. This group of insulin analogs is designated
herein as "deletion analogs." For example, insulin analogs
with deletion of one or more amino acids at positions B1-B3
are active. Likewise, insulin analogs with deletion of one
or more amino acids at positions B28-B30 are active.
Examples of "deletion analogs" include des(B30)-human
insulin, desPhe(B1)-human insulin, des(B27)-human insulin,
des(B28-B30)-human insulin, and des(B1-B3)-human insulin.
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The deletion analogs employed as derivatives in the present
crystals, or employed un-derivatized in the solution phase
of suspension formulations, are properly cross-linked at the
same positions as is human insulin.
Amidated amino acids, and particularly asparagine
residues in insulin, are known to be chemically unstable
[Jorgensen, K. H., et al. U.S. Patent No. 5,008,241, issued
16 April, 1991; Dorschug, M., U.S. Patent No. 5,656,722,
issued 12 August, 1997]. Particularly, they are prone to
l0 deamidation and various rearrangement reactions under
certain conditions that are well-known. Therefore,
optionally, an insulin analog may be insulin or an insulin
analog that has one or more of its amidated residues
replaced with other amino acids for the sake of chemical
stability. For example, Asn or Gln may be replaced with a
non-amidated amino acid. Preferred amino acid replacements
for Asn or Gln are Gly, Ser, Thr, Asp or Glu. It is
preferred to replace one or more Asn residues. In
particular, AsnAl8, AsnA2l, or AsnB3, or any combination of
2o those residues may be replaced by Gly, Asp, or Glu, for
example. Also, G1nA15 or GlnB4, or both, may be replaced by
either Asp or Glu. Preferred replacements are Asp at B21,
and Asp at B3. Also preferred are replacements that do not
change the charge on the protein molecule, so that
replacement of Asn or Gln with neutral amino acids is also
preferred.
The term "proinsulin" means a single-chain peptide
molecule that is a precursor of insulin. Proinsulin may be
converted to insulin or to an insulin analog by chemical or,
3o preferably, enzyme-catalyzed reactions. In proinsulin,
proper disulfide bonds are formed as described herein.
Proinsulin comprises insulin or an insulin analog and a
connecting bond or a connecting peptide. A connecting
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peptide has between 1 and about 35 amino acids. The
connecting bond or connecting peptide connects to a terminal
amino acid of the A-chain and to a terminal amino acid of
the B-chain by an a-amide bond or by two a-amide bonds,
respectively. Preferably, none of the amino acids in the
connecting peptide is cysteine. Preferably, the C-terminal
amino acid of the connecting peptide is Lys or Arg.
Proinsulin may have the formula X-B-C-A-Y or may have the
formula X-A-C-B-Y, wherein X is hydrogen or is a peptide of
1o from 1 to about 100 amino acids that has either Lys or Arg
at its C-terminal amino acid, Y is hydroxy, or is a peptide
of from 1 to about 100 amino acids that has either Lys or
Arg at its N-terminal amino acid, A is the A-chain of
insulin or the A-chain of an insulin analog, C is a peptide
of from 1 to about 35 amino acids, none of which is
cysteine, wherein the C-terminal amino acid is Lys or Arg,
and B is the B-chain of insulin or the B-chain of an insulin
analog.
A "pharmaceutically acceptable salt" means a salt
formed between any one or more of the charged groups in a
protein and any one or more pharmaceutically acceptable,
non-toxic cations or anions. Organic and inorganic salts
include, for example, those prepared from acids such as
hydrochloric, sulfuric, sulfonic, tartaric, fumaric,
hydrobromic, glycolic, citric, malefic, phosphoric, succinic,
acetic, nitric, benzoic, ascorbic, p-toluenesulfonic,
benzenesulfonic, naphthalenesulfonic, propionic, carbonic,
and the like, or for example, ammonium, sodium, potassium,
calcium, or magnesium.
3o The verb "acylate" means to form the amide bond
between a fatty acid and an amino group of a protein. A
protein is "acylated" when one or more of its amino groups
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is combined in an amide bond with the acid group of a fatty
acid.
The term "fatty acid" means a saturated or
unsaturated, straight chain or branched chain fatty acid,
having from one to eighteen carbon atoms.
The term "C1 to C18 fatty acid" refers to a
saturated, straight chain or branched chain fatty acid
having from one to eighteen carbon atoms.
The term "divalent metal cation" refers to the ion
or ions that participate to form a complex with a
multiplicity of protein molecules. The transition metals,
the alkaline metals, and the alkaline earth metals are
examples of metals that are known to form complexes with
insulin. The transitional metals are preferred. Zinc is
particularly preferred. Other transition metals that may be
pharmaceutically acceptable for complexing with insulin
proteins include copper, cobalt, and iron.
The term "complex" has two meanings in the present
invention. In the first, the term refers to a complex
2o formed between one or more atoms in the proteins that form
the complex and one or more divalent metal cations. The
atoms in the proteins serve as electron-donating ligands.
The proteins typically form a hexamer complex with divalent
transition metal cations. The second meaning of "complex"
in the present invention is the association between the
complexing compound and hexamers. The "complexing compound"
is an organic molecule that typically has a multiplicity of
positive charges that binds to, or complexes with hexamers
in the insoluble composition, thereby stabilizing them
3o against dissolution. Examples of complexing compounds
suitable in the present invention include protamine, surfen,
various globin proteins [Brange, J. , Galenics of Insulin,
Springer-Verlag, Berlin Heidelberg (1987)], and various
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polycationic polymer compounds known to complex with
insulin.
The term "protamine" refers to a mixture of
strongly basic proteins obtained from fish sperm. The
average molecular weight of the proteins in protamine is
about 4,200 [Hoffmann, J. A., et al., Protein Expression and
Purification, 1:127-133 (1990)]. "Protamine" can refer to a
relatively salt-free preparation of the proteins, often
called "protamine base." Protamine also refers to
to preparations comprised of salts of the proteins. Commercial
preparations vary widely in their salt content.
Protamines are well-known to those skilled in the
insulin art and are currently incorporated into NPH insulin
products. A pure fraction of protamine is operable in the
z5 present invention, as well as mixtures of protamines.
Commercial preparations of protamine, however, are typically
not homogeneous with respect to the proteins present. These
are nevertheless operative in the present invention.
Protamine comprised of protamine base is operative in the
20 present invention, as are protamine preparations comprised
of salts of protamine, and those that are mixtures of
protamine base and protamine salts. Protamine sulfate is a
frequently used protamine salt. All mass ratios referring
to protamine are given with respect to protamine free base.
25 The person of ordinary skill can determine the amount of
other protamine preparations that would meet a particular
mass ratio referring to protamine.
The term "suspension" refers to a mixture of a
liquid phase and a solid phase that consists of insoluble or
3o sparingly soluble particles that are larger than colloidal
size. Mixtures of NPH microcrystals and an aqueous solvent
form suspensions. The term "suspension formulation" means a
pharmaceutical composition wherein an active agent is
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present in a solid phase, for example, a microcrystalline
solid, an amorphous precipitate, or both, which is finely
dispersed in an aqueous solvent. The finely dispersed solid
is such that it may be suspended in a fairly uniform manner
throughout the aqueous solvent by the action of gently
agitating the mixture, thus providing a reasonably uniform
suspension from which a dosage volume may be extracted.
Examples of commercially available insulin suspension
formulations include, for example, NPH, PZI, and Ultralente.
to A small proportion of the solid matter in a microcrystalline
suspension formulation may be amorphous. Preferably, the
proportion of amorphous material is less than 100, and most
preferably, less than 1% of the solid matter in a
microcrystalline suspension. Likewise, a small proportion
of the solid matter in an amorphous precipitate suspension
may be microcrystalline.
"NPH insulin" refers to the "Neutral Protamine
Hagedorn" preparation of insulin. Synonyms include human
insulin NPH and insulin NPH, among many others. Humulin~ N
2o is a commercial preparation of NPH insulin. A related term
is "NPL" which refers to an NPH-like preparation of
LysB28,ProB29-human insulin analog. The meaning of these
terms, and the methods for preparing them will be familiar
to the person of ordinary skill in the insulin formulation
art .
The term "aqueous solvent" refers to a liquid
solvent that contains water. An aqueous solvent system may
be comprised solely of water, may be comprised of water plus
one or more miscible solvents, and may contain solutes. The
3o more commonly-used miscible solvents are the short-chain
organic alcohols, such as, methanol, ethanol, propanol,
short-chain ketones, such as acetone, and polyalcohols, such
as glycerol.
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An "isotonicity agent" is a compound that is
physiologically tolerated and imparts a suitable tonicity to
a formulation to prevent the net flow of water across cell
membranes that are in contact with an administered
formulation. Glycerol, which is also known as glycerin, is
commonly used as an isotonicity agent. Other isotonicity
agents include salts, e.g., sodium chloride, and
monosaccharides, e.g., dextrose and lactose.
The crystals and co-crystals of the present
to invention contain a hexamer-stabilizing compound. The term
"hexamer-stabilizing compound" refers to a non-
proteinaceous, small molecular weight compound that
stabilizes the protein or derivatized protein in a hexameric
association state. Phenolic compounds, particularly
i5 phenolic preservatives, are the best known stabilizing
compounds for insulin and insulin derivatives. Hex~iner-
stabilizing compounds stabilize the hexamer by binding to it
through specific inter-molecular contacts. Examples of
hexamer-stabilizing agents include: various phenolic
2o compounds, phenolic preservatives, resorcinol, 4'-
hydroxyacetanilide, 4-hydroxybenzamide, and 2,7-
dihyroxynaphthalene. Multi-use formulations of the
insoluble compositions of the present invention will contain
a preservative, in addition to a hexamer-stabilizing
25 compound. The preservative used in formulations of the
present invention may be a phenolic preservative, and may be
the same as, or different from the hexamer-stabilizing
compound.
The term "preservative" refers to a compound added
3o to a pharmaceutical formulation to act as an anti-microbial
agent. A parenteral formulation must meet guidelines for
preservative effectiveness to be a commercially viable
multi-use product. Among preservatives known in the art as
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being effective and acceptable in parenteral formulations
are benzalkonium chloride, benzethonium, chlorohexidine,
phenol, m-cresol, benzyl alcohol, methylparaben,
chlorobutanol, o-cresol, p-cresol, chlorocresol,
phenylmercuric nitrate, thimerosal, benzoic acid, and
various mixtures thereof. See, e.g., Wallhausser, K.-H.,
Develop. Biol. Standard, 24:9-28 (1974) (S. Krager, Basel).
The term "phenolic preservative" includes the
compounds phenol, m-cresol, o-cresol, p-cresol,
l0 chlorocresol, methylparaben, and mixtures thereof. Certain
phenolic preservatives, such as phenol and m-cresol, are
known to bind to insulin-like molecules and thereby to
induce conformational changes that increase either physical
or chemical stability, or both [Birnbaum, D. T., et al.,
Pharmaceutical. Res. 14:25-36 (1997); Rahuel-Clermont, S.,
et al., Biochemistry 36:5837-5845 (1997)].
The term "buffer" or "pharmaceutically acceptable
buffer" refers to a compound that is known to be safe for
use in insulin formulations and that has the effect of
2o controlling the pH of the formulation at the pH desired for
the formulation. The pH of the formulations of the present
invention is from about 6.0 to about 8Ø Preferably the
formulations of the present invention have a pH between
about 6.8 and about 7.8. Pharmaceutically acceptable
buffers for controlling pH at a moderately acidic pH to a
moderately basic pH include such compounds as phosphate,
acetate, citrate, arginine, TRIS, and histidine. "TRIS"
refers to 2-amino-2-hydroxymethyl-1,3,-propanediol, and to
any pharmacologically acceptable salt thereof. The free
3o base and the hydrochloride form are two common forms of
TRIS. TRIS is also known in the art as trimethylol
aminomethane, tromethamine, and
tris(hydroxymethyl)aminomethane. Other buffers that are
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pharmaceutically acceptable, and that are suitable for
controlling pH at the desired level are known to the chemist
of ordinary skill.
The term "administer" means to introduce a
formulation of the present invention into the body of a
patient in need thereof to treat a disease or condition.
The term "treating" refers to the management and
care of a patient having diabetes or hyperglycemia, or other
condition for which insulin administration is indicated for
1o the purpose of combating or alleviating symptoms and
complications of those conditions. Treating includes
administering a formulation of present invention to prevent
the onset of the symptoms or complications, alleviating the
symptoms or complications, or eliminating the disease,
condition, or disorder.
The abbreviations "MMAD" and "MMEAD" are w211-
known in the art, and stand for "mass median aerodynamic
diameter" and "mass median equivalent aerodynamic diameter,"
respectively. The terms are substantially equivalent. The
"aerodynamic equivalent" size of a particle is the diameter
of a unit density sphere which exhibits the same aerodynamic
behavior as the particle, regardless of actual density or
shape. MMAD is determined using a cascade impactor, which
measures the particle size as a function of the aerodynamic
behavior of the particle in a high velocity airstream. The
median (500) particle size is obtained from a linear
regression analysis of the cumulative distribution data.
The abbreviation "VMSED" stands for "volume mean
spherical equivalent diameter." This term is well-known in
3o the art of particle sizing.
The crystals and co-crystals of the present
invention have rod-like morphology or an irregular
morphology. Preferably, the crystals or co-crystals are
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comprised of acylated insulin or acylated insulin analog,
zinc ions, which are present at about 0.3 to about 0.7 mole
per mole of total protein, a phenolic preservative selected
from the group consisting of phenol, m-cresol, o-cresol, p-
cresol, chlorocresol, and mixtures thereof and is present in
sufficient proportions with respect to total protein to
stabilize the T3R3 or R6 hexamer conformation, and
protamine, which is present at about 0.15 to about 0.7 mg
per 3.5 mg of total protein.
1o A preferred group of insulin analogs for preparing
derivatized insulin analogs used to form crystals and co-
crystals consists of animal insulins, deletion analogs, and
pI-shifted analogs. A more preferred group consists of
animal insulins and deletion analogs. Deletion analogs are
yet more preferred.
Another preferred group of insulin analogs-for use
in the crystals and co-crystals of the present invention
consists of the monomeric insulin analogs. Particularly
preferred are those monomeric insulin analogs wherein the
amino acid residue at position B28 is Asp, Lys, Leu, Val, or
Ala, the amino acid residue at position B29 is Lys or Pro,
the amino acid residue at position B10 is His or Asp, the
amino acid residue at position B1 is Phe, Asp or deleted
alone or in combination with a deletion of the residue at
position B2, the amino acid residue at position B30 is Thr,
Ala, Ser, or deleted, and the amino acid residue at position
B9 is Ser or Asp; provided that either position B28 or B29
is Lys.
Another preferred group of insulin analogs for use
3o in the present invention consists of those wherein the
isoelectric point of the insulin analog is between about 7.0
and about 8Ø These analogs are referred to as "pI-shifted
insulin analogs." Examples of pI-shifted insulin analogs
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include, for example, ArgB3l,ArgB32-human insulin,
GlyA2l,ArgB3l,ArgB32-human insulin, ArgAO,ArgB3l,ArgB32-
human insulin, and ArgAO,GlyA2l,ArgB3l,ArgB32-human insulin.
Another preferred group of insulin analogs
consists of LysB28,ProB29-human insulin (B28 is Lys; B29 is
Pro); AspB28-human insulin (B28 is Asp), AspB1-human
insulin, ArgB3l,ArgB32-human insulin, ArgAO-human insulin,
AspBl,G1uB13-human insulin, AlaB26-human insulin, G1yA21-
human insulin, des(ThrB30)-human insulin, and
to GlyA2l,ArgB3l,ArgB32-human insulin.
Especially preferred insulin analogs include
LysB28,ProB29-human insulin, des(ThrB30)-human insulin,
AspB28-human insulin, and AlaB26-human insulin. Another
especially preferred insulin analog is GlyA2l, ArgB3l,
ArgB32-human insulin [Dorschug, M., U. S. Patent No.
5,656,722, 12 August 1997]. The most preferred insulin
analog is LysB28,ProB29-human insulin.
The preferred derivatized proteins are acylated
proteins, and the preferred acylated proteins for the
2o microcrystals and formulations of the present invention are
fatty acid-acylated insulin and fatty acid-acylated insulin
analogs. Fatty acid-acylated human insulin is highly
preferred. Fatty acid-acylated insulin analogs are also
highly preferred.
The particular group used to derivatize insulin or
an insulin analog (collectively, protein) may be any
chemical moiety that does not significantly reduce the
biological activity of the protein, is not toxic when bonded
to the protein, and most importantly, reduces the aqueous
3o solubility, raises the lipophilicity, or decreases the
solubility of zinc/protamine complexes of the derivatized
protein.
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One preferred group of acylating moieties consists
of fatty acids that are straight chain and saturated. This
group consists of methanoic acid (C1), ethanoic acid (C2),
propanoic acid (C3), n-butanoic acid (C4), n-pentanoic acid
(C5), n-hexanoic acid (C6), n-heptanoic acid (C7), n-
octanoic acid (C8), n-nonanoic acid (C9), n-decanoic acid
(C10), n-undecanoic acid (C11), n-dodecanoic acid (C12), n-
tridecanoic acid (C13), n-tetradecanoic acid (C14), n-
pentadecanoic acid (C15), n-hexadecanoic acid (C16), n-
1o heptadecanoic acid (C17), and n-octadecanoic acid (C18).
Adjectival forms are formyl (C1), acetyl (C2), propionyl
(C3), butyryl (C4), pentanoyl (C5), hexanoyl (C6), heptanoyl
(C7), octanoyl (C8), nonanoyl (C9), decanoyl (C10),
undecanoyl (C11), dodecanoyl (C12), tridecanoyl (C13),
tetradecanoyl (C14) or myristoyl, pentadecanoyl (C15),
hexadecanoyl (C16) or palmitic, heptadecanoyl (C17), and
octadecanoyl (C18) or stearic.
A preferred group of fatty acids for forming the
fatty acid-acylated proteins used in the microcrystals of
2o the present invention consists of fatty acids having an even
number of carbon atoms - that is, C2, C4, C6, C8, C10, C12,
C14, C16, and C18 saturated fatty acids.
Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of fatty acids having an
odd number of carbon atoms - that is, C1, C3, C5, C7, C9,
C11, C13, C15, and C17 saturated fatty acids.
Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of fatty acids having more
than 5 carbon atoms - that is, C6, C7, C8, C9, C10, C11,
C12, C13, C14, C15, C16, C17, and C18 saturated fatty acids.
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Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of fatty acids having less
than 9 carbon atoms - that is, C1, C2, C3, C4, C5, C6, C7,
and C8 saturated fatty acids.
Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of fatty acids having
between 6 and 8 carbon atoms - that is, C6, C7, and C8,
to saturated fatty acids.
Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of fatty acids having more
than between 4 and 6 carbon atoms - that is, C4, C5, and C6,
saturated fatty acids.
Another preferred group of fatty acids for-forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of fatty acids having more
than between 2 and 4 carbon atoms - that is, C2, C3, and C4,
2o saturated fatty acids.
Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of fatty acids having less
than 6 carbon atoms - that is, C1, C2, C3, C4, and C5
saturated fatty acids.
Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of fatty acids having less
than 4 carbon atoms - that is, C1, C2, and C3 saturated
fatty acids.
Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of fatty acids having more
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than 9 carbon atoms - that is, C10, C11, C12, C13, C14, C15,
C16, C17, and C18 saturated fatty acids.
Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of fatty acids having an
even number of carbon atoms and more than 9 carbon atoms -
that is, C10, C12, C14, C16, and C18 saturated fatty acids.
Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of fatty acids having 12,
14, or 16 carbon atoms, that is, C12, C14, and C16 saturated
fatty acids.
Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of fatty acids having 14
or 16 carbon atoms, that is, C14 and C16 saturated fatty
acids. Fatty acids with 14 carbons are particularly
preferred. Fatty acids with 16 carbons are also
particularly preferred.
Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of saturated fatty acids
having between 4 and 10 carbon atoms, that is C4, C5, C6,
C7, C8, C9, and C10 saturated fatty acids.
Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of saturated fatty acids
having an even number of carbon atoms between 4 and 10
carbon atoms, that is C4, C6, C8, and C10 saturated fatty
acids .
Another preferred group of fatty acids for forming
the fatty acid-acylated proteins used in the microcrystals
of the present invention consists of fatty acids having 6,
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8, or 10 carbon atoms. Fatty acids with 6 carbons are
particularly preferred. Fatty acids with 8 carbons are also
particularly preferred. Fatty acids with 10 carbons are
particularly preferred.
The skilled person will appreciate that narrower
preferred groups are made by combining the preferred groups
of fatty acids described above.
Another preferred group of acylating moieties
consists of saturated fatty acids that are branched. A
1o branched fatty acid has at least two branches. The length
of a "branch" of a branched fatty acid may be described by
the number of carbon atoms in the branch, beginning with the
acid carbon. For example, the branched fatty acid 3-ethyl-
5-methylhexanoic acid has three branches that are five, six,
and six carbons in length. In this case, the "longest"
branch is six carbons. As another example, 2,3,4,5=
tetraethyloctanoic acid has five branches that are 4, 5, 6,
7, and 8 carbons long. The "longest" branch is eight
carbons. A preferred group of branched fatty acids are
2o those having from three to ten carbon atoms in the longest
branch.
A representative number of such branched,
saturated fatty acids will be mentioned to assure the
reader's comprehension of the range of such fatty acids that
may be used as acylating moieties of the proteins in the
present invention: 2-methyl-propioinic acid, 2-methyl-
butyric acid, 3-methyl-butyric acid, 2,2-dimethyl-propionic
acid, 2-methyl-pentanoic acid, 3-methyl-pentanoic acid, 4-
methyl-pentanoic acid, 2,2-dimethyl-butyric acid, 2,3-
3o dimethyl-butyric acid, 3,3-dimethyl-butyric acid, 2-ethyl-
butyric acid, 2-methyl-hexanoic acid, 5-methyl-hexanoic
acid, 2,2-dimethyl-pentanoic acid, 2,4-dimethyl-pentanoic
acid, 2-ethyl-3-methyl-butyric acid, 2-ethyl-pentanoic acid,
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3-ethyl-pentanoic acid, 2,2-dimethyl-3-methyl-butyric
acid,2-methyl-heptanoic acid, 3-methyl-heptanoic acid, 4-
methyl-heptanoic acid, 5-methyl-heptanoic acid, 6-methyl-
heptanoic acid, 2,2-dimethyl-hexanoic acid, 2,3-dimethyl-
hexanoic acid, 2,4-dimethyl-hexanoic acid, 2,5-dimethyl-
hexanoic acid, 3,3,-dimethyl-hexanoic acid, 3,4-dimethyl-
hexanoic acid, 3,5-dimethyl-hexanoic acid, 4,4-dimethyl-
hexanoic acid, 2-ethyl-hexanoic acid, 3-ethyl-hexanoic acid,
4-ethyl-hexanoic acid, 2-propyl-pentanoic acid, 2-ethyl-
1o hexanoic acid, 3-ethyl-hexanoic acid, 4-ethyl-hexanoic acid,
2-(1-propyl)pentanoic acid, 2-(2-propyl)pentanoic acid, 2,2-
diethyl-butyric acid, 2,3,4-trimethyl-pentanoic acid, 2-
methyl-octanoic acid, 4-methyl-octanoic acid, 7-methyl-
octanoic acid, 2,2-dimethyl-heptanoic acid, 2,6-dimethyl-
heptanoic acid, 2-ethyl-2-methyl-hexanoic acid, 3-ethyl-5-
methyl-hexanoic acid, 3-(1-propyl)-hexanoic acid, 2=(2-
butyl)-pentanoic acid, 2-(2-(2-methylpropyl))pentanoic acid,
2-methyl-nonanoic acid, 8-methyl-nonanoic acid, 6-
ethyl-octanoic acid, 4-(1-propyl)-heptanoic acid, 5-(2-
propyl)-heptanoic acid, 3-methyl-undecanoic acid,2-pentyl-
heptanoic acid, 2,3,4,5,6-pentamethyl-heptanoic acid, 2,6-
diethyl-octanoic acid, 2-hexyl-octanoic acid, 2,3,4,5,6,7-
hexamethyl-octanoic acid, 3,3-diethyl-4,4-diethyl-hexanoic
acid, 2-heptyl-nonanoic acid, 2,3,4,5-tetraethyl-octanoic
acid, 2-octyl-decanoic acid, and 2-(1-propyl)-3-(1-propyl)-
4,5-diethyl-6-methyl-heptanoic acid.
Yet another preferred group of acylating moieties
consists of cyclic alkyl acids having from 5 to 24 carbon
atoms, wherein the cyclic alkyl moiety, or moieties, have 5
3o to 7 carbon atoms. A representative number of such cyclic
alkyl acids will be mentioned to assure the reader's
comprehension of the range of such acids that may be used as
acylating moieties of the proteins in the present invention:
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cyclopentyl-formic acid, cyclohexyl-formic acid, 1-
cyclopentyl-acetic acid, 2-cyclohexyl-acetic acid, 1,2-
dicyclopentyl-acetic acid, and the like.
A preferred group of derivatized proteins consists
of mono-acylated proteins. Mono-acylation at the ~-amino
group is most preferred. For insulin, mono-acylation at
LysB29 is preferred. Similarly, for certain insulin
analogs, such as, LysB28,ProB29-human insulin analog, mono-
acylation at the ~-amino group of LysB28 is most preferred.
1o Mono-acylation at the a-amino group of the B-chain (B1) is
also preferred. Mono-acylation at the a-amino group of the
A-chain (A1) is also preferred.
Another group of acylated proteins consists of di
acylated proteins. The di-acylation may be, for example, at
the E-amino group of Lys and at the a-amino group of the B
chain, or may be at the ~-amino group of Lys and at the a-
amino group of the A-chain, or may be at the oc-amino group
the A-chain and at the oc-amino group of the B-chain.
Another group of acylated proteins consists of
2o tri-acylated proteins. Tri-acylated proteins are those that
are acylated at the ~-amino group of Lys, at the cc-amino
group of the B-chain, and at the a-amino group of the A-
chain.
Aqueous compositions containing water as the major
solvent are preferred. Aqueous suspensions wherein water is
the solvent are highly preferred.
The compositions of the present invention are used
to treat patients who have diabetes or hyperglycemia. The
formulations of the present invention will typically provide
3o derivatized protein at concentrations of from about 1 mg/mL
to about 10 mg/mL. Present formulations of insulin products
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are typically characterized in terms of the concentration of
units of insulin activity (units/mL), such as U40, U50,
U100, and so on, which correspond roughly to about 1.4,
1.75, and 3.5 mg/mL preparations, respectively. The dose,
route of administration, and the number of administrations
per day will be determined by a physician considering such
factors as the therapeutic objectives, the nature and cause
of the patient's disease, the patient's gender and weight,
level of exercise, eating habits, the method of
1o administration, and other factors known to the skilled
physician. In broad range, a daily dose would be in the
range of from about 1 nmol/kg body weight to about 6 nmol/kg
body weight (6 nmol is considered equivalent to about 1 unit
of insulin activity). A dose of between about 2 and about 3
i5 nmol/kg is typical of present insulin therapy.
The physician of ordinary skill in treating
diabetes will be able to select the therapeutically most
advantageous means to administer the formulations of the
present invention. Parenteral routes of administration are
2o preferred. Typical routes of parenteral administration of
suspension formulations of insulin are the subcutaneous and
intramuscular routes. The compositions and formulations of
the present invention may also be administered by nasal,
buccal, pulmonary, or occular routes. The pulmonary route
z5 is particularly advantageous, in that pain and inconvenience
are reduced. The crystals and co-crystals of the present
invention are particularly well-suited for pulmonary
administration.
Glycerol at a concentration of 12 mg/mL to 25
3o mg/mL is preferred as an isotonicity agent. Yet more highly
preferred for isotonicity is to use glycerol at a
concentration of from about 15 mg/mL to about 17 mg/mL.
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M-cresol and phenol, or mixtures thereof, are
preferred preservatives in formulations of the present
invention.
Insulin or insulin analogs used to prepare
derivatized proteins can be prepared by any of a variety of
recognized peptide synthesis techniques including classical
(solution) methods, solid phase methods, semi-synthetic
methods, and more recent recombinant DNA methods. For
example, see Chance, R. E., et al., U.S. Patent No.
5,514,646, 7 May 1996; EPO publication number 383,472, 7
February 1996; Brange, J. J. V., et al. EPO publication
number 214,826, 18 March 1987; and Belagaje, R. M., et al.,
U.S. Patent No. 5,304,473, 19 April 1994, which disclose the
preparation of various proinsulin and insulin analogs.
These references are expressly incorporated herein by
reference.
Generally, derivatized proteins are prepared using
methods known in the art. The publications listed above to
describe derivatized proteins contain suitable methods to
2o prepare derivatized proteins. Those publications are
expressly incorporated by reference for methods of preparing
derivatized proteins. To prepare acylated proteins, the
protein is reacted with an activated organic acid, such as
an activated fatty acid. Activated fatty acids are
derivatives of commonly employed acylating agents, and
include activated esters of fatty acids, fatty acid halides,
activated amides of fatty acids, such as, activated azolide
derivatives [Hansen, L. B., WIPO Publication No. 98/02460,
22 January 1998], and fatty acid anhydrides. The use of
3o activated esters, especially N-hydroxysuccinimide esters of
fatty acids, is a particularly advantageous means of
acylating a free amino acid with a fatty acid. Lapidot, et
a1. describe the preparation of N-hydroxysuccinimide esters
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and their use in the preparation of N-lauroyl-glycine, N-
lauroyl-v-serine, and N-lauroyl-v-glutamic acid. The term
"activated fatty acid ester" means a fatty acid which has
been activated using general techniques known in the art
[Riordan, J. F. and Vallee, B. L., Methods in Enzymology,
XXV:494-499 (1972); Lapidot, Y., et al., J. Lipid Res.
8:142-145 (1967)]. Hydroxybenzotriazide (HOBT), N-
hydroxysuccinimide and derivatives thereof are particularly
well known for forming activated acids for peptide
1o synthesis.
To selectively acylate the ~-amino group, various
protecting groups may be used to block the cc-amino groups
during the coupling. The selection of a suitable protecting
group is known to one skilled in the art and includes p-
methoxybenzoxycarbonyl (pmZ). Preferably, the e-amino group
is acylated in a one-step synthesis without the use of
amino-protecting groups. A process for selective acylation
at the N~-amino group of Lys is disclosed and claimed by
Baker, J. C., et al., U.S. Patent No. 5,646,242, 8 July
1997, the entire disclosure of which is incorporated
expressly by reference. A process for preparing a dry
powder of an acylated protein is disclosed and claimed by
Baker, J. C., et al., U.S. Patent No. 5,700,904, 23 December
1997, the entire disclosure of which is incorporated herein
expressly by reference.
The primary role of zinc in the present invention
is to facilitate formation of Zn(II) hexamers of the protein
and derivatized protein, either separately as mixed
hexamers, or together as hybrid hexamers. Zinc facilitates
3o the formation of hexamers of insulin, and of insulin
analogs. Zinc likewise promotes the formation of hexamers
of derivatized insulin and insulin analogs. Hexamer
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formation is conveniently achieved by bringing the pH of a
solution comprising protein, or derivatized protein, or both
into the neutral region in the presence of Zn(II) ions, or
by adding Zn(II) after the pH has been adjusted to the
neutral region.
For efficient yield of crystals or co-crystals,
the molar ratio of zinc to total protein is bounded at the
lower limit by about 0.33, that is, the approximately two
zinc atoms per hexamer which are needed for efficient
1o hexamerization. Crystals and co-crystals will form suitably
with about 2 to about 4-6 zinc atoms present when no
compound that competes with insulin for zinc binding is
present. Even more zinc may be used during the process if a
compound that competes with the protein for zinc binding,
such as one containing citrate or phosphate, is present.
Excess zinc above the minimum amount needed for efficient
hexamerization may be desirable to more strongly drive
hexamerization. Also, excess zinc above the minimum amount
can be present in a formulation of the present invention,
2o and may be desirable to improve chemical and physical
stability, to improve suspendability, and possibly to
further extend time-action. Consequently, there is a fairly
wide range of zinc: protein ratios allowable in the insoluble
compositions, processes, and formulations of the present
invention.
In accordance with the present invention, zinc is
present in the formulation in an amount of from about 0.3
mole to about 7 moles per mole of total protein and more
preferably about from 0.3 mole to about 1.0 mole of total
3o protein. Yet more highly preferred is a ratio of zinc to
derivatized protein from about 0.3 to about 0.7 mole of zinc
atoms per mole of total protein. Most highly preferred is a
ratio of zinc to total protein from about 0.30 to about 0.55
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mole of zinc atoms per mole of total protein. For higher
zinc formulations that are similar to PzI preparations, the
zinc ratio is from about 5 to about 7 moles of zinc per mole
of total protein.
The zinc compound that provides zinc for the
present invention may be any pharmaceutically acceptable
zinc compound. The addition of zinc to insulin preparations
is known in the art, as are pharmaceutically acceptable
sources of zinc. Preferred zinc compounds to supply zinc
1o for the present invention include zinc chloride, zinc
acetate, zinc citrate, zinc oxide, and zinc nitrate.
A complexing compound is required for the
microcrystals and precipitates of the present invention.
The complexing compound must be present in sufficient
i5 quantities to cause substantial precipitation and
crystallization of the hexamers. Such quantities can be
readily determined for a particular preparation of a
particular complexing compound by simple titration
experiments. Ideally, the complexing compound concentration
20 is adjusted so that there is negligible complexing compound
remaining in the soluble phase after completion of
precipitation and crystallization. This requires combining
the complexing compound based on an experimentally
determined "isophane" ratio. This ratio is expected to be
25 very similar to that of NPH and NPL. However, it may be
slightly different because derivatization may affect the
nature of the protein-protamine interaction.
When protamine is the complexing compound, it is
present in the crystals and co-crystals in an amount of from
30 about 0.15 mg to about 0.5 mg per 3.5 mg of the total
protein. The ratio of protamine to total protein is
preferably from about 0.25 to about 0.40 (mg/3.5 mg). More
preferably the ratio is from about 0.25 to about 0.38
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(mg/3.5 mg). Preferably, protamine is in an amount of 0.05
mg to about 0.2 mg per mg of the total protein, and more
preferably, from about 0.05 to about 0.15 milligram of
protamine per milligram of total protein. Protamine sulfate
is the preferred salt form of protamine for use in the
present invention. When protamine sulfate, or other salt
form of protamine is used, the mass of it to be used would
have to be adjusted with respect to the mass of protamine
free base that would be used for the same application by a
1o factor equal to the ratio of the molecular weights of the
salt form and protamine.
To further extend the time action of the
compositions of the present invention or to improve their
suspendability, additional protamine and zinc may be added
after crystallization. Thus, also within the present
invention are formulations having protamine at higher than
isophane ratios. For these formulations, the protamine
ratio is from 0.25 mg to about 0.5 mg of protamine per mg of
total protein.
2o A required component of the crystals and co-
crystals of the present invention is a hexamer stabilizing
compound. The structures of three hexameric conformations
have been characterized in the literature, and are
designated T6, T3R3, and R6. In the presence of hexamer
stabilizing compound, such as various phenolic compounds,
the R6 conformation is stabilized. Therefore, it is highly
likely that hexamers are in the R6 conformation, or the T3R3
conformation in the crystals and co-crystals produced in the
presence of a hexamer stabilizing compound, such as phenol
or m-cresol, among others. A wide range of hexamer
stabilizing compounds are suitable. They must be present in
sufficient proportions with respect to total protein to
stabilize the R6 hexamer conformation. To accomplish this,
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at least 3 moles of hexamer stabilizing compound per hexamer
are required for effective hexamer stabilization. It is
preferred that more than 3 moles of hexamer stabilizing
compound per hexamer be present in the crystals and co-
y crystals of the present invention. The presence of higher
ratios of hexamer stabilizing compound, at least up to 25 to
50-fold higher, in the solution from which the microcrystals
and precipitates are prepared will not adversely affect
hexamer stabilization.
1o In formulations of the present invention, a
preservative may be present, especially if the formulation
is intended to be sampled multiple times. As mentioned
above, a wide range of suitable preservatives are known.
Preferably, the preservative is present in the solution in
15 an amount suitable to provide an antimicrobial effect
sufficient to meet pharmacopoeial requirements.
Preferred preservatives are the phenolic
preservatives, which are enumerated above. Preferred
concentrations for the phenolic preservative are from about
20 2 mg to about 5 mg per milliliter of the aqueous suspension
formulation. These concentrations refer to the total mass
of phenolic preservatives because mixtures of individual
phenolic preservatives are contemplated. Suitable phenolic
preservatives include, for example, phenol, m-cresol, and
25 methylparaben. Preferred phenolic compounds are phenol and
m-cresol. Mixtures of phenolic compounds, such as phenol
and m-cresol, are also contemplated and highly preferred.
Examples of mixtures of phenolic compounds are 0.6 mg/mL
phenol and 1.6 mg/mL m-cresol, and 0.7 mg/mL phenol and 1.8
3o mg/mL m-cresol.
The crystals and co-crystals of the present
invention are preferably oblong-shaped, also known as "rod-
like", single crystals that are comprised of a derivatized
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protein, a divalent metal cation, and including a complexing
compound and a hexamer-stabilizing compound.
A preferred composition comprises from about 3 mg
to about 6 mg of protamine sulfate per 35 mg of total
protein, and from about 0.1 to about 0.4 mg zinc per 35 mg
of total protein. Another preferred composition comprises
from about 10 mg to about 17 mg of protamine sulfate per 35
mg of total protein, and from about 2.0 to about 2.5 mg zinc
per 35 mg of total protein. Another preferred composition
l0 comprises, per mL, protamine sulfate, 0.34-0.38 mg; zinc,
0.01-0.04 mg; and total protein, 3.2-3.8 mg.
Both an un-derivatized protein and a derivatized
protein are required for the present co-crystals. The ratio
between the masses of these proteins determines the degree
of time extension of the preparations. A preferred ratio of
the number of moles of the protein to the number of moles of
the derivatized protein is between about 1:100 and about
100:1. A further preferred ratio of the number of moles of
the protein to the number of moles of the derivatized
2o protein is between about 1:1 and about 100:1. Another
preferred ratio of the number of moles of the protein to the
number of moles of the derivatized protein is between about
1:1 and about 20:1. Yet other preferred ratios of the
number of moles of the protein to the number of moles of the
derivatized protein are: between about 2:1 and about 20:1;
between about 2:1 and 10:1; between about 2:1 and 5:1;
between about 3:1 and 5:1; between 1:1 and 1:20; between 1:1
and 1:10; between about 1:2 and about 1:20; between about
1:2 and 1:10; between about 1:2 and 1:5; between about 1:3
3o and 1:5; between about 10:1 and about 1:10; between about
9:1 and about 1:9; between about 5:1 and about 1:5; and
between about 3:1 and about 1:3.
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The present invention provides processes for
preparing the crystals and co-crystals. In summary,
suitable processes are comprised generally of the steps in
one of the following sequences: solubilization (if starting
with dry material), hexamerization, homogenization,
complexation, precipitation, crystallization, and optionally
formulation; or solubilization (if starting with dry
material), homogenization, hexamerization, complexation,
precipitation, crystallization, and optionally formulation.
l0 Solubilization means the dissolution of
derivatized protein and protein sufficiently to allow them
to form hexamers. Hexamerization refers to the process
wherein molecules of protein and derivatized protein bind
with zinc(II) atoms to form hexamers. Complexation denotes
the formation of insoluble complexes between the hexamers
and protamine. Precipitation results typically from the
formation of insoluble complexes. Crystallization involves
the conversion of precipitated hexamer/protamine complexes
into crystals, typically, rod-like crystals.
2o Solubilization is carried out by dissolving the
derivatized protein and protein in an aqueous solvent. The
aqueous solvent may be, for example, an acidic solution, a
neutral solution, or a basic solution. The aqueous solvent
may be comprised partially of a miscible organic solvent,
such as ethanol, acetonitrile, dimethylsulfoxide, and the
like. Acidic solutions may be, for example, solutions of
HC1, advantageously from about 0.01 N HCl to about 1.0 N
HCl. Other acids that are pharmaceutically acceptable may
be employed as well. Basic solutions may be, for example,
solutions of NaOH, advantageously from about 0.01 N NaOH to
about 1.0 N NaOH, or higher. Other bases that are
pharmaceutically acceptable may be employed as well. For
the sake of protein stability, the concentration of acid or
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base is preferably as low as possible while still being
effective to adequately dissolve the protein and derivatized
protein.
Most proteins (insulin and insulin analogs) and
many derivatized proteins may be dissolved to suitable
concentrations at neutral pH. Solutions to dissolve
derivatized proteins at neutral pH may contain a buffer and
optionally, one or more additional solutes such as salts,
phenolic compounds, zinc, and isotonicity agents.
l0 When hexamerization occurs before homogenization,
two populations of homogenous hexamers are formed first, and
then the populations are mixed, thereby forming mixed
hexamers. When homogenization occurs first, hexamerization
yields hybrid hexamers. As mentioned above, to prepare
i5 insoluble compositions comprised of hybrid hexamers, protein
and derivatized protein are homogenized under conditions
favoring dissociation to monomer or dimer aggregation states
prior to hexamerization with a divalent metal cation. To
achieve the necessary dissociation, the protein and
2o derivatized protein may be mixed under strongly acidic or
strongly basic conditions. The degree of dissociation, and
therefore, homogenization is influenced by the solution
conditions chosen for this step. Insulin and related
proteins readily self-associate in a series of reactions
25 producing dimers, hexamers, and other associated forms. The
distribution of these association forms at equilibrium is
dependent on many parameters, including pH. These
association reactions are commonly thought to involve
primarily monomer-dimer-hexamer assembly. Consequently,
3o depending on the solution conditions chosen, homogenization
should accomplish the mixing of monomers, dimers, or a
mixture thereof. Homogenization in 1 N HCl, for example,
could involve a higher fraction of monomer mixing than in
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0.1 N HCl, which would probably involve more dimer mixing.
For the preparation of compositions comprised of hybrid
hexamers, the homogenization process will be effective
provided that only a very small or negligible fraction of
homogeneous hexamers of the protein or derivatized protein
exist under the homogenization conditions employed.
Compositions comprised of mixed hexamers
incorporate predominantly two types of hexamers, namely
hexamers of the protein, and hexamers of the derivatized
1o protein. In this case, the homogenization step occurs after
the hexamerization step, and achieves the homogenization of
the hexamers prior to complexation with the complexing
compound. Consequently, the homogenization step is
performed under solution conditions that stabilize the
Zn(II)-insulin hexamer. Solution conditions that stabilize
insulin hexamers are well known in the literature.
The solution conditions required for
hexamerization are those that allow the formation of the
hybrid hexamers or mixed hexamers in solution. These
2o conditions will be identical or very similar to the
conditions under which insulin or insulin analogs are made
to hexamerize. Typically, hexamerization requires zinc and
a neutral to slightly basic pH, which is taken to be from
about pH 6.8 to about pH 8.4. The presence of a hexamer-
stabilizing compound advantageously influences
hexamerization by promoting the R6 or the T3R3 conformations
of the derivatized protein, and in certain instances, of the
protein also. For certain monomeric insulin analogs, a
hexamer-stabilizing compound is required to form hexamers.
3o For compositions comprised of hybrid hexamers,
seven hexameric species are expected: P6, P5D1, P4D2, P3D3,
P2D4, P1D5, and D6, where P represents the protein monomer,
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and D represents the derivatized protein monomer. The
statistical distribution of hexamers is expected to conform
to a Poisson distribution, and will be influenced by the
relative proportion of protein and derivatized protein, and
by the degree of dissociation prior to hexamerization. For
example, from a homogenized solution constituted
predominantly of dimers, four major hybrid hexamer species
are expected: P6, P4D2, P2D4, and D6. For compositions
comprised of mixed hexamers, only two hexameric species are
1o expected to predominate: P6 and D6.
The complexation step must involve the combination
a complexing compound with hexamer under solution conditions
where each is initially soluble. This could be accomplished
by combining separate solutions of hexamers and of
protamine, or by first forming a solution of protein,
derivatized protein, and protamine at acidic or basic pH,
and then shifting the pH to the neutral range.
During crystallization, the solution conditions
must stabilize the crystallizing species, and promote the
conversion of precipitate to solute to crystal. Thus, the
solution conditions will determine the rate and outcome of
crystallization. Crystallization likely involves a complex
equilibrium involving non-crystalline precipitate, dissolved
hexamer-protamine complexes, and crystal. To obtain
microcrystals, the conditions chosen for crystallization
must drive the equilibrium toward crystal formation. Also,
in light of the hypothesized equilibrium, the solubility of
the derivatized protein is expected to profoundly affect
crystallization rate and size because lower solubility will
likely slow the net conversion from precipitate to solute to
crystal. Furthermore, it is well-recognized that slowing
the rate of crystallization often results in larger
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crystals. Thus, the crystallization rate and crystal size
are thought to depend on the size and nature of the
derivatizing moiety on the derivatized protein.
Crystallization parameters that had been
previously thought to influence the crystallization rate and
the size of crystals of the present invention are (see
Brader I and Brader II): acyl group size and nature;
temperature; the presence and concentration of "competing
compounds" that compete with the protein and derivatized
1o protein for zinc, such as citrate, phosphate, and the like;
the nature and concentration of phenolic compound(s); zinc
concentration; the presence and concentration of a miscible
organic solvent; the time permitted for crystallization; the
pH and ionic strength; buffer identity and concentration;
the concentration of precipitants; the presence of seeding
materials; the shape and material of the container; the
stirring rate; and the total protein concentration.
Temperature and the concentration of competing compounds
were thought to be of particular importance.
2o It has now been discovered quite surprisingly that
chloride ions, apart from their influence on ionic strength,
play a significant role in determining the size of crystals
and co-crystals. The exact nature of chloride's effect on
crystal and co-crystal size is not known. A specific link
between chloride ion concentration and the rate of
crystallization has not been previously described for these
crystals and co-crystals.
Competing compounds, such as citrate, may affect
the rate at which crystals form, and indirectly, crystal
size and quality. These compounds may exert their effect by
forming coordination complexes with zinc in solution, thus
competing with the relatively weak zinc binding sites on the
surface of the hexamer for zinc. Occupation of these weak
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surface binding sites probably impedes crystallization.
Additionally, many derivatized proteins are partially
insoluble in the presence of little more than 0.333 zinc per
mole of derivatized protein, and the presence of competing
compounds restores solubility, and permits crystallization.
The optimum concentration of competing compound can be
determined using routine techniques for any combination of
protein and derivatized protein. As an upper limit, of
course, is the concentration at which zinc is precipitated
1o by the competing compound, or the concentration at which
residual competing compound would be pharmaceutically
unacceptable, such as, when it would cause pain or
irritation at the site of administration.
An example of a process for preparing the
i5 precipitates and crystals of the present invention follows.
A measured amount of the derivatized protein and a measured
amount of the protein are dissolved in, or are combined to
form a solution in an aqueous solvent containing a hexamer-
stabilizing compound, such as a phenolic compound. To this
2o solution is added a solution of zinc as one of its soluble
salts, for example Zn(II)C12, to provide from about 0.3
moles of zinc per mole of derivatized insulin to about 0.7
moles, or to as much as 1.0 moles, of zinc per mole of total
protein (protein + derivatized protein). Absolute ethanol,
z5 or another miscible organic solvent, may optionally be added
to this solution in an amount to make the solution from
about 5% to about 10% by volume organic solvent. This
solution may then be filtered through a 0.22 micron, low-
protein binding filter. A protamine solution is prepared by
3o dissolving a measured amount of protamine in an aqueous
solvent. This solution may be filtered through a 0.22
micron, low-protein binding filter. The solution of protein
and derivatized protein and the protamine solution are
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combined, whereupon a precipitate forms initially. The
resulting suspension is stirred slowly at room temperature
(typically about 20-25°C), whereupon microcrystals are
formed within a period from about 4 hours to about 10 days.
The microcrystals may then be separated from the
mother liquor and introduced into a different solvent, for
storage and administration to a patient. Examples of
appropriate aqueous solvents are as follows: water for
injection containing 25 mM TRIS, 5 mg/mL phenol and 16 mg/mL
1o glycerol; water for injection containing 2 mg/mL sodium
phosphate dibasic, 1.6 mg/mL m-cresol, 0.65 mg/mL phenol,
and 16 mg/mL glycerol; and water for injection containing 25
mM TRIS, 5 mg/mL phenol, 0.1 M trisodium citrate, and 16
mg/mL glycerol.
In another process for preparing the insoluble
compositions of the present invention, for example, a
measured mass of dry derivatized protein and a measured mass
of dry protein are dissolved together in an acidic aqueous
solvent, such as 0.1 N - 1.0 N HCl. This solution is
2o stirred to insure thorough mixing of derivatized protein and
protein. The ratio of derivatized protein powder to protein
powder in this mixture is predefined to achieve a similar
ratio of derivatized protein to protein in the insoluble
composition to be produced. A separately prepared aqueous
solution comprised of a phenolic preservative and,
optionally, a pharmaceutically acceptable buffer, is
combined with the acidic solution of the proteins. The pH
of the resulting solution is then adjusted to about 6.8 to
about 8.4, preferably from about 6.8 to about 8.0, or
3o preferably to a pH of from about 7.2 to about 7.8, and most
preferably from about 7.4 to about 7.8. To this solution is
added a solution of zinc as one of its soluble salts, for
example Zn(II)C12, to provide from about 0.3 moles of zinc
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per mole of total insulin to about 4 moles of zinc per mole
of total insulin. This solution is adjusted to a pH as
given above, and preferably to about 7.4 -7.6, and may then
be filtered through a 0.22 micron, low-protein binding
filter. A solution of protamine is prepared by dissolving a
measured mass of protamine in an aqueous solvent. The
protamine solution may be filtered through a 0.22 micron,
low-protein binding filter. The solution of protein and
derivatized protein and the protamine solution are combined,
1o whereupon a precipitate forms initially. The resulting
suspension is stirred slowly at room temperature (typically
about 20-25°C), whereupon microcrystals are formed within a
period from about 4 hours to about 10 days.
In another process for preparing the insoluble
compositions of the present invention, a measured amount of
a derivatized protein is first dissolved in an aqueaus
solvent containing a phenolic preservative. To this
solution is added a solution of zinc as one of its soluble
salts, for example Zn(II)C12, to provide from about 0.3
2o moles of zinc per mole of derivatized protein to about 4
moles of zinc per mole of derivatized protein. The pH of
the resulting solution is then adjusted to about 6.8 to
about 8.4, preferably from about 6.8 to about 8.0, or
preferably to a pH of from about 7.2 to about 7.8, and most
preferably from about 7.4 to about 7.8. A second solution
is prepared separately wherein a measured amount of a
protein selected from the group consisting of insulin,
insulin analogs, and proinsulin is dissolved in an aqueous
solvent containing a phenolic preservative. To this
3o solution is added a solution of zinc as one of its soluble
salts, for example Zn(II)C12, to provide from about 0.3
moles of zinc per mole of protein to about 4 moles of zinc
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per mole of protein. The pH of the resulting solution is
then adjusted to about 6.8 to about 8.4, preferably from
about 6.8 to about 8.0, or preferably to a pH of from about
7.2 to about 7.8, and most preferably from about 7.4 to
about 7.8, or 7.4 - 7.6. Portions of the derivatized
protein solution and the protein solution are combined in a
ratio that is predefined in order to achieve a similar ratio
of derivatized protein to protein in the insoluble
composition. This solution is stirred to insure thorough
1o mixing of derivatized protein and protein. This solution is
then adjusted to a pH of about 7.6, and may then be filtered
through a 0.22 micron, low-protein binding filter. A
protamine solution is prepared separately by dissolving a
measured amount of protamine in an aqueous solvent. This
z5 protamine solution may be filtered through a 0.22 micron,
low-protein binding filter. The solution of protein and
derivatized protein and the protamine solution are combined,
whereupon a precipitate forms initially. The resulting
suspension is stirred slowly at room temperature (typically
20 about 20-25°C), whereupon microcrystals are formed within a
period from about 4 hours to about 10 days.
While not describing all of the very many types of
processes that will produce the insoluble compositions of
the present invention in any way, the following are yet
25 further processes of the present invention:
dissolving a protein, a derivatized protein, a
hexamer-stabilizing compound, and a divalent metal cation in
an aqueous solvent having a pH that will permit the
formation of hexamers, and adding a complexing compound;
30 dissolving a protein, a derivatized protein, a
hexamer-stabilizing compound, and a divalent metal cation in
an aqueous solvent having a pH that will not permit the
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formation of hexamers, adjusting the pH to between about 6.8
and about 7.8, and adding a complexing compound;
dissolving a protein, a hexamer-stabilizing
compound, and a divalent metal cation in an aqueous solvent
having a pH that will permit the formation of hexamers,
separately, dissolving a derivatized protein, a hexamer-
stabilizing compound, and a divalent metal can on in an
aqueous solvent having a pH that will permit the formation
of hexamers, thoroughly mixing together these two solution,
1o and then adding a complexing compound;
dissolving a protein, a hexamer-stabilizing
compound, a divalent metal cation, and a complexing compound
in an aqueous solvent, wherein the resulting solution has a
pH at which precipitation does not occur, separately,
dissolving a derivatized protein, a hexamer-stabilizing
compound, a divalent metal cation, and a complexing compound
in an aqueous solvent, wherein the resulting solution has a
pH at which precipitation does not occur, thoroughly mixing
together these two solutions, and adjusting the pH of the
2o solution to a value at which precipitation occurs;
dissolving a protein, a derivatized protein, a
hexamer-stabilizing compound, a divalent metal cation, and a
complexing compound in an aqueous solvent, wherein the
resulting solution has a pH at which precipitation does not
occur and adjusting the pH of the solution to a value at
which precipitation occurs;
dissolving a protein, a derivatized protein, a
hexamer-stabilizing compound, and a divalent metal cation,
in an aqueous solvent, wherein the resulting solution has a
3o pH at which precipitation will not occur when a complexing
agent is added, adding a complexing compound, and adjusting
the pH of the solution to a value at which precipitation
occurs;
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dissolving a protein, a hexamer-stabilizing
compound, and a divalent metal ration in an aqueous solvent,
wherein the resulting solution has a pH at which
precipitation will not occur when a complexing compound is
added, separately, dissolving a derivatized protein, a
hexamer-stabilizing compound, and a divalent metal ration in
an aqueous solvent, wherein the resulting solution has a pH
at which precipitation will not occur when a complexing
compound is added, thoroughly mixing together these two
l0 solutions, adding complexing compound to the solution, and
adjusting the pH to a value at which precipitation occurs;
dissolving a protein, a protein derivative, a
hexamer-stabilizing compound, and a divalent metal ration in
an aqueous solvent, wherein the resulting solution has a pH
at which precipitation will not occur when a complexing
compound is added, adjusting the pH of the solution to a
value at which precipitation will occur when a complexing
compound is added, and adding a complexing compound to the
solution;
dissolving a protein, a hexamer-stabilizing
compound, and a divalent metal ration in an aqueous solvent,
wherein the resulting solution has a pH at which
precipitation will not occur when a complexing compound is
added, separately, dissolving a derivatized protein, a
hexamer-stabilizing compound, and a divalent metal ration in
an aqueous solvent, wherein the resulting solution has a pH
at which precipitation will not occur when a complexing
compound is added; thoroughly mixing together these two
solutions, adjusting the pH of the solution to a value at
which precipitation will occur when a complexing compound is
added, and adding a complexing compound to the solution;
In a preferred embodiment, the microcrystals are
prepared in a manner that obviates the need to separate the
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microcrystals from the mother liquor. Thus, it is preferred
that the mother liquor itself be suitable for administration
to the patient, or that the mother liquor can be made
suitable for administration by dilution with a suitable
diluent. The term diluent will be understood to mean a
solution comprised of an aqueous solvent in which is
dissolved various pharmaceutically acceptable excipients,
including without limitation, a buffer, an isotonicity
agent, zinc, a preservative, protamine, and the like.
1o In addition to the protein, derivatized protein,
divalent cation, complexing compound, and hexamer-
stabilizing compound, pharmaceutical compositions adapted
for parenteral administration in accordance with the present
invention may employ additional excipients and carriers such
as water miscible organic solvents such as glycerol, sesame
oil, aqueous propylene glycol and the like. When present,
such agents are usually used in an amount less than about
2.0o by weight based upon the final formulation. For
further information on the variety of techniques using
2o conventional excipients or carriers for parenteral products,
please see Remington's Pharmaceutical Sciences, 17th
Edition, Mack Publishing Company, Easton, PA, USA (1985),
which is incorporated herein by reference.
In the broad practice of the present invention, it
is also contemplated that a formulation may contain a
mixture of the microcrystals and a soluble fraction of a
protein selected from insulin, derivatized insulin, insulin
analogs, and derivatized insulin analogs. Examples of such
pharmaceutical compositions include sterile, isotonic,
3o aqueous saline solutions of insulin, an insulin analog, a
derivatized insulin, or a derivatized insulin analog,
buffered with a pharmaceutically acceptable buffer and
pyrogen-free. Preferred for the soluble phase are insulin
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or a rapid-acting insulin analog, such as, LysB28,ProB29
human insulin, or AspB28-human insulin. Such mixtures are
designed to provide a combination of meal-time control of
glucose levels, which is provided by the soluble insulin,
and basal control of glucose levels, which is provided by
the insoluble insulin. The ratio of total protein (protein
plus derivatized protein) in the insoluble phase and total
protein in the soluble phase is in the range of about 9:1 to
about 1:9. A preferred range of this ratio is from about
9:1 to about 1:1, and more preferably, about 7:3. Other
ratios are 1:1, and 3:7.
An effective dose of crystals or co-crystals for
inhalation requires inhalation of from about 0.5 ~g/kg to
about 200 ~g/kg total protein. Preferably the dose is about
5 ~g/kg to about 100 ~g/kg, about 10 ~g/kg to about 100
~g/kg, about 20 ~g/kg to about 100 ~g/kg, or about 30 ~g/kg
to about 100 ~g/kg. More preferably, the dose is from about
10 ~,g/kg to about 60 ~g/kg, 20 ~g/kg to about 60 ~g/kg, or
30 ~g/kg to about 60 ~g/kg. A therapeutically effective
amount can be determined by a knowledgeable practitioner,
who will take into account factors including insulin protein
level, the physical condition of the patient, the patient's
pulmonary status, the potency and bioavailability of the
proteins, whether the total proteins are administered
together with another insulin, such as a fast-acting, or
meal-time insulin, or with other therapeutic agents, or
other factors known to the medical practitioner. Effective
starting therapy can include "titration" of the patient,
that is, starting at a low dose, monitoring blood glucose
levels, and increasing the dose as required to achieve
desired blood glucose levels.
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According to the invention, crystals or co-
crystals are delivered by inhalation to achieve advantageous
slow uptake of insulin protein compared both to inhalation
of non-derivatized insulin protein and inhalation of
derivatized, but soluble, protein. Administration by
inhalation results in pharmacokinetics comparable to
subcutaneous administration of crystalline insulins.
According to the present invention, crystals or
co-crystals are delivered by any of a variety of inhalation
1o devices and methods known in the art for administration of
insulin, or other proteins, by inhalation [Rubsamen, U.S.
Patent No. 5,364,838, issued 15 November, 1994; Rubsamen,
U.S. Patent No. 5,672,581, issued September 30, 1997; Platz,
et al., WIPO publication No. W096/32149, published October
17, 1996; Patton, et al., WIPO publication No. W095/24183,
published September 14, 1995; Johnson, et al., U.S.~Patent
No. 5,654,007, issued August 5, 1997; Goodman, et al., U.S.
Patent No. 5,404,871, issued April 11, 1995; Rubsamen, et
al., U.S. Patent No. 5,672,581, issued September 30, 1997;
2o Gonda, et al., U.S. Patent No. 5,743,250, issued April 28,
1998; Rubsamen, U.S. Patent No. 5,419,315, issued May 30,
1995; Rubsamen, et al., U.S. Patent No. 5,558,085, issued
September 24, 1996; Gonda, et al., WIPO publication No.
W098/33480, published August 6, 1998; Rubsamen, U.S. Patent
No. 5,364,838, issued November 15, 1994; Laube, et al., U.S.
Patent No. 5,320,094, issued June 14, 1994; Eljamal, et a1.
U.S. Patent No. 5,780,014, issued July 14, 1998; Backstrom,
et al., U.S. Patent NO. 5,658,878, issued August 19, 1997;
Backstrom, et al., 5,518,998, issued May 21, 1996;
3o Backstrom, et al., 5,506,203, issued April 9, 1996; Meezan,
et al., U.S. Patent No. 5,661,130, issued August 26, 1997;
Hodson, et al., U.S. Patent No. 5,655,523, issued August
12, 1997; Schultz, et al., U.S. Patent No. 5,645,051, issued
CA 02370302 2001-10-29
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July 8, 1997; Eisele, et al., U.S. Patent No. 5,622,166,
issued April 22, 1997; Mecikalski, et al., U.S. Patent No.
5,577,497, November 26, 1996; Mecikalski, et al., U.S.
Patent No. 5,492,112, issued February 20, 1996; Williams, et
al., U.S. Patent No. 5,327,883, issued July 12, 1994;
Williams, U.S. Patent No. 5,277,195, issued January 11,
1994] .
Included among the devices used to administer
crystals and co-crystals according to the present invention
1o are those well-known in the art, such as, metered dose
inhalers, liquid nebulizers, dry powder inhalers, sprayers,
thermal vaporizers, and the like, and those provided by
developing technology, including the AERx~ pulmonary drug
delivery system being developed by Aradigm Corporation, the
dry powder formulation and delivery devices being developed
by Inhale Therapeutic Systems, Inc., and the Spiros~ dry
powder inhaler system being developed by Dura
Pharmaceuticals, Inc. Other suitable technology includes
electrohydrodynamic aerosolizers. The inhalation device
2o should deliver small particles, e.g., less than about 10 ~.m
MMAD, preferably about 1-5 ~tm MMAD, for good respirability,
and more preferably in the range of about 1 to about 3 ~.m
N.~1AD, and most preferably from about 2 to about 3 dun MMAD.
In addition, the inhalation device must be
practical, in the sense of being easy to use, small enough
to carry conveniently, capable of providing multiple doses,
and durable. Some specific examples of commercially
available inhalation devices suitable for the practice of
this invention are Turbohaler (Astray, Rotahaler (Glaxo),
3o Diskus (Glaxo), the Ultravent nebulizer (Mallinckrodt), the
Acorn II nebulizer (Marquest Medical Products), the Ventolin
metered dose inhaler (Glaxo), the Spinhaler powder inhaler
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(Fisons), or the like. Both insulin and fatty acid-acylated
insulin insulin can be advantageously delivered by a dry
powder inhaler or a sprayer. There are several desirable
features of a dry powder inhalation device for administering
crystals or co-crystals. For example, delivery by such
inhalation devices is advantageously reliable, reproducible,
and accurate.
As those skilled in the art will recognize, the
nature and quantity of the pharmaceutical composition, and
so the duration of administration of a single dose depend on
the type of inhalation device employed. For some aerosol
delivery systems, such as nebulizers, the frequency of
administration and length of time for which the system is
activated will depend mainly on the concentration of
crystals or co-crystals in the aerosol. For example,
shorter periods of administration can be used at higher
concentrations of crystals or co-crystals in the nebulizer
solution. Devices such as metered dose inhalers can produce
higher aerosol concentrations, and can be operated for
2o shorter periods to deliver the desired amount of crystals or
co-crystals. Devices such as dry powder inhalers deliver
active agent until a given charge of agent is expelled from
the device. In this type of inhaler, the amount of crystals
or co-crystals in a given quantity of the powder determines
the dose delivered in a single administration.
The particle size of the crystals and co-crystals
delivered by the inhalation device determines the extent to
which the particles are conveyed into the lower airways or
alveoli, where deposition is most advantageous because of
3o the large surface area. Preferably, at least about 100 of
the crystals or co-crystals are deposited in the lower lung,
preferably about 10o to about 20o, or more. It is known
that the maximum efficiency of pulmonary deposition for
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mouth-breathing humans is obtained at about 2 ~m to about 3
~.m MMAD. Above about 5 E.tm MMAD, pulmonary deposition
decreases substantially. Below about 1 ~.un MMAD pulmonary
deposition decreases, and it becomes difficult to deliver
particles with sufficient mass to be therapeutically
effective. Preferably, the crystals and co-crystals have a
particle size less than about 10 dun, preferably in the range
of about 1 ~m to about 5 ~.m MMAD, and more preferably in the
range of about 1 to about 3 ~tm MMAD, and most preferably
1o from about 2 to about 3 ~,un NINIAD.
Dry powder generation typically employs a method
such as a scraper blade or an air blast to generate
particles from a solid formulation of fatty acid-acylated
insulin protein. The particles are generally generated in a
container and then transported into the lung of a patient
via a carrier air stream. Typically, in current dry powder
inhalers, the force for breaking up the solid and air flow
is provided solely by the patient's inhalation. One
suitable dry powder inhaler is the Turbohaler manufactured
by Astra. Administration by dry powder inhaler is a
preferred method for crystals or co-crystals.
Inhalation delivery of the crystals and co-
crystals of the present invention can be accomplished using
inhaler devices such as, but not limited to, jet nebulizers,
dry powder inhalers, ultrasonic nebulizers, piston pump, or
piezoelectric nebulizers. The liquid solutions for the
nebulizers might also contain agents such as, but not
limited to, buffering agents, preservatives, or surfactants.
Dry powder formulations might include spray dried powders
3o from solutions of sugars or polyols such as, but not limited
to sucrose, lactose, dextrose, mannitol, trehalose, starch,
as well as buffering agents.
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Formulations of crystals or co-crystals for
administration from a dry powder inhaler typically include a
finely divided dry powder of the crystals or co-crystals,
preferably produced without resort to milling or other
mechanical operations. The powder can also include an un-
derivatized insulin or insulin analog to provide relatively
rapid onset, and short duration of action, a bulking agent,
buffer, carrier, excipient, another additive, or the like.
Additives can be included in a dry powder formulation of
1o crystals and co-crystals, for example, to dilute the powder
as required for delivery from the particular powder inhaler,
to facilitate processing of the formulation, to provide
advantageous powder properties to the formulation, to
facilitate dispersion of the powder from the inhalation
i5 device, to stabilize the formulation (e.g., antioxidants or
buffers), to provide taste to the formulation, or tire like.
Advantageously, the additive does not adversely
affect the patient's airways. The crystals or co-crystals
can be mixed with an additive so that the solid formulation
2o includes crystal or co-crystal particles mixed with or
coated on particles of the additive. Typical additives
include mono-, di-, and polysaccharides; sugar alcohols and
other polyols, such as, for example, lactose, glucose,
raffinose, melezitose, lactitol, maltitol, trehalose,
25 sucrose, mannitol, starch, or combinations thereof;
surfactants, such as sorbitols, diphosphatidyl choline, or
lecithin; or the like. Typically an additive, such as a
bulking agent, is present in an amount effective for a
purpose described above, often at about 50o to about 90o by
3o weight of the formulation. Additional agents known in the
art for formulation of a protein can also be included in the
formulation. See, for example, Japanese Patent No.
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J04041421, published February 12, 1992 (Taisho
Pharmaceutical).
Advantageously for administration as a dry powder,
the crystals or co-crystals have an MMAD of less than about
10 microns, preferably about 1 to about 5 microns, and more
preferably in the range of about 1 to about 3 ~m MMAD, and
most preferably, from about 2 to about 3 ~.un NIMAD. The
preferred particle size is effective for efficient delivery
to and deposition in the alveoli of the patient's lung.
1o Preferably, the dry powder is largely composed of particles
produced so that a majority of the particles have a size in
the desired range.
A spray including crystals or co-crystals can be
produced by forcing a suspension of crystals or co-crystals
through a nozzle under pressure. The nozzle size and
configuration, the applied pressure, and the liquid feed
rate can be chosen to achieve the desired output and
particle size. An electrospray can be produced by an
electric field in connection with a capillary or nozzle
2o feed. Advantageously, particles delivered by a sprayer have
a particle size less than about 10 ~t.m, preferably in the
range of about 1 ~,un to about 5 ~m MMAD, and more preferably
in the range of about 1 to about 3 Eun MMAD, and most
preferably from about 2 to about 3 ~tm NAIAD. Administration
as a spray is a preferred method for crystals and co-
crystals.
Formulations of crystals and co-crystals suitable
for use with a sprayer typically include crystals or co-
crystals in an aqueous solution at a concentration of about
1 mg to about 20 mg of total protein per mL of solution.
The formulation can include agents such as an excipient, a
buffer, an isotonicity agent, a preservative, a surfactant,
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and zinc. The formulation can also include an excipient or
agent for stabilization of the protein, such as a buffer, a
reducing agent, a bulk protein, or a carbohydrate. Bulk
proteins useful in formulating crystals or co-crystals
include albumin, protamine, or the like. Typical
carbohydrates useful in spray formulations include sucrose,
mannitol, lactose, trehalose, glucose, or the like. The
spray formulation can also include a surfactant, which can
reduce or prevent surface-induced aggregation of the
1o crystals or co-crystals caused by atomization of the
solution in forming an aerosol. Various conventional
surfactants can be employed, such as polyoxyethylene fatty
acid esters and alcohols, and polyoxyethylene sorbitol fatty
acid esters. Amounts will generally range between 0.0010
and 4o by weight of the formulation. Especially preferred
surfactants for purposes of this invention are
polyoxyethylene sorbitan monooleate, polysorbate 80,
polysorbate 20, or the like. Additional agents known in the
art for formulation of a protein also be included in the
2o formulation.
Crystals or co-crystals can be administered by a
nebulizer, such as jet nebulizer or an ultrasonic nebulizer.
Typically, in a jet nebulizer, a compressed air source is
used to create a high-velocity air jet through an orifice.
As the gas expands beyond the nozzle, a low-pressure region
is created, which draws a suspension of crystals or co-
crystals through a capillary tube connected to a liquid
reservoir. The suspension streaming from the capillary tube
is sheared into unstable filaments and droplets as it exits
3o the tube, creating an aerosol. A range of configurations,
flow rates, and baffle types can be employed to achieve the
desired performance characteristics from a given jet
nebulizer. In an ultrasonic nebulizer, high-frequency
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electrical energy is used to create vibrational, mechanical
energy, typically by employing a piezoelectric transducer.
This energy is transmitted to the suspension of crystals or
co-crystals either directly or through a coupling fluid,
creating an aerosol. Advantageously, particles containing
crystals or co-crystals delivered by a nebulizer have a
particle size less than about 10 ~.un., preferably in the range
of about 1 ~m to about 5 ~,un MMAD, and more.preferably in the
range of about 1 to about 3 E,im NAIAD, and most preferably
so from about 2 to about 3 ~.tm N~iAD .
Formulations of crystals suitable for use with a
nebulizer, either jet or ultrasonic, typically include
crystals or co-crystals in an aqueous solution at a
concentration of about 1 mg to about 20 mg of total protein
per mL of solution. The formulation can include agents such
as an excipient, a buffer, an isotonicity agent, a
preservative, a surfactant, and, preferably, zinc. The
formulation can also include an excipient or agent for
stabilization of the proteins, such as a buffer, a reducing
2o agent, a bulk protein, or a carbohydrate. Bulk proteins
useful in formulating include albumin, protamine, or the
like. Typical carbohydrates useful in formulating include
sucrose, mannitol, lactose, trehalose, glucose, or the like.
The formulation can also include a surfactant, which can
reduce or prevent surface-induced aggregation of the fatty
acid-acylated insulin protein caused by atomization of the
solution in forming an aerosol. Various conventional
surfactants can be employed, such as polyoxyethylene fatty
acid esters and alcohols, and polyoxyethylene sorbital fatty
acid esters. Amounts will generally range between 0.001 and
4o by weight of the formulation. Especially preferred
surfactants for purposes of this invention are
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polyoxyethylene sorbitan monooleate, polysorbate 80,
polysorbate 20, or the like.
In a metered dose inhaler (MDI), a propellant,
suspension of crystals or co-crystals, and any excipients or
other additives are contained in a canister as a mixture
including a liquefied compressed gas. Actuation of the
metering valve releases the mixture as an aerosol,
preferably with a MMAD in the range of less than about 10
~.un, preferably about 1 ~m to about 5 ~.m, and more preferably
to in the range of about 1 to about 3 ~.un MMAD, and, most
preferably from about 2 to about 3 ~m MMAD. The desired
aerosol particle size can be obtained by employing a
formulation of crystals or co-crystals produced by various
methods known to those of skill in the art, including jet-
milling, spray drying, critical point condensation, or the
like. Preferably, mechanical methods are avoided by
controlled crystallization according to the present
processes. Preferred metered dose inhalers include those
manufactured by 3M or Glaxo and employing a
2o hydrofluorocarbon propellant.
Formulations of crystals or co-crystals for use
with a metered-dose inhaler device will include the crystals
or co-crystals as a finely divided powder, in a suspension
in a non-aqueous medium, for example, suspended in a
z5 propellant with the aid of a surfactant. The propellant may
be any conventional material employed for this purpose, such
as chlorofluorocarbon, a hydrochlorofluorocarbon, a
hydrofluorocarbon, or a hydrocarbon, including
trichlorofluoromethane, dichlorodifluoromethane,
3o dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane,
HFA-134a (hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-
227), or the like. Preferably the propellant is a
hydrofluorocarbon. The surfactant can be chosen to
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stabilize the crystals or co-crystals as a suspension in the
propellant, to protect the active agent against chemical
degradation, and the like. Suitable surfactants include
sorbitan trioleate, Soya lecithin, oleic acid, or the like.
Additional agents or excipients can also be included in the
formulation.
The present invention may be better understood
with reference to the following examples. These examples
are intended to be representative of specific embodiments of
1o the invention, and are not intended as limiting the scope of
the invention.
Particle sizes are determined as follows. The
following equipment may used to determine particle diameters
for the crystals and co-crystals: Coulter~ Multisizer 646 or
equivalent (Coulter Corporation, Hialeah, FL), a Sampling
Stand II, Model 999 or equivalent (Coulter Corporation,
Hialeah, FL), a 50 ~m Coulter~ aperture tube, a pH meter,
calibrated with pH buffers that bracket the desired pH
value.
2o A stock diluent (at 2 X concentration) is prepared
containing in one liter of water, 7.56 g of dibasic sodium
phosphate crystals, 3.2 g of m-cresol, 32 g of glycerin and
1.46 g of phenol. The pH is adjusted to 7.35-7.45 with 5N
HCl or 5N NaOH, and the diluent is filtered using a 0.22 um
or smaller pore size filter. Store at room temperature. A
working diluent (at 1X concentration) is prepared by
combining one volume of the stock diluent with one volume of
water, and again filtering.
The sample to be tested is resuspended. If in a
vial, resuspend by 10 palm rolls and 10 inversions. If in a
cartridge, resuspend by three cycles of 10 palm rolls and 10
inversions. Pipette 0.25 mL of the sample into 100 mL of
working diluent. This generally gives a particle count in
CA 02370302 2001-10-29
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the range of 90,000 to 300,000 counts over 5C second
sampling time. If the particle count is less than this
range, the sample might be too dilute so a volume greater
than 0.25 mL sample will have to be used to get the
appropriate number of counts. Add sufficient volume of the
suspension to give a particle count in the range of 100,000
to 300,000. The coincidence correction should not exceed
150. For more concentrated samples, a volume less than 0.25
mL may need to be used. Place the beaker containing this
1o diluted sample on the sampling stand making sure that the
outer electrode is submerged. Perform one measurement per
sample. Measurements are made with continuous, slow
stirring over a sampling time of 50 seconds.
The following parameters are typical for the
instrument:
Page One
Orifice Diameter: 50 um
Orifice Length: 53 ~,m
Set-up: Manual
2o Analysis: Sample
Calibration: Recall
Kd: 505.00 (default value) will vary according to
each calibration.
Size: 5
Units : ~,m
Page Two
Current and Gain: Manual
Aperture Current: 400 ~,A
3o Gain : 4
Polarity: +
Instrument control: Time
Time: 50 s
Channel count: 0
Total count: Ot
Page Three
Channels: 256
Autoscaling: On
4o Edit: Off
Coincidence correction: On
Analytical volume:
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Particle relative density: 1
Diff values: o
End tone: On
Page Four
Communications Set-up
RS232C Serial output
Baud rate: 9600
Device: Computer
Autoplot: No
Print/Plot Options
Channel Data: No
Analog Plot: No
Screen Dump: Yes
Overlay Mode: No
Format: Enhanced
2o Computer Options
Send STX/ETX: No
End Field Char: 59
End Line Char(s): CR
Loading Zeros: No
The Coulter~ Multisizer is used for particle
characterization, namely, particle number and size
determination. This instrument operates on the principle
that when a particle suspended in a conductive liquid passes
through a small orifice having electrodes on either side, a
3o change in electrical resistance occurs. The change in
resistance is related to the particle volume, and causes a
short electrical pulse that is essentially proportional to
the particle volume. The measurement of particle volume
allows calculation of the equivalent volume diameter. The
series of pulses is electronically sorted to produce a size
distribution curve. The software associated with the
instrument provides the number and volume statistics and
their distributions.
Abbreviations used in the following Preparations
4o and Examples are as follows:
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BHI: biosynthetic human insulin, which is human
insulin synthesized biosynthetically in an
organism tranformed with recombinant DNA
Zn-BHI: zinc crystals of biosynthetic human insulin,
containing approximately 0.33 zinc atoms per
molecule of human insulin
C6-BHI: B29-N~-hexanoyl-human insulin
C8-BHI: B29-N~-octanoyl-human insulin
C10-BHI: B29-N~-decanoyl-human insulin
to VMSED: volume mean spherical equivalent diameter of the
particle size distribution of crystals or co-
crystals; units are microns
S.D.: standard deviation of the particle size
distribution of crystals or co-crystals; units
are microns
Preparation 1
A stock solution of 40 mg/mL C8-BHI was prepared
by dissolving lyophilized C8-BHI powder at pH 1.2. To 1 mL
of 40 mg/mL C8-BHI was added 25 ~.L 12.44 mg/mL zinc oxide.
To 1 mL of this solution was added 4 mL of crystallization
buffer containing 40 mg/mL glycerin, 4.4 mg/mL m-cresol, 1.8
mg/mL phenol, 9.375 mg/mL dibasic phosphate and 7.35 mg/mL
trisodium citrate. The pH of the resulting solution was
adjusted with 5N NaOH to 7.6. The solution was filtered
with a Millipore Millex-GV filter and mixed with an equal
volume of 0.64 mg/mL protamine sulfate. A precipitate
formed immediately. The sample was stored undisturbed at a
controlled temperature of 25°C. After 24 hours, some rod-
3o shaped crystals were observed with some amorphous material.
CA 02370302 2001-10-29
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The particle size distribution was broad, with a volume mean
spherical equivalent diameter (VMSED) of 9.7 microns.
i
2s ________ _ _.-., j,,
z
,o
d 1.5
E
' 1
0
n5
I o0('~1ON(~(~LO(htD07
(~ (O m I~ (D 07 O 0 OD (~ I~ ~ (D
V m N ~ ~ ~ p ~ N V 1n f~
.- N C f~ CO ~ ~ ~ .- .-
Pwtide 3me (rrioar)
Preparation 2
The procedure of Preparation 1 was carried out,
except that the crystallization buffer contained 7.3 mg/mL
sodium chloride. A precipitate formed immediately after
adding the protamine sulfate. The sample was stored
1o undisturbed at a controlled temperature of 25°C. After 24
hours, the amorphous precipitate converted to rod-shaped
crystalline material. The particle size distribution was
much narrower than in the absence of added sodium chloride,
with a VMSED of 5.8 microns. This also provides a means to
prepare 1000 C8-BHI/protamine crystals.
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Preparation 3
Stock solutions of C8-BHI and Zn-BHI both at 40
mg/mL were prepared at pH 1.2. 0.85 mL of 40 mg/mL C8-BHI
was mixed with 0.15 mL of 40 mg/mL Zn-BHI. To this insulin
solution was added 21.25 ~tL 12.44 mg/mL zinc oxide. To 1 mL
of this solution was added 4 mL of crystallization buffer
containing 40 mg/mL glycerin, 4.4 mg/mL m-cresol, 1.8 mg/mL
phenol, 9.375 mg/mL dibasic phosphate and 7.35 mg/mL
trisodium citrate. The pH of the resulting solution was
adjusted with 5N NaOH to 7.6. The solution was filtered
with Millipore Millex-GV filter and mixed with an equal
volume of 0.64 mg/mL protamine sulfate. A precipitate
formed immediately. The sample was stored undisturbed at a
controlled temperature of 25°C. After 24 hours, the
z5 amorphous precipitate converted to rod-shaped crystalline
material. The VMSED was 7.7 microns.
Preparation 4
The procedure of Preparation 3 was carried out,
except that the crystallization buffer contained 7.3 mg/mL
sodium chloride. A precipitate formed immediately after
adding the protamine sulfate. The sample was stored
undisturbed at a controlled temperature of 25°C. After 24
hours, the amorphous precipitate converted to rod-shaped
CA 02370302 2001-10-29
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crystalline material. The effect of adding sodium chloride
was clear and significant. Again the particle size
distribution was much narrower and the VMSED was reduced to
4.8 microns.
Preparation 5
Stock solutions of C8-BHI and Zn-BHI both ~t 40
1o mg/mL were prepared at pH 1.2. 0.75 mL of 40 mg/mL C8-BHI
was mixed with 0.25 mL of 40 mg/mL Zn-BHI. To this insulin
solution was added 18.75 ~L 12.44 mg/mL zinc oxide. To 1 mL
of this solution was added 4 mL of crystallization buffer
containing 40 mg/mL glycerin, 4.4 mg/mL m-cresol, 1.8 mg/mL
z5 phenol, 9.375 mg/mL dibasic phosphate and 7.35 mg/mL
trisodium citrate. The pH of the resulting solution was
adjusted with 5N NaOH to 7.6. The solution was filtered
with Millipore Millex-GV filter and mixed with an equal
volume of 0.64 mg/mL protamine sulfate. A precipitate
2o formed immediately. The sample was stored undisturbed at a
controlled temperature of 25°C. After 24 hours, the
amorphous precipitate converted to rod-shaped crystalline
material. The VMSED was 6.2 microns.
CA 02370302 2001-10-29
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v.
3
2
1
0
O (~ I~ N V f~ 0 ~ 07 (O ('~ N u7 ('7 ~ I
f~ V W ~ (O (D ~ V I~ O N 1O 07
N 47 ~,.~ O M ~ ~ ~ ~ N V M CO h
N 47 (O N .-
Preparation 6
The procedure of Preparation 5 was carried out,
except that the crystallization buffer contained 7.3 mg/mL
sodium chloride. A precipitate formed immediately after
adding the protamine sulfate. The sample was stored
undisturbed at a controlled temperature of 25°C. After 24
hours, the amorphous precipitate converted to rod-shaped
crystalline material. The effect of adding sodium chloride
1o was clear and significant. Again the particle size
distribution was much narrower and the VMSED was reduced to
4.2 microns.
Preparation 7
Stock solutions of C8-BHI and Zn-BHI both at 40
mg/mL were prepared at pH 1.2. 0.65 mL of 40 mg/mL C8-BHI
was mixed with 0.35 mL of 40 mg/mL Zn-BHI. To this insulin
2o solution was added 16.25 ~tL 12.44 mg/mL zinc oxide. To 1 mL
of this solution was added 4 mL of crystallization buffer
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containing 40 mg/mL glycerin, 4.4 mg/mL m-cresol, 1.8 mg/mL
phenol, 9.375 mg/mL dibasic phosphate and 7.35 mg/mL
trisodium citrate. The pH of the resulting solution was
adjusted with 5N NaOH to 7.6. The solution was filtered
with Millipore Millex-GV filter and mixed with equal volume
of 0.64 mg/mL protamine sulfate. A precipitate formed
immediately. The sample was stored undisturbed at a
controlled temperature of 25°C. After 24 hours, the
amorphous precipitate had converted to rod shaped
1o crystalline material. The VMSED was 5.0 microns.
Preparation 8
The procedure of Preparation 7 was carried out,
except that the crystallization buffer contained 7.3 mg/mL
sodium chloride. A precipitate formed immediately after
adding the protamine sulfate. The sample was stored
undisturbed at a controlled temperature of 25°C. After 24
hours, the amorphous precipitate had converted to rod-shaped
2o crystalline material. The effect of adding sodium chloride
was clear and significant. Again the particle size
distribution was much narrower and the VMSED was reduced to
3.8 microns.
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Preparation 9
The procedure of Preparation 1 was carried out,
except that the C8-BHI was initially dissolved at pH 2.4
instead of pH 1.2. After 24 hours, the material was still
largely amorphous with a few rod-shaped crystals observed.
The particle size distribution was multi-modal.
Preparation 10
The procedure of Preparation 9 was carried out,
except that the crystallization buffer contained 7.3 mg/mL
sodium chloride. A precipitate formed immediately after
adding the protamine sulfate. The sample was stored
undisturbed at a controlled temperature of 25°C. After 24
hours, unlike the preparation without added sodium chloride,
the amorphous precipitate had converted to rod-shaped
crystalline material. The VMSED was 8.4 microns. This
o m n N o n v v rn co c~ co m c~
r v m w co co ~ v n o cyn w a;
W 7 ~,j O (~ r m ~ ~ N V 1n t0 I~
'- N ~ tD N ~ '- ~ .-
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represented an example of controlling the preparation of
1000 C8-BHI protamine crystals to achieve a Gaussian
distribution having a narrower distribution of particle
sized, and a lower mean.
J
i
p
'~. O c~ I~ N 0 I~ V V O> CO (~ r. CO ~ (~
~, I~ V N m (D tD ~ ~ V 1~ O N ~ c0
', N ~ ~ O ('J ~ 07 p ~ N V ~ (D h
N n17 (O fD
Preparation 11
The procedure of Preparation 3 was carried out,
1o except that the C8-BHI was initially dissolved at pH 2.4
instead of pH 1.2. After 24 hours, the amorphous
precipitate had converted to rod shaped crystalline
material. The VMSED was 11.0 microns.
Preparation 12
The procedure of Preparation 11 was carried out,
except that the crystallization buffer contained 7.3 mg/mL
sodium chloride. A precipitate formed immediately after
2o adding the protamine sulfate. The sample was stored
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undisturbed at a controlled temperature of 25°C. After 24
hours, the amorphous precipitate had converted to rod-shaped
crystalline material. The effect of adding sodium chloride
was clear and significant. Again the particle size
distribution was much narrower and the VMSED was reduced to
6.5 microns.
1o Preparation 13
The procedure of Preparation 5 was carried out,
except that the C8-BHI was initially dissolved at pH 2.4
instead of pH 1.2. After 24 hours, the amorphous
precipitate had converted to rod-shaped crystalline
material. The VMSED was 8.7 microns.
o c~ n N v r v v o~ co c~ m m c~
n v co rn ~ co ~ v n o N m m
cu p ~ o c~ ~ oo p ~ N v u~ c~ ~
N tn (O OD r
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Preparation 14
The procedure of Preparation 13 was carried out,
except that the crystallization buffer contained 7.3 mg/mL
sodium chloride. A precipitate formed immediately after
adding the protamine sulfate. The sample was stored
undisturbed at a controlled temperature of 25°C. After 24
hours, the amorphous precipitate had converted to rod-shaped
crystalline material. The effect of adding sodium chloride
was clear and significant. Again the particle size
to distribution was much narrower and the VMSED was reduced to
6.0 microns.
Preparation 15
The procedure of Preparation 7 was carried out,
except that the C8-BHI was initially dissolved at pH 2.4
instead of pH 1.2. After 24 hours, the amorphous
precipitate had converted to rod-shaped crystalline
material. The VMSED was 7.0 microns.
CA 02370302 2001-10-29
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i 3.
1
0
o c~ n N v n v v o~ ~ c~ ~. w u~ c~
n v m o> co c~ ~ ~ v n o cyn m
N N ~ O CJ n O7
N u7 CD N
i
Preparation 16
The procedure of Preparation 15 was carried out,
except that the crystallization buffer contained 7.3 mg/mL
sodium chloride. A precipitate formed immediately after
adding the protamine sulfate. The sample was stored
undisturbed at a controlled temperature of 25°C. After 24
hours, the amorphous precipitate had converted to rod-shaped
1o crystalline material. The effect of adding sodium chloride
was clear and significant. Again the particle size
distribution was much narrower and the VMSED was reduced to
4.7 microns.
Preparation 17
A C8-BHI solution was prepared by dissolving 61.4
mg lyophilized powder of C8-BHI in 1.54 mL 0.1 N HC1. A Zn-
BHI solution was prepared by dissolving 20.5 mg of Zn-BHI
crystals in 0.51 mL 0.1 N HCl. To the Zn-BHI solution was
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added 1.5 mL of the C8-BHI solution. To the Zn-BHI and C8-
BHI solution was added 49 ~L 12.44 mg/mL Zinc oxide stock
solution. To this solution was added 8 mL of
crystallization buffer containing 40 mg/mL glycerin, 4.4
mg/mL m-cresol, 1.8 mg/mL phenol, 9.375 mg/mL dibasic
phosphate and 7.35 mg/mL trisodium citrate. The pH of the
resulting solution was adjusted with 5N NaOH to 7.6. The
solution was filtered with Millipore Millex-GV filter and
mixed with equal volume of 0.64 mg/mL protamine sulfate. A
to precipitate formed immediately. The suspension was equally
divided and stored undisturbed at controlled temperatures of
20, 25 and 30°C. After 24 hours, the amorphous precipitates
had in each sample converted to rod-shaped crystalline
material. The effect of temperature was clear and
significant. The VMSED was 7.8, 6.9 and 5.5 microns at 20,
and 30°C respectively.
Summary of Preparations 1 - 17
Day Day
1 30
Preparation C8- NaCl Disso- VMSED S.D. VMSED S.D.
BHI added lution
~ pH
1 100 None 1.2 9.65 2.68 9.60 2.58
2 100 50mM 1.2 5.78 1.55 6.70 1.62
3 85 None 1.2 7.68 2.00 8.68 2.01
4 85 50mM 1.2 4.79 1.20 5.72 1.28
5 75 None 1.2 6.24 1.69 7.08 1.62
6 75 50mM 1.2 4.21 1.12 4.70 1.06
7 65 None 1.2 4.99 1.20 5.97 1.24
8 65 50mM 1.2 3.85 1.21 4.45 1.23
9 100 None 2.4 - - 10.91 6.61
10 100 50mM 2.4 8.38 2.05 8.88 2.24
11 85 None 2.4 11.03 2.93 11.50 2.93
12 85 50mM 2.4 6.52 1.53 7.25 1.50
13 75 None 2.4 8.73 2.31 9.35 2.21
14 75 50mM 2.4 6.01 1.42 6.74 1.36
15 65 None 2.4 7.05 1.71 7.75 1.68
16 65 50mM 2.4 4.67 1.14 5.30 1.20
Temp.
17 75 None 20C 7.80 2.28 - -
17 75 None 25C 6.93 1.84 - -
17 75 None 30C 5.52 1.58 - -
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The following conclusions are drawn on the basis
of Preparations 1 - 17, above. First, at the same
percentage of C8-BHI, pH 1.2 dissolution gives smaller and
tighter particle size distribution than pH 2.4 dissolution.
Second, a lower percentage of C8-BHI gives smaller co-
crystals and a smaller standard deviation. Third, adding
sodium chloride also yields smaller co-crystals and a
smaller standard deviation. This clearly provides a means
to control the particle size with possible different
1o variations in formulations and manufacturing processes.
Finally, adding sodium chloride also clearly provides a
means to prepare 1000 C8-BHI crystals.
Preparation 18
A 50 mL stock solution of C8-BHI at approximately
26.5 mg/mL and Zn-BHI at 8.9 mg/mL was prepared at pFt 1.2.
To this insulin solution was added approximately 1mL of
12.44 mg/mL zinc oxide. To this solution was added 200 mL
of crystallization buffer containing 40 mg/mL glycerin, 4.4
2o mg/mL m-cresol, 1.8 mg/mL phenol, 9.375 mg/mL dibasic
phosphate and 7.35 mg/mL trisodium citrate. The pH of the
resulting solution was adjusted with 5N NaOH to 7.6. The
solution was filtered with Millipore Millex-GV filter and
mixed with equal volume of 0.64 mg/mL protamine sulfate. A
precipitate formed immediately. The suspension was stored
undisturbed at a controlled temperature of 25°C. After 24
hours, the amorphous precipitate had converted to rod-shaped
crystalline material. The VMSED was 7.8 microns.
Preparation 19
An approximately 120 mL stock solution of C8-BHI
at approximately 10.5 mg/mL and approximately 3.5 mg/mL Zn-
BHI was prepared at pH 2.4. To this insulin solution was
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added approximately 1 mL of 12.44 mg/mL zinc oxide. To this
solution was added approximately 125 mL of crystallization
buffer containing 64 mg/mL glycerin, 7.0 mg/mL m-cresol, 2.9
mg/mL liquefied phenol, 15 mg/mL dibasic sodium phosphate
and 11.76 mg/mL trisodium citrate. The pH of the resulting
solution was adjusted with 5N NaOH to 7.6. Water was added
to bring the volume to 250 mL. The solution was filtered
with Millipore Millex-GV filter and mixed with an equal
volume of 0.64 mg/mL protamine sulfate. A precipitate
formed immediately. The suspension was stored undisturbed
at a controlled temperature of 25°C. After 24 hours, the
amorphous precipitate had converted to rod-shaped
crystalline material. The VMSED was 9.2 microns.
Preparation 20
The procedure of Preparation 19 was followed,
except that the crystallization buffer also contained 11.69
mg/mL sodium chloride. After 24 hours, the amorphous
precipitate had converted to rod-shaped crystalline
2o material. The VMSED was 5.3 microns.
Preparation 21
A 25 mL stock solution of C8-BHI at approximately
10.5 mg/mL and approximately 3.5 mg/mL Zn-BHI was prepared
at pH 2.5. To 2.5 mL of this insulin solution was added
approximately 0.24 ~L of 12.44 mg/mL zinc oxide. To this
solution was added approximately 2.5 mL of crystallization
buffer containing 64 mg/mL glycerin, 7.0 mg/mL m-cresol, 2.9
mg/mL liquefied phenol, 15 mg/mL dibasic sodium phosphate
3o and 11.76 mg/mL trisodium citrate. The pH of the resulting
solution was adjusted with 5N NaOH to 7.6. The solution was
filtered with Millipore Millex-GV filter and mixed with
equal volume of 0.64 mg/mL protamine sulfate. A precipitate
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formed immediately. The suspension was stored undisturbed
at a controlled temperature of 25°C. After 24 hours, the
amorphous precipitate had converted to rod-shaped
crystalline material. The VMSED was 7.09 microns.
Preparation 22
The procedure of Preparation 21 was followed,
except that the crystallization buffer also contained 11.69
mg/mL sodium chloride. After 24 hours, the amorphous
1o precipitate had converted to rod-shaped crystalline
material. The VMSED was 5.72 microns.
Preparation 23
The procedure of Preparation 21 was followed,
except that the crystallization buffer also contained 14.91
mg/mL potassium chloride. After 24 hours, the amorphous
precipitate had converted to rod-shaped crystalline
material. The VMSED was 5.39 microns.
2o Preparation 24
The procedure of Preparation 21 was followed,
except that the crystallization buffer also contained 16.41
mg/mL sodium acetate. After 24 hours, the amorphous
precipitate had converted to rod-shaped crystalline
material. The VMSED was 7.39 microns.
Preparation 25
The procedure of Preparation 21 was followed,
3o except that the crystallization buffer also contained 46.02
mg/mL sodium tartrate. After 24 hours, the amorphous
precipitate had converted to rod-shaped crystalline
material. The VMSED was 8.07 microns.
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Preparation 26
The procedure of Preparation 21 was followed,
except that the crystallization buffer also contained 28.41
mg/mL sodium sulfate. After 24 hours, the amorphous
precipitate had converted to rod-shaped crystalline
material. The VMSED was 7.07 microns.
Preparation 27
l0 The procedure of Preparation 21 was followed,
except that the crystallization buffer also contained 13.6
mg/mL sodium formate. After 24 hours, the amorphous
precipitate had converted to rod-shaped crystalline
material. The VMSED was 6.90 microns.
Summary of Preparations 18 - 27
PreparationScale Salt added VMSED SD
(50 mM)
18 500mL None 7.81 1.77
19 500mL None 9.24 2.46
500mL NaCl 5.31 1.32
21 lOmL None 7.09 1.85
22 lOmL NaCl 5.72 1.45
23 lOmL KC1 5.39 1.40
24 lOmL Na acetate 7.39 2.07
lOmL Na tartrate 8.07 2.21
26 lOmL Na sulfate 7.07 1.70
27 lOmL Na formate 6.90 1.88
The following conclusions are drawn from
2o Preparations 18 - 27. First, the scale at which the
crystallization is carried out affects the mean particle
size only when no sodium chloride is added. The addition of
sodium chloride, or just chloride anion, will permit more
reproducible results at a larger scale, using a
25 manufacturable process. The effect of sodium chloride is
due to chloride, not sodium. Other anions tested had little
or no effect. These results also show that the effect of
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sodium chloride is not due to an increase in ionic strength,
but rather to the specific effect of the chloride anion.
Preparation 28
100% C8-BHI Crystal Formulations For Rat Intratracheal
Instillation Experiments
A dry lyophilized powder of C8-BHI (471 mg) was
dissolved in 11.77 mL of 0.1 N HCl. To this solution was
1o added 3.532 mL of a 1000 ppm solution of zinc nitrate and
stirred. To this solution was added 47.08 mL of an aqueous
solution comprised of 9.5 mg/mL disodium phosphate
heptahydrate, 0.375 M NaCl, 4 mg/mL m-cresol, 1.65 mg/mL
phenol, and 16 mg/mL glycerol at pH 7.63. The pH was
adjusted to 7.6 with small quantities of 1N HC1 and 1N NaOH.
This solution was then filtered through a 0.2 micron low
protein binding filter. A second solution was prepared by
dissolving 86.8 mg of a dry powder of protamine sulfate in
115.73 mL of water and then filtered through a 0.2 micron
low protein binding filter. 55.95 mL of C8-BHI solution was
mixed with 55.95 mL of protamine sulfate solution. A white
precipitate formed. This suspension was stirred gently to
complete mixing. The preparation was allowed to stand
undisturbed for 24 hours in a 30°C water bath. Inspection
under an optical microscope revealed the presence of
microcrystalline solid comprising rod-like crystals.
Measurement of particle size distribution by the Coulter
technique revealed a VMSED of 4.7 microns.
The mother liquor of this preparation was
3o exchanged with an aqueous solution comprised of 0.7 mg/mL
phenol, l.6mg/mL m-cresol and 0.3 mg/mL of disodium
phosphate heptahydrate by the following procedure. 50 mL of
suspension was centrifuged at 3000 rpm for 12 minutes at
23°C, 40 mL of mother liquor was removed without disturbing
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the solid phase and replaced with the aqueous solution
comprised of 0.7 mg/mL phenol, l.6mg/mL m-cresol and 0.3
mg/mL of disodium phosphate heptahydrate. This procedure
was repeated twice more. Following this exchange of mother
liquor, the microcrystals were again analyzed using the
Coulter counter, and were found to have a VMSED of 3.5
microns.
Analytical characterization by HPLC revealed that
almost all of the insulin and protamine sulfate was present
to in the solid phase. For rat intratracheal instillation
experiments, these formulations were diluted to the required
concentration by appropriate dilution with an aqueous
solution containing 0.7mg/mL phenol, l.6mg/mL m-cresol and
0.3 mg/mL of disodium phosphate heptahydrate.
Preparation 29
75°o C8-BHI Crystal Formulations For Rat Intratracheal
Instillation Experiments
2o A dry lyophilized powder of C8-BHI (330 mg) was
dissolved in 8.25 mL of 0.1 N HC1. A dry powder of human
insulin-Zn crystals (116 mg) was dissolved in 2.9 mL of 0.1
N HC1. 2.75 mL of the later solution was mixed with C8-BHI
solution to produce a mixture of C8-BHI and human insulin in
approximate weight ratio of 75:25. This solution was
stirred to mix. To this solution was added 2.866 mL of a
1000 ppm solution of zinc nitrate and stirred. To this
solution was added 47 mL of an aqueous solution comprised of
9.5 mg/mL disodium phosphate heptahydrate, 0.375 M NaCl, 4
3o mg/mL m-cresol, 1.65 mg/mL phenol, and 16 mg/mL glycerol at
pH 7.63. The pH was adjusted to 7.6 with small quantities
of 1N HCl and 1N NaOH. This solution was then filtered
through a 0.2 micron low protein binding filter. A second
solution was prepared by dissolving 86.8 mg of a dry powder
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of protamine sulfate in 115.73 mL of water and then filtered
through a 0.2 micron low protein binding filter. 50mL of
the solution of the mixture of C8-BHI and human insulin was
mixed with 50 mL of protamine sulfate solution. A white
precipitate formed. This suspension was stirred gently to
complete mixing. The preparation was allowed to stand
undisturbed for 24 hours at 30°C. Inspection under an
optical microscope revealed the presence of microcrystalline
solid comprising rod-like crystals. Measurement of particle
size distribution by Coulter technique revealed a VMSED of
4.4 microns.
The mother liquor of this preparation was
exchanged with an aqueous solution comprised of 0.7 mg/mL
phenol, l.6mg/mL m-cresol and 0.3 mg/mL of disodium
phosphate heptahydrate as described above in Preparation 28.
Following the exchange of mother liquor, the microcrystals
were determined to have VMSED of 3.9 microns.
Analytical characterization by HPLC revealed that
almost all of the insulin and protamine sulfate was present
2o in the solid phase. For rat intratracheal instillation
experiments, these formulations were diluted to the required
concentration by appropriate dilution with an aqueous
solution containing 0.7 mg/mL phenol, l.6mg/mL m-cresol and
0.3 mg/mL of disodium phosphate heptahydrate.
The procedures described in Preparations 28 and 29
demonstrate that crystals comprised of a derivatized
insulin, together with zinc and protamine, can be produced
in a size that is preferred for optimizing deposition in the
deep lung (preferably <5 microns, and more preferably <3
3o microns) when the crystallization conditions are properly
controlled. The critical parameters of these preparations
were the concentration of NaCl present during
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crystallization (150 mM) and the temperature during
crystalli zation ( 3 0°C ) .
Preparation 30
The mother liquors of Preparation 28 and
Preparation 30 were exchanged with water by the following
procedure. 5mL of suspension was centrifuged at 3000 rpm
for 12 minutes at 23°C. 4 mL of mother liquor was removed
without disturbing the solid phase and replaced with water.
1o This procedure was repeated twice more. The resulting
suspension was freeze dried. The solid obtained after
freeze drying was reconstituted in 5 mL of water. The
resulting suspension contained small microcrystals when
examined under an optical microscope.
Preparation 31
The mother liquors of Preparation 28 and
Preparation 29 were exchanged as described in Preparation
30, except that a solution of 0.9 g NaCl per 100 mL was used
2o to wash the crystals. After three washes, the resulting
suspension was freeze dried. The solid obtained after
freeze drying was reconstituted in 5mL of water. The
resulting suspension contained small microcrystals when
examined under an optical microscope.
The procedures described in Preparations 30 and 31
demonstrate that the microcrystals obtained by the processes
of the present invention (i.e., for example, Preparations 28
and 29) can be freeze-dried without loss of their
crystalline nature. Such microcrytalline powders can be
3o used in dry powder inhalers for pulmonary delivery. These
results also demonstrate that these freeze-dried
microcrystals can be mixed with water to form stable aqueous
suspensions with minimal excipients. Such aqueous
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suspensions can be used in nebulizers for pulmonary
delivery.
Preparation 32
After crystallization according to the procedure
described for either one of Preparations 28 or 29, as
determined by optical microscopy, the microcrystals are
separated from the mother liquor and are recovered by
conventional solid/liquid separation methods. The recovered
1o microcrystals are then resuspended in a solution comprised
of a buffer (e. g., 0.3 mg/mL dibasic sodium phosphate
heptahydrate), a anti-microbial preservative (e. g., 0.65
mg/mL phenol and/or 1.6 mg/mL m-cresol), and an isotonicity
agent (e.g., 16 mg/mL glycerol or 9 mg/mL NaCl), and the pH
is adjusted to 6.8.
Preparation 33
After crystallization according to the procedure
described for either one of Preparations 28 or 29, as
2o determined by optical microscopy, the microcrystals are
separated from the mother liquor and are recovered by
conventional solid/liquid separation methods. The recovered
microcrystals are then resuspended according to Preparation
32, except that the resuspension solution contains no
antimicrobial preservative. Such a preparation might be
more suitable when the absence of irritants or unneeded
excipients is desired.
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Preparation 34
Dissolution of 75% C8-BHI co-crystals having VMSED of 2.1
microns
A preparation of 75o C8-BHI cocrystals was made by
a procedure similar to preparation 29, on a 10 mL scale.
The crystals prepared by this procedure had a VMSED of 2.1
microns.
The in-vitro dissolution rate of these crystals
1o was examined using a spectrophotometric dissolution assay.
3 mL of phosphate buffered saline was placed in a quartz
cuvette with a small stir bar. The solution was stirred at
a fixed rate. The absorbance was zeroed. Five microliters
of uniformly suspended formulation was quickly suspended at
z5 the bottom of the cuvette. One minute after the addition of
the formulation, absorbance data at 305 nm was collected as
a function of time. Change of absorbance was followed as a
function of time.
The absorbance decreases as the scattering
2o particles dissolve. The time for the absorbance to decrease
by half of its full decrease is denoted by the parameter
t1/2. This parameter is useful for comparing the rates of
dissolution of different crystalline preparations. The
greater t1/2, the slower is the dissolution.
25 In the dissolution test described above, t1/2 for
the co-crystals prepared according to Preparation 34 was
determined to be 50 minutes. Under similar conditions of
dissolution, t1/2 for NPH-human insulin crystals was
typically about 10 minutes or less. Thus, despite its
3o smaller VMSED and expected concomitant increase in surface
area, the co-crystals of Preparation 34 dissolved
significantly slower than NPH. This observation supports
the conclusion that the smaller microcrystals of the present
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invention will provide more sustained release in vivo as
compared with NPH-human insulin crystals.
Preparations 35-40
The procedures of Preparations 28 and 29 above
were followed at the 10 mL scale to produce three samples of
crystals each, with the following exceptions: the
crystallization buffer for the three samples contained
1o either no sodium chloride, 75 mM sodium chloride, or 150 mM
sodium chloride; and the temperature was controlled at 30°C.
Very small particles, with approximately 2 micron mean
particle diameter and a narrow size distribution were
obtained for both 75o and 1000 C8-BHI compositions when 150
mM NaCl was present during crystallization.
Preparation Composition [NaCl] VMSED S.D.
(mM) (microns)(MicYons)
35 100 C8-BHI 0 4.4 2.9
36 100 C8-BHI 75 4.1 1.3
37 100 C8-BHI 150 2.1 0.7
38 75~ C8-BHI 0 4.2 1.1
39 75o C8-BHI 75 3.6 1.0
40 75o C8-BHI 150 2.2 0.6
Preparations 41-44
2o A dry powder of C8-BHI (24.0 mg) was dissolved in
1.20 mL of 0.1 N HCl. A separate solution was prepared by
dissolving a dry powder of human insulin zinc crystals (8.0
mg) in 0.400 mL of 0.1 N HC1. These two solutions were
combined and mixed to produce 1.60 mL of a solution mixture
of human insulin and C8-BHI. To this solution was added 448
microliters of a 15.3 mM solution of zinc chloride with
stirring. To this solution was added 6.4 mL an aqueous
solvent composed of 10 mg/mL dibasic sodium phosphate
heptahydrate, 7.5 mg/mL trisodium citrate dehydrate, 4 mg/mL
3o m-cresol, 2 mg/mL phenol, and 40 mg/mL glycerol at pH 7.59.
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The pH was adjusted to 7.61 with small quantities of 1 N HCl
and 1 N NaOH. This solution was then filtered through a
0.22 micron low protein binding filter. A second solution
was prepared by dissolving 72.1 mg of protamine sulfate in
96 mL of water and then filtering through a 0.22 micron low
protein binding filter. An 8 mL volume of the insulin
mixture solution was combined with 8 mL of the protamine
sulfate solution. An amorphous precipitate formed. This
suspension was stirred gently to complete mixing. The
to preparation was divided into four volumes of 4 mL which were
allowed to stand undisturbed at temperatures of 15 °C, 25
°C, 30 °C and 35 °C, respectively, for 90 hours.
Inspection
under an optical microscope (1000x) revealed that in each
case the amorphous precipitate had converted to a
microcrystalline solid of uniform appearance, comprising
single, rod-like crystals. The preparations corresponding
to temperatures of 15 °C , 2 5 °C , 3 0 °C and 3 5
°C were
designated Preparations 41, 42, 43, and 44 respectively.
Each sample was centrifuged at 3000 rpm for 12
2o minutes to sediment the crystals. For each Preparation, 3.2
mL of supernatant was decanted off and replaced with 3.2 mL
of an aqueous diluent comprising 4 mg/mL dibasic sodium
phosphate heptahydrate, 3 mg/mL trisodium citrate dehydrate,
0.8 mg/mL phenol, and 16 mg/mL glycerol at pH 7.61. This
diluent exchange process was repeated a second and a third
time, except on the third occasion the 3.2 mL was replaced
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with 2.8 mL of the aforementioned aqueous diluent. The
VMSED of these preparations are shown in the table below.
Preparation Crystallization VMSED
Temperature
( ~C ) ( mi Irons )
41 15 9.0
42 25 6.0
43 30 5.5
44 35 4.9
Preparations 45 - 48
A dry powder of C8-BHI (24.0 mg) was dissolved in
0.60 mL of 0.1 N HC1. A separate solution was prepared by
dissolving a dry powder of human insulin zinc crystals (8.2
mg) in 0.20 mL of 0.1 N HC1. These two solutions were
1o combined and mixed to produce 0.80 mL of a solution mixture
of human insulin and C8-BHI. To this solution was added 300
microliters of a 15.3 mM solution of zinc chloride with
stirring. This solution was divided into four separate
volumes of 0.275 mL. Four different crystallization buffers
z5 were prepared, each of which contained 35 mM dibasic sodium
phosphate heptahydrate, 4 mg/mL m-cresol, 1.6 mg/mL phenol,
and 40 mg/mL glycerol at pH 7.6, and each of which differed
in sodium citrate concentrations, which were 0 mM, 12.5 mM,
37.5 mM, and 87.5 mM, respectively, for Preparations 45, 46,
20 47, and 48. To each of the four samples of 0.275 mL of the
protein solution was added 1.1 mL of a crystallization
buffer. The pH of each solution was adjusted to 7.6 with
small quantities of 1 N HCl and 1 N NaOH. Each solution was
then filtered through a 0.22 micron low protein binding
25 filter. To 1 mL of each solution was added 1 mL of a
protamine sulfate solution prepared by dissolving 37.6 mg of
protamine sulfate in 50 mL of water and then filtering
through a 0.22 micron low protein binding filter. An
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amorphous precipitate formed in each case. Each preparation
was stirred gently to complete mixing. The four preparations
were allowed to stand undisturbed at 25°C for 23 hours.
Inspection under an optical microscope (1000x) revealed that
in each case the amorphous precipitate had converted to a
microcrystalline solid of uniform appearance, comprising
single, rod-like crystals and that the mean crystal size of
each preparation was different as tabulated:
Preparation [Citrate] Crystal length
(microns)
45 0 <3
46 5 3-6
47 15 5-10
48 35 >10
Preparation 49
A dry powder of C10-BHI (60.7 mg) was dissolved in
1.50 mL of 0.1 N HC1. To this solution was added 600
microliters of a 15.3 mM solution of zinc chloride with
stirring. To 0.70 mL of this solution was added 2 mL of an
aqueous solvent composed of 50 mM TRIS, 10 mg/mL phenol, 30
mg/mL trisodium citrate dehydrate, and 31 mg/mL glycerol at
pH 7.60. The pH was adjusted to 7.61 with small quantities
of 1 N HCl and 1 N NaOH. This solution was then filtered
2o through a 0.22 micron low protein binding filter. A second
solution was prepared by dissolving 37.8 mg of protamine
sulfate in 50 mL of water and then filtering through a 0.22
micron low protein binding filter. A 2.5 mL volume of the
C10-BHI mixture solution was combined with 2.5 mL of the
protamine sulfate solution. An amorphous precipitate
formed. This suspension was stirred gently to complete
mixing. The preparation was allowed to stand undisturbed at
a temperature of 25°C for 60 hours. Inspection under an
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optical microscope (1000x) revealed that the amorphous
precipitate had converted to a microcrystalline solid of
uniform appearance, comprising small rod-like crystals with
an estimated approximate mean particle size of 2 microns.
Preparation 50
A dry powder of C6-BHI(39.2 mg) was dissolved in
1000 parts by volume of 0.1 N HCl. To this solution was
added 400 microliters of a 15.3 mM solution of zinc chloride
l0 with stirring. To this solution was added 4 mL an aqueous
solvent comprised of 5 mg/mL dibasic sodium phosphate
anhydrous, 25 mM trisodium citrate, 1.6 mg/mL phenol, 4
mg/mL m-cresol and 40 mg/mL glycerol at pH 7.6. The pH was
adjusted to 7.60 with small quantities of 1 N HCl and 1 N
NaOH. This solution was then filtered through a 0.22 micron
low protein binding filter. A second solution was prepared
by dissolving 37.3 mg of protamine sulfate in 50 mL of water
and then filtering through a 0.22 micron low protein binding
filter. A 5 mL volume of the C6-BHI solution was combined
with 5 mL of the protamine sulfate solution. An amorphous
precipitate formed. This suspension was stirred gently to
complete mixing. The preparation was allowed to stand
undisturbed at a temperature of 25°C for 47 hours.
Inspection under an optical microscope (1000x) revealed that
the amorphous precipitate had converted to a
microcrystalline solid of uniform appearance, comprising
single, rod-like crystals possessing an approximate mean
length of 2 microns.
3o Preparations 51 - 54
The procedure of either Preparation 1 or
Preparation 19 was followed as indicated in the table below
to prepare 75o C8-BHI co-crystals, except that the
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crystallization buffer was prepared so that the final
citrate and sodium chloride concentrations in the four
samples were as follows:
PreparationProcess Final Final VMSED S.D.
used
# [citrate) [NaCl)(microns) (microns)
(mM) (mM)
51 Preparation10 0 9.243 2.46
19
52 Preparation10 50 5.314 1.32
19
53 Preparation2 20 6.67 1.69
19
54 Preparation10 0 7.812 1.77
1
After 24 hours, the amorphous precipitate had
converted to rod-shaped crystalline material in each case.
The VMSED and S.D. for each preparation are given in the
table above.
Preparation 55
Formulation of Microcrystals
The microcrystals prepared according to any of
Preparations 1 - 51 are separated from the mother liquor and
are recovered by conventional solid/liquid separation
methods. The recovered microcrystals are then suspended in
a solution consisting of 2 mg/mL sodium phosphate dibasic,
1.6 mg/mL m-cresol, 0.65 mg/ml phenol, and 16 mg/ml
glycerol, pH 6.8, so that the final concentration of insulin
activity is about 100 U/mL.
Preparation 56
Formulation of Microcrystals
The microcrystals prepared according to any of
Preparations 1 - 51 are separated from the mother liquor and
are recovered by conventional solid/liquid separation
methods. The recovered microcrystals are then suspended in
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a solution consisting of 0.65 mg/ml phenol in water so that
the final concentration of insulin activity is about 100
U/mL. A pH adjustment to approximately 6.8 is performed
with 1 N HCl and 1 N NaOH.
Preparations 57 - 83
All preparations produced co-crystals of 75o C8-
BHI. For all but three preparations (57, 62, and 63), the
procedure of Preparation 19 was essentially followed, except
1o that the crystallization buffer was adjusted as needed to
achieve the final citrate and sodium chloride concentrations
indicated. The scale was 32 - 50 mL. The pH at which
dissolution of the proteins was carried out is indicated in
the table. These preparations provide further support for
the conclusions drawn above, and additionally, reveal that
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VMSED is inversely related to the citrate concentration at
least in the range of 0 to 10 mM.
Preparation CitrateNaCl dissolutionVMSED S.D.
No. (mM) (mM) pH (microns) (micron
s)
57 10 0 1.2 7.87 1.62
58 10 0 2.1 8.88 2.02
59 0 0 2.1 8.08 2.21
60 0 50 2.1 5.83 1.4
61 10 50 2.1 5.42 1.25
62 10 50 1.2 5.17 1.21
63 10 50 2.5 6.52 1.94
64 10 0 2.1 8.59 2.03
65 10 15 2.1 6.66 1.49
66 10 30 2.1 5.95 1.39
67 10 50 2.1 5.42 1.55
68 0 0 2.1 7.81 2.25
69 0 15 2.1 6.65 1.76
70 0 30 2.1 6.32 1.79
71 0 50 2.1 Amorphous
72 4 0 2.1 7.60 2.22
73 4 15 2.1 6.43 1.57
74 4 30 2.1 6.01 1.47
75 4 50 2.1 5.89 1.34
76 0 20 2.2 5.94 1.85
77 1 20 2.2 6.27 1.94
78 2 20 2.2 6.29 1.84
79 4 20 2.2 6.36 1.75
80 0 50 2.2 5.02 1.78
81 1 50 2.2 6.52 2.07
82 2 ~ 50 2.2 6.55 2.17
83 4 50 2.2 6.31 T 1.88
Preparations 84 - 89
Effect of Zinc
All preparations produced co-crystals of 75o C8-
BHI. For all preparations the procedure of Preparation 19
1o was essentially followed with the final citrate
concentration of 10 mM and the final sodium chloride
concentration of 50 mM. The scale was 20 mL. The pH at
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which dissolution of the proteins was carried out was 2.5.
Zinc was added as indicated in the table. The zinc
concentration is expressed as a weight proportion with
respect to the total protein (insulin plus insulin
derivative). Zinc at greater than 0.1o is required, and
between 0.5o and 5.80, zinc concentration no consistent
effect on VMSED.
PreparationZinc (o) VMSED S.D.
No. (microns) (microns)
84 0.1 Amorphous -
85 0.5 5.40 1.42
86 0.8 5.99 1.67
87 1.6 6.63 1.76
88 3.0 6.89 1.90
89 5.8 5.87 1.85
(hazy, pH
7.8)
Preparation 90
Preparation of 85o N8-octanoyl-LysB29 Human Insulin and
Human Insulin cocrystallized with protamine and Zn
A dry lyophilized powder of NE-octanoyl-LysB29 Human
Insulin (1678.5 mg) was dissolved in 42.5 mL of 0.1 N HC1.
A dry powder of human insulin-Zn crystals (286 mg) was
dissolved in 7.5 mL of 0.1 N HC1. 7.5 mL of the later
solution was mixed with NE-octanoyl-LysB29 Human Insulin
solution to produce a mixture of Ns-octanoyl-LysB29 Human
2o Insulin and human insulin in approximate weight ratio of
75:25. This solution was stirred to mix. To this solution
was added 1.38 grams of a 10 mg/mL solution of Zn. To this
solution was added 200 mL of an aqueous solution composed of
9.5 mg/mL disodium phosphate heptahydrate, 0.375 M NaCl, 4
mg/mL m-cresol, 1.65 mg/mL phenol, and 16 mg/mL glycerol at
pH 7.63. The pH was adjusted to 7.6 with small quantities
of 5N HCl and 5N NaOH. This solution was then filtered
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through a 0.2 micron low protein binding filter. A second
solution was prepared by dissolving 262.9 mg of a dry powder
of protamine sulfate in 305.69 grams of water and then
filtered through a 0.2 micron low protein binding filter.
262.84 grams of solution of a mixture of N~-octanoyl-LysB29
human insulin and human insulin was mixed with 259.6 grams
of protamine sulfate solution. A white precipitate formed.
This suspension was stirred gently to complete mixing. The
preparation was allowed to stand undisturbed for 24 hours at
32°C. Inspection under an optical microscope revealed the
presence of microcrystalline solid. Measurement of particle
size distribution by Coulter technique revealed a mean
particle diameter of 2.5 microns. The mother liquor of this
preparation was exchanged with an aqueous solution composed
of 0.04 mg/mL phenol, 0.11 mg/mL m-cresol and 0.3 mg/mL of
Disodium phosphate heptahydrate and 24 mg/mL of glycerin by
the following procedure. 450 mL of supernatant was removed
by aspiration from a well settled suspension without
disturbing the solid phase and replaced with the aqueous
2o solution described above. Analytical characterization by
HPLC revealed a composition of 85.20 C8-BHI and 14.80 BHI.
For dog intrabronchial instillation experiments, these
formulations were diluted to the required concentration by
appropriate dilution with the aqueous solution used for
exchange described above. The microcrystals in this
preparation had a mean particle diameter of 2.5 microns by
Coulter multisizing. This preparation was used for
intrabronchial instillation in dogs.
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Preparation 91
Isolation of 85o cocrystal formulation (from preparation 90
above) in powder form (Approximately 1 gram scale)
Approximately 400 mL of the crystalline suspension from
Preparation 90 was filtered through a 0.22 micron filtration
apparatus by application of vacuum. The solid was washed
with approximately 5 mL of absolute ethanol and air-dried by
the application vacuum to the filtration apparatus. The
to agglomerated powder was collected using a clean spatula.
Preparation 92
In vitro dissolution properties of 85o cocrystal formulation
(from preparation 90 and preparation 91 above)
The in vitro dissolution rate of the microcrystalline
suspension from preparation 90 was measured in phosphate
buffered saline (PBS) at pH 7.4 and a temperature of 25
degrees centigrade. The PBS buffer contained 1 mg/mL of
2o bovine serum albumin to minimize adsorption loss of
insulins. A volume of suspension that contains 1.8 mg of
total insulin is suspended in 200 mL of buffer solution and
stirred at a constant rate of 180 rpm. At regular interval
aliquots of the solution was filtered through a 0.2 micron
low protein binding filter and assayed to determine total
dissolved insulins. Unfiltered control samples were also
analyzed to determine total available insulins. Based on
this assay, preparation 90 required approximately six hours
for complete dissolution. In comparison, NPH crystals
dissolved in about 2 minutes. Furthermore, as preparation
90 dissolved, it released a constant ratio of C8-BHI to BHI,
confirming the co-crystalline nature of preparation 90. The
isolated powder (preparation 91) retained the slow
dissolution properties of preparation 90 suggesting that the
process of isolating the powder and drying did not affect
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the dissolution properties. For the powder, approximately
50% dissolution occurred in about three hours. During this
period, as it dissolved, the powder released a constant
ratio of about 85% C8-BHI and 15o BHI. This property of
homogeneous dissolution further confirms that the process of
filtration and air-drying does not alter the fundamental
nature of the microcrystals.
These in-vitro dissolution data suggest that these
microcrystals with mean particle diameter of about 2.5
1o microns dissolve very slowly compared to NPH, despite their
increased surface area. These data support the potential of
Preparation 90 to serve as a sustained release formulation.
These data further support that the process o- isolation and
drying do not alter the unique dissolution characteristics
of the suspension.
Example 1
Two types of crystalline insulin were studied,
1000 C8-BHI crystals and 75o C8-BHI:25% BHI co-crystals.
2o For both of these, intratracheally instilled compounds
produced blood levels of immunoreactive insulin that were
higher for a longer sustained period than seen in previous
studies conducted in rats with regular insulin and
comparable to results with subcutaneously delivered NPH-
human insulin. Interestingly, when lung lavage fluid was
examined at 4 and 8 hours after lung instillation, insulin
crystals were observed primarily free and intact in the lung
lavage fluid with only relatively few crystals seen within
alveolar macrophages. The expectation was that most of the
3o crystalline material would have been taken up by the
alveolar macrophages and digested since the literature shows
that particle ingestion by macrophages in the lung is
largely complete within a few hours. This surprising
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finding suggests that if crystalline insulin is insoluble
enough not to dissolve too quickly in lung fluids it will
remain available for slow dissolution and absorption into
the blood. Therefore, lung delivery of these two
crystalline insulins may be a non-invasive approach for
sustained release of insulin to provide basal control of
glucose.
Glucose levels were measured in these rat
experiments and they were depressed commensurate with the
1o absorbed levels of insulin into blood. These experiments
show that lung delivery is a feasible method for delivering
insoluble insulin crystals to the lung for sustained release
of insulin into the blood. Aerosol inhalation will be the
means used in clinical use to delivery the crystalline
insulin to the lungs of patients to obviate the need for
injections and improve patient compliance.
Example 2
Twelve male F344 rats/group were used in this
2o study. The dose groups were as follows:
Group 01 Intratracheal Instillation of 1 mg/kg of
100% C8-BHI, prepared according to
Preparation 28, above.
Group 02 Intratracheal Instillation of 1 mg/kg of
75% C8-BHI:25% BHI, prepared according
to Preparation 29, above.
Group 03 Subcutaneous Administration of 1 mg/kg
of 75o C8-BHI:25% BHI, prepared
according to Preparation 29, above.
3o Group 04 Subcutaneous Administration of 1 mg/kg
of NPH insulin
Blood samples were collected at 0 (pre), 0.5, 1,
4, 8, 16, and 24 hours after dosing. These blood samples
were centrifuged and the serum was collected to determine
blood levels of the test article and glucose concentrations.
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The results of the glucose determinations are
presented in Figure 1. These preparations instilled into
rat trachea provided lowering of blood glucose, sustained
release comparable with subcutaneous delivery of NPH-human
insulin, and surprisingly high activity.
These data create a reasonable expectation that
these crystals and co-crystals can be administered by
inhalation, that particles having properties known to make
them likely to deposit in the lungs of a patient would be
to deposited in the lung of a human patient in need of insulin
to control blood glucose, and that these crystals or co-
crystals would dissolve within the lung, and the insulin
activity would be absorbed into the patient's blood from the
deposited particles.
Example 3
Physical Stability and Resuspendability Testing
This study evaluated physical stability under
physical stress conditions for four different 75o C8-BHI co-
2o crystal formulations in 3.0-mL Cartridges. Preparations 51-
54 were filled into 3.0 mL cartridges and placed on an
accelerated physical stability test. Forty-four cartridges
were filled for each Preparation.
Thirty-eight cartridges of each Preparation were
placed in an insulin agitator system at a constant
temperature of 37°C for 14 days. During this period the
materials were agitated by rotation at 30 rpm, 4 hours per
day. The cartridges were inspected for fibrillation
(aggregation or agglomeration) and physical changes were
3o noted on days 0, 2, 5, 7, 9, 11, and 14. A set of control
cartridges (6 for each Preparation) were stored at 5°C
(vertical cap side up) without agitation (except for
resuspension) and were inspected every 7 days.
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Cartridges were failed for the appearance of any
of the following conditions, as determined by visual
examination by trained personnel: conversion prom a milky
white, uniform suspension with slow sedimentation to one
exhibiting discrete particles having more rapid
sedimentation; film on cartridge walls; frost; clumping
(large aggregates); loss of resuspension; or any combination
of the above reasons.
After 14 days of accelerated physical stability
l0 testing, Preparations 51 and 54 had roughly ttie same number
of failed cartridges. Preparation 53 exhibited the least
number, and Preparation 52 had only slightly more failures
than Preparation 53, but less than either Preparations 52 or
55.
Example 4
Glucose Response to Powders of 85o C8 Insulin:l5oBHI Co-
Crvstals Insufflated into the Lungs of F344 Rats.
2o To determine the effects of airborne powders delivered
to the lung, an experiment was carried out in which 85o C8
insulin powders (preparations 90-92) were blown into the
lungs of rats using an insufflation method. A dose of 2
mg/kg was used.
Fasted male Fischer 344 rats (200-250 gm) were briefly
anesthetized with isoflurane and intratracheally intubated.
Pre-weighed aliquots of powder were prepared with inhalation
grade lactose employing geometric dilution to assure uniform
mixing of the insulin crystals at a concentration of 5o by
3o weight with the carrier lactose. 10 mg of powder was filled
into size 00 capsules that were then loaded into a Penn
Century insufflation device. The Penn Century device was
introduced into the intratracheal cannula and the powder
blown into the lungs by rapidly expelling 3 ml of air from a
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hand-operated syringe. Dosing was synchronized with the
animal's inspiration. Blood samples were taken at 0 (pre-
dose), and 0.25, 0.5, 1, 3, 4, 6, 8, and 12 hours after
dosing for glucose measurements.
The figure below shows the glucose response (mean ~ SE)
following insufflation of powders of 85o CBinsulin:l5o BHI
crystals into rat lungs. The data point at 8 hrs is
considered as an outlier because for some unknown reason
there were two rats in this group that had glucose levels
1o that were more than 500 of their baseline levels. Ignoring
this data point, the glucose responses show an extended
time-action profile quite similar to the data obtained
following instillation of 75o C8 Insulin:25% BHI crystal
suspensions obtained in the previous experiment above. These
data show both the airborne powders introduced into the lung
and the liquid suspensions produce similar responses.
Previous rat experiments had shown qualitatively similar
glucose response following instillation of the various types
of crystalline insulins. These results, in aggregate,
2o suggest that the results from instillation studies should be
good predictors of results from airborne powders introduced
into the lung. As shown in this experiment one crystalline
insulin showed extended time action of glucose response
following introduction airborne powder into the lung and
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similar behavior is expected for the other crystalline
insulins when introduced into the lung as airborne powders.
Changes in Glucose Following Intratracheal Insufflation of
85~'c C8-insulin Co-crystals and Intratracheal Instillation
of 75 ~/c C8-insulin Co-crystals in F344 Rats
14(
12(
10(
L
8(
J
4(
21
Example 5
Comparison of 3 Crystalline Formulations of Insulin
Delivered Intrabronchially to Beagle Dogs
The present study was designed to compare the kinetics
following subcutaneous (sc) administration of NPH crystals
to the kinetics following intrabronchial (IB) administration
of NPH crystals, MicroUltralente crystals, and 85o C8-
insulin co-crystals.
The live phase of the study was performed at Lilly's
i5 Toxicology Research Laboratories. Two male and three female
adult beagle dogs were used in this study. Weights ranged
from 7.1 to 14.6 kg at the start of the study.
The test articles used were 85o C8-insulin co-crystals,
MicroUltralente crystals, and NPH crystals. Each test
2o article was specifically formulated for pulmonary
administration using modified crystallization conditions to
Time (hoursl
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produce 2-3 micrometer particles. Specific vehicles were
used for each crystalline insulin formulation as shown in
Table 1.
Table 1. Crystalline insulin formulations and their
associated vehicles.
Compound Vehicle
85o C8- Aqueous solution containing 0.7
insulin mg/ml phenol,
1.6 mg/ml metacresol, 0.3 mg/ml
phosphate
MicroUltra- Water
lente
NPH Water, glycerin, and trace amounts
of phenol and metacresol
Anesthetized, fasted animals were dosed with 85o C8-
insulin co-crystals, MicroUltralente crystals, and NPH
to crystals via pulmonary or subcutaneous routes of
administration. Pulmonary doses, as shown in Table 2, were
delivered intrabronchially via a fiberoptic bronchoscope
with instillation volumes of 0.25 ml/kg. For comparative
purposes, each dog was dosed with 0.75 U/kg of NPH crystals
subcutaneously. At least 7 days elapsed between each
treatment. Blood samples were collected pre-treatment, and
at 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, and 24 hours
after administration. For the subcutaneous and pulmonary
doses of NPH, blood samples were collected at pre-treatment,
2o and at 0.5, 1, 2, 4, 6, 8, 10, 12, 16, and 24 hours after
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dosing. Serum obtained from these samples was analyzed for
glucose concentrations.
Table 2. Delivered Doses
Compound Dose Route
(Units/kg)
NPH crystals 0.75 Subcutane
ous
NPH crystals 1.5 Intrabron
chial
85o C8-insulin 1.5 Intrabron
co-crystals chial
MicroUltralente 0.75 Intrabron
chial
Results and Discussion
Intrabronchial instillation of all crystalline insulin
formulations produced a significant suppression of serum
glucose levels (Table 3, Figure 1). The average blend
1o glucose level reached an initial low point at 2 hours for
all formulations.
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Table 3.
Blood glucose (percent of baseline) after intrabronchial or
subcutaneous administration of crystalline
insulin
formulations in
Beagle
dogs.
es o
CB-insulin Micro- sc IH
Timeco-crystalsStd UltralenteStd NPH Std NPH Std
(hrs)(n=5) Error(n=3) Error (n=5) Error (n=5) Error
0 100 0 100 0 100 0 100 0
0.5 89 2 96 8 91 5 80 10
1 50 7 72 9 56 5 45 12
2 40 4 32 6 40 4 37 5
3 41 8 39 4
4 50 7 44 6 47 5 46 10
34 6 31 5
6 46 13 44 5 51 7 35 5
7 46 14 38 3
8 45 14 36 6 50 8 33 5
9 46 16 40 4
63 17 49 4 77 7 56 7
12 71 21 80 20 93 3 95 17
16 89 13 93 4 102 7 111 6
24 102 7 98 0 92 4 102 4
5
Glucose levels remained markedly depressed up to
approximately 10 hours following intrabronchial or
subcutaneous administration for all formulations. The time-
action for sustained release was similar for intrabronchial
1o instillation of all formulations and was at least as long as
subcutaneous administration of NPH in all cases. Two dogs
following intrabronchial administration of NPH and 85o C8-
insulin co-crystals experienced critically low glucose
levels between 7 and 10 hours post-dose and were rescued
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using 50o dextrose administered either orally or
intravenously. These episodes artificially increased serum
glucose levels in the rescued animals and therefore
shortened potential extended release action and the overall
mean of the group.
Changes in Serum Glucose Levels (mean +/- SE) Following Subcutaneous
Administration of NPH or Intrabronchial Administration of NPH,
MicroUltralente, and 85 % C8-insulin Co-crystals
140
120
100
0
L
w
L'
O
U 80
0
a
L 6O
47
pr
-~ 1.5 U/kg 8~~Io C8-insulin (IB)
40 ~ 0.75 U/kg Microllltralente (IB)
f ~-- 0.75 U/k~ NPH (scv
-p- 1.5 U/kg NPH (1B>
0 5 10 15 20 25 30
Time (hours)
to Example 6
Aerosol Characterization for 3 Crystalline
Formulations of Insulin
Three dry powder formulations of crystalline insulin
15 were aerosolized using a Wright Dust Feed (WDF) generator
operated at 10 LPM. The aerosol generated by the WDF either
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passed through a cyclone designed to remove large particles
before entering a 12-L head-dome exposure system or passed
directly from the WDF into a head-dome The mass median
aerodynamic diameter (MMAD) was determined by using a Sierra
Model 218K Cascade Impactor fitted with Gelman Type A/E
glass fiber filters. Airflow through the Cascade Impactor
was 3 L/minute with sample times ranging from 5 to 50
minutes. Aerosol concentration was determined by collecting
a gravimetric sample during the exposure.
rlo~"'I r
Particle size and gravimetric data for the 3 crystalline
insulins are shown in Table 1.
Table 1. Aerosol Characterization
Mean ~( SE)
Aerosol
Test Articles MMEAD GSD Concentration
(mg/L)
85% C8-insulin
w/ cyclone 1.15 3.98 NA
w/o cyclone 1.30 4.03 0.069
10% MicroUltralente/90%
Lactose
w/ cyclone 2.74 1.61 0.280
w/o cyclone 4.07 1.97 0.104
Spray-dried
MicroUltralente
w/ cyclone 1.79 2.90 0.088
w/o cyclone 2.31 3.04 0.040
These data show that highly respirable particle size
distribution result upon aerosol generation of these
crystalline insulins.
The data from Examples 1-5 are convincing that
instillation studies are predictive for effects of inhaled
dry powders. Studies have been conducted in beagle dogs to
compare glucose responses following a) inhalation of
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solution aerosols of regular insulin, b) inhalation of
powders of regular insulin, and c) instillation of a
solution of regular insulin. The accompanying figure shows
that the time-action profile of the glucose response is
rapid in all cases and the results are similar for all 3
types of dose administration. The time to minimum glucose
levels is less than 2 hours in all cases, and responses are
returning to near baseline within 4-5 hours. This profile
is also similar to that achieved in humans following
1o inhalation of regular insulin either as liquid aerosols
(Laube BL, Georgopoulus, Adams GK. 1992. Aerosolized
insulin delivered through the lungs is effective in
normalizing plasma glucose levels in non-insulin dependent
diabetes. J. Biopharm. Sci 3 (1992) 163-169) or powders
(Patton J, Bukar J., and Najarajan S. 1999. Inhaled
insulin. Adv. Drug Delivery Rev. 35: 235-247). These
results lend confidence to predicting similar results in
inhalation studies to those achieved with instillation
studies. The similar glucose response in rats following
2o admistration of intratracheally instilled suspensions of C8
co-crystals compared to insufflation of powders of the same
C8 co-crystals also directly supports this view.
The invention has been described with reference to
various specific and preferred embodiments and techniques.
However, it should be understood that many variations and
modifications may be made while remaining within the spirit
and scope of the invention. All publications and patent
applications in this specification are indicative of the
level of ordinary skill in the art to which this invention
pertains.
