U.S. patent application number 09/209799 was filed with the patent office on 2001-08-16 for glucagon-like peptide-1 crystals.
Invention is credited to HERMELING, RONALD NORBERT, HOFFMANN, JAMES ARTHUR, NARASIMHAN, CHAKAVARTHY.
Application Number | 20010014666 09/209799 |
Document ID | / |
Family ID | 22090845 |
Filed Date | 2001-08-16 |
United States Patent
Application |
20010014666 |
Kind Code |
A1 |
HERMELING, RONALD NORBERT ;
et al. |
August 16, 2001 |
GLUCAGON-LIKE PEPTIDE-1 CRYSTALS
Abstract
The invention provides individual tetragonal flat rod shaped or
plate-like crystals of glucagon-like peptide-1 related molecules,
processes for their preparation, compositions and methods of use.
The crystal preparations exhibit extended time action in vivo and
are useful for treating diabetes, obesity and related
conditions.
Inventors: |
HERMELING, RONALD NORBERT;
(INDIANAPOLIS, IN) ; HOFFMANN, JAMES ARTHUR;
(GREENWOOD, IN) ; NARASIMHAN, CHAKAVARTHY;
(CARMEL, IN) |
Correspondence
Address: |
ROBERT A CONRAD
ELI LILLY AND COMPANY
PATENT DIVISION/RSM
LILLY CORPORATE CENTER
INDIANAPOLIS
IN
46285
|
Family ID: |
22090845 |
Appl. No.: |
09/209799 |
Filed: |
December 11, 1998 |
Current U.S.
Class: |
514/5.3 ;
435/212; 514/11.7; 514/6.9; 530/308; 530/412 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 3/10 20180101; C07K 14/605 20130101; A61P 3/04 20180101 |
Class at
Publication: |
514/12 ; 530/308;
530/412; 435/212 |
International
Class: |
A61K 038/00; C07K
017/00; C07K 016/00; C07K 014/00; C07K 001/00; A23J 001/00 |
Claims
We claim:
1. A process for preparing single rod-shaped or plate-like crystals
of a glucagon-like peptide-1 related molecule (GLP) which comprises
preparing a crystallization solution comprising a GLP, a buffering
agent, an alcohol or a mono or disaccharide, and optionally,
ammonium sulfate or zinc.
2. The process of claim 1 wherein the GLP is at a final
concentration of between about 1-10 mg/ml and is selected from the
group consisting of a GLP-1 analog, a GLP-1 derivative, a
dipeptidyl-peptidase-IV (DPP-IV) protected GLP, a GLP-1 peptide
analog, or a biosynthetic GLP-1 analog, and wherein the buffering
agent is about 10 to 50 mM, and about pH 6-7 and is selected from
the group consisting of Tris, ammonium acetate, sodium acetate, or
Bis-Tris, and wherein the alcohol or mono or disaccharide is
selected from the group consisting of methanol, ethanol, propanol,
glycerol, trehalose, mannitol, glucose, erythrose, ribose,
galactose, fructose, maltose, sucrose, and lactose, and,
optionally, wherein about 1% ammonium sulfate is present.
3. The process of claim 1 wherein total zinc is in a 0.5-1.7 molar
ratio to the GLP which is at a final concentration of between about
1-20 mg/ml and is selected from the group consisting of a GLP-1
analog, a GLP-1 derivative, a DPP-IV protected GLP, a GLP-1 peptide
analog, or a biosynthetic GLP-1 analog, and wherein the buffering
agent is about 10 to 100 mM, and about pH 7-10 and is selected from
the group consisting of glycine, aspartic acid or Tris, and wherein
the alcohol or mono or disaccharide is selected from the group
consisting of methanol, ethanol, propanol, glycerol, trehalose,
mannitol, glucose, erythrose, ribose, galactose, fructose, maltose,
sucrose, and lactose.
4. The process of claim 1 wherein the GLP is selected from the
group consisting of a DPP-IV protected GLP, or a biosynthetic
GLP.
5. The process of claim 1 wherein the GLP is a DPP-IV protected GLP
selected from the group consisting of Val-8-GLP-1(7-37)OH,
Thr-8-GLP-1(7-37)OH, Gly-8-GLP-1(7-37)OH, or
Met-8-GLP-1(7-37)OH.
6. The process of claim 1 having the additional step of soaking the
GLP crystals in a zinc containing solution.
7. GLP crystals having tetragonal flat rod shaped or plate-like
morphology selected from the group consisting of a GLP-1 analog, a
GLP-1 derivative, a DPP-IV protected GLP, a GLP-1 peptide analog,
or a biosynthetic GLP-1 analog.
8. The crystals of claim 7 wherein the GLP is selected from the
group consisting of DPP-IV protected GLP, or a biosynthetic
GLP.
9. The crystals of claim 7 wherein the GLP is selected from the
group consisting of Val-8-GLP-1(7-37)OH, Thr-8-GLP-1(7-37)OH,
Gly-8-GLP-1(7-37)OH, or Met-8-GLP-1(7-37)OH.
10. GLP crystals whenever prepared by the process of claim 1.
11. A substantially homogenous composition of GLP crystals.
12. The composition of claim 11 wherein the GLP crystals are
selected from the group consisting of a GLP-1 analog, a GLP-1
derivative, a DPP-IV protected GLP, a GLP-1 peptide analog, or a
biosynthetic GLP-1 analog.
13. The composition of claim 11 wherein the GLP is selected from
the group consisting of DPP-IV protected GLP, or a biosynthetic
GLP.
14. The composition of claim 11 wherein the GLP is selected from
the group consisting of Val-8-GLP-1(7-37)OH, Thr-8-GLP-1(7-37)OH,
Gly-8-GLP-1(7-37)OH, or Met-8-GLP-1(7-37)OH.
15. A pharmaceutical formulation comprising a GLP crystal as
claimed in claim 7 together with one or more pharmaceutically
acceptable diluents, carriers or excipients therefor.
16. A pharmaceutical formulation comprising a GLP crystal as
claimed in claim 8 together with one or more pharmaceutically
acceptable diluents, carriers or excipients therefor.
17. A pharmaceutical formulation comprising a GLP crystal as
claimed in claim 9 together with one or more pharmaceutically
acceptable diluents, carriers or excipients therefor.
18. A pharmaceutical formulation comprising a GLP crystal as
claimed in claim 10 together with one or more pharmaceutically
acceptable diluents, carriers or excipients therefor.
19. The pharmaceutical formulation of claim 15 wherein the
formulation is prepared by additions to and/or modifications of the
post-crystallization mother liquor without separating the GLP
crystals from the mother liquor.
20. The pharmaceutical formulation of claim 16 wherein the
formulation is prepared by additions to and/or modifications of the
post-crystallization mother liquor without separating the GLP
crystals from the mother liquor.
21. The pharmaceutical formulation of claim 17 wherein the
formulation is prepared by additions to and/or modifications of the
post-crystallization mother liquor without separating the GLP
crystals from the mother liquor.
22. The pharmaceutical formulation of claim 18 wherein the
formulation is prepared by additions to and/or modifications of the
post-crystallization mother liquor without separating the GLP
crystals from the mother liquor.
23. A method of treating diabetes, obesity or related conditions in
a mammal in need thereof, which comprises administering to said
mammal a GLP crystal of claim 7.
24. A method of treating diabetes, obesity or related conditions in
a mammal in need thereof, which comprises administering to said
mammal a composition of claim 11.
25. A method of treating diabetes, obesity or related conditions in
a mammal in need thereof, which comprises administering to said
mammal a pharmaceutical formulation of claim 15.
Description
FIELD OF INVENTION
[0001] The present invention relates to peptide chemistry as it
applies to pharmaceutical research and development. The invention
provides individual tetragonal flat rod shaped or plate-like
crystals of glucagon-like peptide-1 related molecules, processes
for their preparation, compositions and uses for these improved
crystal forms.
BACKGROUND OF THE INVENTION
[0002] GLP-1, a 37 amino acid peptide naturally formed by
proteolysis of the 160 amino acid precursor protein preproglucagon,
was first identified in 1987 as an incretin hormone. GLP-1 is
secreted by the L-cells of the intestine in response to food
ingestion and has been found to stimulate insulin secretion
(insulinotropic action) causing glucose uptake by cells which
decreases serum glucose levels (see, e.g., Mojsov, S., Int. J.
Peptide Protein Research, 40:333-343 (1992)). GLP-1 is poorly
active. A subsequent endogenous cleavage between the 6.sup.th and
7.sup.th position produces a more potent biologically active
GLP-1(7-37)OH peptide. Approximately 80% of the GLP-1(7-37)OH so
produced is amidated at the C-terminal in conjunction with removal
of the terminal glycine residue in the L-cells and is commonly
referred to GLP-1(7-36)NH.sub.2. Molecules which are reasonably
homologous to, or are derived from, or based on these native forms
will generally be referred to as GLP's in this specification.
[0003] The biological effects and metabolic turnover of the free
acid, the amide form, and many of the numerous known GLP's are
similar and show promise as agents for the treatment of diabetes,
obesity, and related conditions, including but not limited to
impaired glucose tolerance and insulin resistance. However, many
GLP's suffer from extremely short biological half lives, some as
short as 3-5 minutes, which makes them unattractive for use as
pharmaceutical agents. Presently, the activity of
dipeptidyl-peptidase-IV (DPP-IV) is believed to readily inactivate
many GLP's and is in part responsible for the very short serum half
lives observed. Rapid absorption and clearance following parenteral
administration are also factors. Thus, there is a need to find a
means for prolonging the action of these promising agents.
[0004] One such approach has been to modify these molecules to
protect them from in vivo cleavage by DPP-IV. For example, see U.S.
Pat. No. 5,512,549. In the insulin arts, it has long been known
that extended time action can be achieved by administering
crystalline protein formulations into the subcutis which act like
depots, paying out soluble protein over time.
[0005] Heterogeneous micro crystalline clusters of GLP-1(7-37)OH
have been grown from saline solutions and examined after crystal
soaking treatment with zinc and/or m-cresol (Kim and Haren, Pharma.
Res. Vol. 12 No. 11 (1995)). Also, crude crystalline suspensions of
GLP(7-36)NH.sub.2 containing needle-like crystals and amorphous
precipitation have been prepared from phosphate solutions
containing zinc or protamine (Pridal, et. al., International
Journal of Pharmaceutics Vol. 136, pp. 53-59 (1996)). Also, EP 0
619 322 A2 describes the preparation of micro-crystalline forms of
GLP-1(7-37)OH by mixing solutions of the protein in pH 7-8.5 buffer
with certain combinations of salts and low molecular weight
polyethylene glycols (PEG). However, such crystalline clusters and
crude suspensions are less than ideal for preparing long acting
pharmaceutical formulations of GLP's since they are loosely bound
heterogeneous clusters of crystals or amorphous-crystalline
suspensions which tend to trap impurities and are otherwise
difficult to reproducibly manufacture and administer.
[0006] Most unexpectedly it was discovered that single tetragonal
flat rod shaped or plate-like crystals of various GLP's could be
reproducibly formed from a mother liquor containing a GLP dissolved
in a buffered solution and a C.sub.1-3 alcohol, or optionally a
mono or disaccharide, over a wide range of pH conditions. The
resulting single flat rod shaped or plate-like crystals are
superior to, and offer significant advantages over, the
GLP-1(7-37)OH crystal clusters or crude suspensions known in the
art.
[0007] The single tetragonal flat rod shaped or plate-like crystals
of the present invention are less prone to trap impurities and
therefore may be produced in greater yields and administered more
reproducibly than the known heterogeneous clusters. The crystal
compositions of the present invention are pharmaceutically
attractive because they are relatively uniform and remain in
suspension for a longer period of time than the crystalline
clusters or amorphous crystalline suspensions which tend to settle
rapidly, aggregate or clump together, clog syringe needles and
generally exacerbate unpredictable dosing. Most importantly, the
crystal compositions of the present invention display extended,
uniform, and reproducible pharmacokinetics which can be modulated
by adding zinc using conventional crystal soaking techniques or,
alternatively, by including zinc in the crystallization
solution.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention includes processes for preparing
single rod-shaped or plate-like crystals of glucagon-like peptide-1
related molecules (GLP's) which comprises preparing a
crystallization solution comprising a purified GLP, a buffering
agent containing an alcohol or a mono or di saccharide, and
optionally, ammonium sulfate or zinc. In another embodiment the GLP
crystals having tetragonal flat rod shaped or plate-like morphology
selected from the group consisting of a GLP-1 analog, a GLP-1
derivative, a DPP-IV protected GLP, a GLP-1 peptide analog, or a
biosynthetic GLP-1 analog are claimed. The invention also includes
substantially homogenous compositions of GLP crystals,
pharmaceutical formulations and processes for preparing such
formulations, and methods for treating diabetes, obesity and
related conditions.
DETAILED DESCRIPTION OF THE INVENTION
[0009] By custom in the art, the amino terminus of GLP-1(7-37)OH
has been assigned number residue 7 and the carboxy-terminus, number
37. This nomenclature carries over to other GLP's. When not
specified, the C-terminal is usually considered to be in the
traditional carboxyl form. The amino acid sequence and preparation
of GLP-1(7-37)OH is well-known in the art. See U.S. Pat. No.
5,120,712, the teachings of which are herein incorporated by
reference. For the convenience of the reader the sequence is
provided below.
[0010]
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-
-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-COOH (SEQ
ID NO:1)
[0011] "-GLP-1 analog" is defined as a molecule having one or more
amino acid substitutions, deletions, inversions, or additions
relative to GLP-1(7-37) and may include the d-amino acid forms.
Numerous GLP-1 analogs are known in the art and include, but are
not limited to, GLP-1(7-34), GLP-1(7-35), GLP-1(7-36)NH.sub.2,
Gln.sup.9-GLP-1(7-37), d-Gln.sup.9-GLP-1(7-37),
Thr.sup.16-Lys.sup.18-GLP-1(7-37), and Lys.sup.18-GLP-1(7-37),
Gly.sup.8-GLP-1(7-36)NH.sub.2, Gly.sup.8-GLP-1(7-37)OH,
Val.sup.8-GLP-1(7-37)OH, Met.sup.8-GLP-1(7-37)OH- ,
acetyl-Lys.sup.9-GLP-1(7-37), Thr.sup.9-GLP-1(7-37),
D-Thr.sup.9-GLP-1(7-37), Asn.sup.9-GLP-1(7-37) ,
D-Asn.sup.9-GLP-1(7-37),
Ser.sup.22-Arg.sup.23-Arg.sup.24-Gln.sup.26-GLP-1(7-37),
Arg.sup.23-GLP-1(7-37), Arg.sup.24-GLP-1(7-37),
.alpha.-methyl-Ala.sup.8-- GLP-1(7-36)NH.sub.2, and
Gly.sup.8-Gln.sup.21-GLP-1(7-37)OH, and the like.
[0012] Other GLP-1 analogs consistent with the present invention
are described by the formula:
[0013]
R.sub.1-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Y-Gly-Gln-
-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R.sub.2 (SEQ ID
NO:2)
[0014] wherein: R.sub.1 is selected from the group consisting of
L-histidine, D-histidine, desamino-histidine, 2-amino-histidine,
beta-hydroxy-histidine, homohistidine,
alpha-fluoromethyl-histidine, and alpha-methyl-histidine; X is
selected from the group consisting of Ala, Gly, Val, Thr, Met, Ile,
and alpha-methyl-Ala; Y is selected from the group consisting of
Glu, Gln, Ala, Thr, Ser, and Gly; Z is selected from the group
consisting of Glu, Gln, Ala, Thr, Ser, and Gly; and R.sub.2 is
selected from the group consisting of NH.sub.2, and Gly-OH.
[0015] GLP-1 analogs have also been described in WO 91/11457, and
include GLP-1(7-34), GLP-1(7-35), GLP-1(7-36), or GLP-1(7-37), or
the amide form thereof, and pharmaceutically-acceptable salts
thereof, having at least one modification selected from the group
consisting of:
[0016] (a) substitution of glycine, serine, cysteine, threonine,
asparagine, glutamine, tyrosine, alanine, valine, isoleucine,
leucine, methionine, phenylalanine, arginine, or D-lysine for
lysine at position 26 and/or position 34; or substitution of
glycine, serine, cysteine, threonine, asparagine, glutamine,
tyrosine, alanine, valine, isoleucine, leucine, methionine,
phenylalanine, lysine, or a D-arginine for arginine at position
36;
[0017] (b) substitution of an oxidation-resistant amino acid for
tryptophan at position 31;
[0018] (c) substitution of at least one of: tyrosine for valine at
position 16; lysine for serine at position 18; aspartic acid for
glutamic acid at position 21; serine for glycine at position 22;
arginine for glutamine at position 23; arginine for alanine at
position 24; and glutamine for lysine at position 26; and
[0019] (d) substitution of at least one of: glycine, serine, or
cysteine for alanine at position 8; aspartic acid, glycine, serine,
cysteine, threonine, asparagine, glutamine, tyrosine, alanine,
valine, isoleucine, leucine, methionine, or phenylalanine for
glutamic acid at position 9; serine, cysteine, threonine,
asparagine, glutamine, tyrosine, alanine, valine, isoleucine,
leucine, methionine, or phenylalanine for glycine at position 10;
and glutamic acid for aspartic acid at position 15; and
[0020] (e) substitution of glycine, serine, cysteine, threonine,
asparagine, glutamine, tyrosine, alanine, valine, isoleucine,
leucine, methionine, or phenylalanine, or the D- or N-acylated or
alkylated form of histidine for histidine at position 7; wherein,
in the substitutions in (a), (b), (d), and (e), the substituted
amino acids can optionally be in the D-form and the amino acids
substituted at position 7 can optionally be in the N-acylated or
N-alkylated form.
[0021] A "GLP-1 derivative" is defined as a molecule having the
amino acid sequence of GLP-1(7-37) or of a GLP-1 analog, but
additionally having chemical modification of one or more of its
amino acid side groups, .alpha.-carbon atoms, terminal amino group,
or terminal carboxylic acid group. A chemical modification
includes, but is not limited to, adding chemical moieties, creating
new bonds, and removing chemical moieties. Modifications at amino
acid side groups include, without limitation, acylation of lysine
.epsilon.-amino groups, N-alkylation of arginine, histidine, or
lysine, alkylation of glutamic or aspartic carboxylic acid groups,
and deamidation of glutamine or asparagine. Modifications of the
terminal amino include, without limitation, the des-amino, N-lower
alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of
the terminal carboxy group include, without limitation, the amide,
lower alkyl amide, dialkyl amide, and lower alkyl ester
modifications. Lower alkyl is C.sub.1-C.sub.4 alkyl. Furthermore,
one or more side groups, or terminal groups, may be protected by
protective groups known to the ordinarily-skilled protein chemist.
The .alpha.-carbon of an amino acid may be mono- or
dimethylated.
[0022] Other GLP-1 derivatives are claimed in U.S. Pat. No.
5,188,666, which is expressly incorporated by reference. Such
molecules are selected from the group consisting of a peptide
having the amino acid sequence:
[0023]
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-
-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-X (SEQ ID NO:3)
[0024] and pharmaceutically-acceptable salts thereof, wherein X is
selected from the group consisting of Lys-COOH and Lys-Gly-COOH;
and a derivative of said peptide, wherein said peptide is selected
from the group consisting of: a pharmaceutically-acceptable lower
alkyl ester of said peptide; and a pharmaceutically-acceptable
amide of said peptide selected from the group consisting of amide,
lower alkyl amide, and lower dialkyl amide.
[0025] Yet other GLP-1 derivatives consistent for use in the
present invention include compounds claimed in U.S. Pat. No.
5,512,549, which is expressly incorporated herein by reference,
described by the formula:
[0026]
R.sup.1-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-
-Gln-Ala-Ala-Xaa-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R.sup.3
(SEQ ID NO:4) R.sup.2
[0027] wherein R.sup.1 is selected from the group consisting of
4-imidazopropionyl, 4-imidazoacetyl, or 4-imidazo-.alpha., .alpha.
dimethyl-acetyl; R.sup.2 is selected from the group consisting of
C.sub.6-C.sub.10 unbranched acyl, or is absent; R.sup.3 is selected
from the group consisting of Gly-OH or NH.sub.2; and, Xaa is Lys or
Arg, may be used in present invention.
[0028] "DPP-IV protected GLP's" refers to GLP-1 analogs which are
resistant to the action of DPP-IV. These include analogs having a
modified or d amino acid residue in position 8. These also include
biosynthetic GLP-1 analogs having Gly or the 1 amino acid residues
Val, Thr, Met, Ser, Cys, or Asp in position 8. Other DPP-IV
protected GLP's include des amino His.sup.7 derivatives.
[0029] "GLP-1 peptide analogs" are defined as GLP-1 analogs or
derivatives which exclude acylated forms.
[0030] "Biosynthetic GLP-1 analogs" are defined as any GLP-1
analogs or derivatives which contain only naturally occurring amino
acid residues and are thus capable of being expressed by living
cells, including recombinant cells and organisms.
[0031] "Treating" is defined as the management and care of a
patient for the purpose of combating the disease, condition, or
disorder and includes the administration of a compound of present
invention to prevent the onset of the symptoms or complications,
alleviating the symptoms or complications, or eliminating the
disease, condition, or disorder. Treating diabetes therefore
includes the maintenance of physiologically desirable blood glucose
levels in patients in need thereof.
[0032] The flat rod shaped or plate-like GLP crystals of the
present invention, which are prepared using the claimed process,
vary in size and shape to some degree. Generally, they range in
size from approximately 2-25 microns (.mu.m) by 10-150 .mu.m and
are flat, having a depth of approximately 0.5-5 .mu.m. These single
crystals form from a single nucleation point and do not appear as
multiple spiked star-like clusters known in the art.
[0033] Given the sequence information herein disclosed and the
state of the art in solid phase protein synthesis, GLP's can be
obtained via chemical synthesis. However, it also is possible to
obtain some GLP's by enzymatically fragmenting proglucagon using
techniques well known to the artisan. Moreover, well known
recombinant DNA techniques may be used to express GLP's consistent
with the invention.
[0034] The principles of solid phase chemical synthesis of
polypeptides are well known in the art and may be found in general
texts in the area such as Dugas, H. and Penney, C., Bioorganic
Chemistry (1981) Springer-Verlag, New York, pgs. 54-92, Merrifield,
J. M., Chem. Soc., 85:2149 (1962), and Stewart and Young, Solid
Phase Peptide Synthesis, pp. 24-66, Freeman (San Francisco,
1969).
[0035] Likewise, the state of the art in molecular biology provides
the ordinarily skilled artisan another means by which GLP's can be
obtained. Although GLP's may be produced by solid phase peptide
synthesis, recombinant methods, or by fragmenting glucagon,
recombinant methods are preferable when producing biosynthetic
GLP-1 analogs because higher yields are possible.
[0036] For purposes of the present invention, GLP-1 peptide analogs
and biosynthetic GLP-1 peptide analogs are preferred. More
preferred are the DPP-IV protected GLP's, More highly preferred are
biosynthetic GLP-1 peptide analogs. Another preferred group of
GLP-1 peptide analogs are those which contain a single amino acid
substitution at the 8 position which may include d and modified
amino acid residues. More highly preferred biosynthetic GLP-1
peptide analogs are those which contain a single amino acid
substitution at the 8 position, more preferably those which contain
Gly or the 1 amino acid residues Val, Thr or Met in the 8
position.
[0037] The present invention provides a process for producing
individual tetragonal rod shaped GLP crystals from a mother liquor.
Under low to neutral pH conditions ranging from about pH 6-7,
preferably about 6.4 .+-. about 0.2, the crystallization solution,
or mother liquor, contains a final GLP concentration of about 1-10
mg/ml, preferably 2-7 mg/ml.
[0038] A number of conventional buffer solutions containing an
alcohol or mono or disaccharide are suitable in the practice of the
invention. 10 to 50 mM Tris, ammonium acetate, sodium acetate, or
Bis-Tris is preferred. The concentration of alcohol ranges from
about 2-15% (v/v), preferably 3-13%. Preferred alcohols are
selected from the group containing methanol, ethanol, propanol, or
glycerol, ethanol being most preferred.
[0039] Optionally, the addition of approximately 1% (w/v) ammonium
sulfate to the mother liquor will generally increase the yield of
crystals. The skilled artisan will also recognize the benefits of
adding a preservative such as sodium azide and other such
preservatives to the mother liquor to prevent bacterial growth.
[0040] In another embodiment, mono or disaccharides may be
substituted for the alcohol in the same ratios on a weight to
volume basis. Mono or disaccharides suitable for use in the
presently claimed process include trehalose, mannitol, glucose,
erythrose, ribose, galactose, fructose, maltose, sucrose, and
lactose, though trehalose is preferred.
[0041] In yet another embodiment of the present invention, the
process may be carried out in a neutral or high pH, zinc-containing
environment ranging from about pH 7-10, preferably about pH
7.2-9.7. Under these conditions, the GLP concentration is in the
range of approximately 1-20 mg/ml, preferably about 2-10 mg/ml.
Total zinc, in a molar ratio to GLP, ranges from about 0.5-1.7,
preferably 0.6-1.5.
[0042] Under such neutral or high pH conditions with zinc, suitable
buffers and salts range in concentration from about 10-100 mM
glycine and 0-200 mM NaCl, preferably 40-60 mM glycine and 0-150 mM
NaCl. Preferred buffers are glycine, aspartic acid and Tris. The
alcohol or sugar conditions are as stated previously.
[0043] Once the mother liquor is prepared, it is allowed to stand
at approximately 15-37.degree. C., preferably about 18-25.degree.
C., for 12-48 hours until crystallization occurs. The crystals may
then be transferred or otherwise handled without any noticeable
deleterious effects to the crystalline morphology suggesting that
such crystals may be stored for prolonged periods without suffering
structural damage.
[0044] In another embodiment, a pharmaceutical formulation may be
prepared by adding pharmaceutically acceptable excipients,
carriers, preservatives, and diluents directly to the mother liquor
after the cystals have formed. In this embodiment, crystallization
and subsequent additions are performed under sterile conditions.
Zinc may be added directly to the mother liquor to effect the
incorporation of zinc into the crystals. Preservatives may be added
to the mother liquor to provide formulations of crystals suitable
for multiple injections from the same container. Other excipients,
such as antioxidants, buffers, acids and bases for pH adjustments,
isotonicity agents and the like, may also be added directly to the
mother liquor after the crystals have formed.
[0045] In another embodiment, the invention provides homogenous
compositions of individual tetragonal flat rod shaped or plate-like
crystal of GLP's. Prior to the processes herein disclosed and
claimed, such compositions could not be achieved. The compositions
of the invention are useful in manufacturing processes and for
preparing pharmaceutical formulations having extended time action
for the treatment or prevention of diabetes, obesity and related
conditions.
[0046] The claimed GLP crystals and compositions may optionally be
treated with zinc using conventional crystal soaking techniques. By
soaking the crystals in about a 0.5 mg/ml solution of zinc,
complexes of crystals are formed which serve to extend the time
action of the administered GLP. Also, by varying the zinc
concentration, the complex composition can be altered leading to
longer or shorter time actions.
[0047] As noted the invention provides pharmaceutical formulations,
which are comprised of single tetragonal flat rod shaped or
plate-like crystal of a GLP, together with one or more
pharmaceutically acceptable diluents, carriers, or excipients. The
crystals can be formulated for parenteral administration for the
therapeutic or prophylactic treatment of diabetes, obesity or
related conditions. For example, the crystals of the present
invention can be admixed with conventional pharmaceutical carriers
and excipients. The formulations comprising the claimed crystals
contain from about 0.5 to 50 mg/ml of the active GLP, and more
specifically from about 1.0 to 10 mg/ml. Furthermore, the crystals
of the present invention may be administered alone or in
combination with other antidiabetic agents. For subcutaneous or
intramuscular preparations, a sterile formulation of the crystals
of the present invention can be administered as a suspension in the
original or modified crystallization mother liquor or in a
pharmaceutical diluent such as pyrogen-free distilled water,
physiological saline, or 5% glucose solution. A suitable
formulation of the crystals of the present invention may be
prepared and administered as a suspension in an aqueous base or a
pharmaceutically acceptable oil base, e.g., an ester of a
long-chain fatty acid such as ethyl oleate.
[0048] Pharmaceutically acceptable preservatives such as an
alkylparaben, particularly methylparaben, ethylparaben,
propylparaben, or butylparaben or chlorobutanol, phenol or
meta-cresol are preferably added to the formulation to allow
multi-dose use.
[0049] The formulation may also contain an isotonicity agent, which
is an agent that is tolerated physiologically and imparts a
suitable tonicity to the formulation to prevent the net flow of
water across the cell membrane. Compounds, such as glycerin, are
commonly used for such purposes at known concentrations. Other
possible isotonicity agents include salts, e.g., NaCl, dextrose,
mannitol, and lactose. Glycerin is the preferred isotonicity agent.
The concentration of the isotonicity agent is in the range known in
the art for parenteral formulations, and for glycerin, is
preferably about 16 mg/mL to about 25 mg/mL.
[0050] The formulation may also contain a pharmaceutically
acceptable buffering agent to control the pH at a desired level.
The pH is ideally such as to be acceptable to the patient upon
administration, yet one at which the formulation is sufficiently
stable, both physically and chemically. Preferably, the pH is
controlled from a mildly acidic pH to a mildly basic pH, such as,
between about pH 5 and pH 9. More preferably, the pH is between
about pH 6 and pH 8. Buffering agents include but are not limited
to citrate, acetate, phosphate, Tris, or a basic amino acid, such
as, lysine or arginine, which are known to be pharmaceutically
acceptable in these pH ranges. Other pharmacologically acceptable
buffers for buffering at pH in these ranges are known in the art.
The selection and concentration of buffer is well within the skill
of the art.
EXAMPLE 1
pH 6.4 with 1.0% Ammonium Sulfate
[0051] 12.5 mg of chemically synthesized GLP-1(7-37)OH analog
having Val substituted for Ala in position 8 (V8-GLP-1) was weighed
into a 3.0 ml glass vial and treated with 2.0 ml of 10 mM Tris-HCl,
0.02% NaN.sub.3, pH 6.4, to give a clear solution at pH 3.6. The pH
of the solution was adjusted to 8.7 with 2N NaOH and then lowered
to pH 6.4 with 1N HCl. The solution remained clear during the pH
adjustments. The solution was filtered through a 0.22 micron Millex
GV13 syringe filter (Millipore, Bedford Mass.) into a new 3.0 ml
glass vial. The concentration of the V8-GLP-1 stock solution was
4.76 mg/ml as determined from the absorbance at 280 nm and using an
extinction coefficient of 2.015 for a 1.0 mg/ml V8-GLP-1 solution
in a 1 cm cell. A 0.25 ml aliquot of this V8-GLP-1 stock solution
was transferred to a 2.0 ml glass vial. To this solution was added
0.25 ml of a 10 mM Tris-HCl, 0.02% NaN.sub.3, pH 6.4, buffer
containing 2.0% (NH.sub.4).sub.2SO.sub.4. The vial was sealed,
gently swirled, and then placed at 18.degree. C. After 36 hours
crystalline clusters were identified at 200.times. magnification.
For quantitation, a portion of the mother liquor was removed and
centrifuged at 16,000.times. g. The V8-GLP-1 content remaining in
the clear supernatant was determined from the absorbance at 280 nm
as cited above. The crystalline yield was quantitated by
subtracting the V8-GLP-1 level in the supernatant from the V8-GLP-1
level in the starting solution. This sample showed a
crystallization yield of 63.9%.
EXAMPLE 2
pH 6.4 with 1% Ethanol and 1.0% Ammonium Sulfate
[0052] A 0.25 ml aliquot of the V8-GLP-1 stock solution was
transferred to a 2.0 ml glass vial as in Example 1. To this
solution was added 0.25 ml of a 10 mM Tris-HCl, 0.02% NaN.sub.3, pH
6.4, buffer containing 2.0% (NH.sub.4).sub.2SO.sub.4and 2.0%
ethanol. The solution was then treated and evaluated as in Example
1. This sample generated crystalline clusters and a few single
tetragonal crystals. The yield was 73.1%.
EXAMPLE 3
pH 6.4 with 5% Ethanol and 1.0% Ammonium Sulfate
[0053] A 0.25 ml aliquot of the V8-GLP-1 stock solution was
transferred to a 2.0 ml glass vial as in Example 1. To this
solution was added 0.25 ml of a 10 mM Tris-HCl, 0.02% NaN.sub.3, pH
6.4, buffer containing 2.0% (NH.sub.4).sub.2SO.sub.4 and 10.0%
ethanol. The solution was then treated and evaluated as in Example
1. This sample generated crystalline clusters, single tetragonal
crystals, and some rods. The yield was 80.3%.
EXAMPLE 4
pH 6.4 with 10% Ethanol and 1.0% Ammonium Sulfate
[0054] A 0.25 ml aliquot of the V8-GLP-1 stock solution was
transferred to a 2.0 ml glass vial as in Example 1. To this
solution was added 0.25 ml of a 10 mM Tris-HCl, 0.02% NaN.sub.3, pH
6.4, buffer containing 2.0% (NH.sub.4).sub.2SO.sub.4 and 20.0%
ethanol. The solution was then treated and evaluated as in Example
1. This sample generated single tetragonal crystals and rods. The
yield was 81.9%.
EXAMPLE 5
pH 6.4 with 1% Ethanol
[0055] A 0.25 ml aliquot of the V8-GLP-1 stock solution was
transferred to a 2.0 ml glass vial as in Example 1. To this
solution was added 0.25 ml of a 10 mM Tris-HCl, 0.02% NaN.sub.3, pH
6.4, buffer containing 2.0% ethanol. The solution was then treated
and evaluated as in Example 1. This sample generated a trace of
crystal clusters. The yield was 8.8%.
EXAMPLE 6
pH 6.4 with 5% Ethanol
[0056] A 0.25 ml aliquot of the V8-GLP-1 stock solution was
transferred to a 2.0 ml glass vial as in Example 1. To this
solution was added 0.25 ml of a 10 mM Tris-HCl, 0.02% NaN.sub.3, pH
6.4, buffer containing 10.0% ethanol. The solution was then treated
and evaluated as in Example 1. This sample generated crystal
clusters, single tetragonal crystals, and rods. The yield was
39.1%.
EXAMPLE 7
pH 6.4 with 10% Ethanol
[0057] A 0.25 ml aliquot of the V8-GLP-1 stock solution was
transferred to a 2.0 ml glass vial as in Example 1. To this
solution was added 0.25 ml of a 10 mM Tris-HCl, 0.02% NaN.sub.3, pH
6.4, buffer containing 20.0% ethanol. The solution was then treated
and evaluated as in Example 1. This sample generated single
tetragonal crystals and rods. The yield was 55.5%.
EXAMPLE 8
Pharmacokinetics
[0058] 28 mg of biosynthetic V8-GLP-1 was weighed into a glass vial
and dispersed in 4.5 ml of 10 mM NH.sub.4OAc to give a turbid
solution with a pH of 5.6. The material was completely solublized
by adjusting the pH to 9.5 with 5N NaOH and remained completely
soluble after the pH of the solution was lowered to 6.4 with 2N
HCl. This solution was filtered through a 0.22 micron Millex GV13
syringe filter into a new glass vial to give a total volume of 4.3
ml. The concentration of the V8-GLP-1 solution was 5.51 mg/ml as
determined from the absorbance at 280 nm of a 20.times. dilution of
the stock solution and using an extinction coefficient of 2.015 for
a 1 mg/ml V8-GLP-1 solution in a 1 cm cell. To this solution was
added 4.3 ml of a 10 mM NH.sub.4OAc, 2.0% (NH.sub.4).sub.2SO.sub.4,
20% ethanol, pH 6.4, precipitant buffer. The vial was sealed, the
solution was gently swirled and then placed at 18.degree. C. After
72 hours single tetragonal crystals were identified at 200.times.
magnification. The crystals were removed from the mother liquor by
low speed centrifugation and resuspended in a 10 mM NH.sub.4OAc, 16
mg/ml glycerin, pH 5.5, buffer (buffer A) to a concentration of
about 4.0 mg/ml. A portion of the mother liquor was centrifuged at
16,000.times. g. The V8-GLP-1 content remaining in the clear
supernatant was determined from the absorbance at 280 nm. The
crystallization yield was quantitated by subtracting the V8-GLP-1
level in the supernatant from the V8-GLP-1 level in the starting
solution. This crystallization gave an 83% yield.
[0059] Calculated aliquots of 4.0 mg/ml V8-GLP-1 crystal
suspensions prepared in a similar manner as above were transferred
to five glass vials and diluted with buffer A to a concentration
slightly above the final target concentration of 2.5 mg/ml
V8-GLP-1. To the crystalline suspensions were added aliquots of a
ZnCl.sub.2 stock solution (33.4 mg/ml Zn.sup.++ in buffer A) to
make final zinc concentrations either 0.5, 1.0, 1.5, or 2.4 mg/ml
zinc. The suspensions were gently swirled and placed at 5.degree.
C. for 18 hours. The final V8-GLP-1 concentration in each vial was
now at the 2.5 mg/ml target concentration. After 18 hours the
crystalline V8-GLP-1 zinc suspensions were transferred to room
temperature, passed through a 30 gauge needle, and adjusted to pH
6.0 with 1N NaOH.
[0060] A 0.1 mg/ml zinc crystalline V8-GLP-1 suspension was
prepared by first treating a 2.5 mg/ml crystal suspension with 0.15
mg/ml zinc in the same manner as described above. After 18 hours at
5.degree. C. the zinc treated crystals were isolated by low speed
centrifugation and transferred to buffer B (buffer A containing 0.1
mg/ml zinc). The final V8-GLP-1 concentration of this suspension
was adjusted to the 2.5 mg/ml target concentration using buffer B.
The suspension was passed through a 30 gauge needle, and the pH
increased to 6.0 with 1N NaOH.
[0061] The five crystalline V8-GLP-1 zinc suspensions described
above, each at 2.5 mg/ml V8-GLP-1 and containing 0.1, 0.5, 1.0,
1.5, or 2.4 mg/ml zinc were tested in overnight-fasted beagle dogs.
Each animal received a single 24 nmole/kg subcutaneous injection of
the crystalline V8-GLP-1 zinc suspension at time zero. Arterial
blood samples (1.5 ml) were withdrawn from the animals at scheduled
times, transferred to tubes pretreated with EDTA and containing 40
ul of Trasylol, and then centrifuged. The plasma portion of each
sample was separated and stored at -80.degree. C. until analysis.
The plasma concentration of immunoreactive V8-GLP-1 in each sample
was measured using a RIA procedure. Table 1 shows the resulting
immunoreactive V8-GLP-1 plasma levels over a 24 hour time period
for each suspension.
1TABLE 1 Immunoreactive V8-GLP-1 Levels (picomolar) in Dog Plasma
0.1 mg/ml 0.5 mg/ml 1.0 mg/ml 1.5 mg/ml 2.4 mg/ml Zinc (n = 5) Zinc
(n = 5) Zinc (n = 3) Zinc (n = 5) Zinc (n = 5) Time V8-GLP-1
V8-GLP-1 V8-GLP-1 V8-GLP-1 V8-GLP-1 (hrs) (pM) SEM (pM) SEM (pM)
SEM (pM) SEM (pM) SEM 0 0 0 0 0 0 0 0 0 0 0 1.5 nd nd 123 41 27 4 7
5 5 5 3.0 nd nd 132 38 38 7 40 13 27 21 4.5 nd nd 196 51 108 41 76
34 83 37 6.0 301 57 264 79 140 55 142 47 143 60 7.5 nd nd 265 71
184 57 198 61 179 63 9.0 nd nd 344 94 220 61 252 64 214 66 10.5 nd
nd 302 80 231 78 250 66 225 50 12.0 nd nd 282 78 236 76 267 60 238
42 13.5 nd nd 238 54 241 97 286 74 236 38 15.0 nd nd 263 67 273 118
325 114 246 28 16.5 nd nd 235 51 234 106 275 77 218 25 18.0 nd nd
210 47 184 62 254 59 211 23 19.5 nd nd 221 54 209 120 278 57 173 9
21.0 nd nd 215 54 219 115 301 48 178 13 22.5 nd nd 224 54 193 72
232 23 167 9 24.0 190 30 210 51 187 72 227 34 166 25 30.0 nd nd nd
nd nd nd nd nd 171 24 SEM = Standard Error of Mean. nd = not
determined
EXAMPLE 9
pH 9.4 with 5% Trehalose and Zinc
[0062] 6.8 mg of lyophilized biosynthetic V8-GLP-1 was weighed into
a 3.0 ml glass vial. Then, 1.0 ml of a 25 mM glycine-HCl, 150 mM
NaCl, 5% trehalose, pH 9.0, buffer was added to dissolve the
peptide. The solution was then adjusted to pH 10.3 with 5N NaOH.
While the solution was gently stirred 9.0 ul of a 10 mg/ml zinc
chloride solution in water was added and the pH adjusted to 9.4
with 2N HCl. The final concentration of V8-GLP-1 was 5.4 mg/ml as
determined from the absorbance at 280 nm of a 10.times. dilution of
the solution. The solution was then filtered with a 0.22 micron
Millex GV13 syringe filter. The vial was capped, gently swirled,
and then placed at ambient temperature. After 24 hours V8-GLP-1
crystal clusters and single rectangular crystals were identified at
430.times. magnification and estimated to be about 40 microns long,
15 microns wide, and 3 microns thick. A portion of the mother
liquor was removed and centrifuged at 16,000.times. g. The V8-GLP-1
content remaining in the clear supernatant was determined from the
absorbance at 280 nm. The crystalline yield was quantitated by
subtracting the V8-GLP-1 level in the supernatant from the V8-GLP-1
level in the starting solution. This sample showed a
crystallization yield of 89.8%. The small rectangular crystal
morphology was not observed in parallel crystallization trials
without trehalose.
EXAMPLE 10
pH 9.4 with 10% Mannitol and Zinc
[0063] 6.8 mg of lyophilized biosynthetic V8-GLP-1 was weighed into
a 3.0 ml glass vial, treated with 1.0 ml of a 25 mM glycine-HCl,
150 mM NaCl, 10% mannitol, pH 9.0, buffer and dispersed to give a
clear solution. The solution was then adjusted to pH 10.3 with 5N
NaOH. While the solution was gently stirred 9.0 ul of 10 mg/ml zinc
chloride solution in water was added and the pH adjusted to 9.4
with 2N HCl. The final concentration of V8-GLP-1 was 5.31 mg/ml as
determined from the absorbance at 280 nm of a 10.times. dilution of
the crystallization solution. The solution was then filtered with a
0.22 micron Millex GV13 syringe filter. The vial was capped, gently
swirled, and then placed at ambient temperature. After 24 hours,
small rectangular plate-like crystals of V8-GLP-1 were identified
at 430.times. magnification and estimated to be about 10 to 30
microns long and 10 microns wide. The yield was determined as in
Example 9. This sample showed a crystallization yield of 35%.
EXAMPLE 11
pH 9.0 with Zinc
[0064] A 1-ml aliquot of a solution of V8-GLP-1 at 3 mg/ml in 50 mM
glycine-150 mM NaCl buffer at pH 9.0 was prepared. To this solution
was added 7.5 .mu.l of a 20.85 mg/ml zinc chloride solution in
water, followed by a pH adjustment back up to pH 9.0. After gentle
swirling, the clear sample in a 3-ml glass vial was stored at
ambient temperature for one day. After this time the crystalline
precipitate was examined under the microscope at 90.times.
magnification, revealing clusters of small plates. For quantitation
of crystallization yield, the entire suspension was passed through
a 0.2 .mu.m filter (Gelman Sciences, Ann Arbor, Mich.). The
V8-GLP-1 content remaining in the clear filtrate was quantitated by
spectroscopic evaluation at a wavelength of 280 nm, using an
extinction coefficient of 2.015 for a 1 mg/ml solution of V8-GLP-1
in a 1 cm cell. The crystallization yield was quantitated by
subtracting the V8-GLP-1 level in the supernatant from the V8-GLP-1
level in the starting solution. This sample showed a
crystallization yield of 5.6%.
EXAMPLE 12
pH 9.0 with 10% Ethanol and Zinc
[0065] A 1-ml aliquot of a solution of V8-GLP-1 was prepared as in
Example 11, except that 110 .mu.l of absolute ethanol was added to
the solution prior to the addition of the zinc chloride solution.
This sample generated large tetragonal crystals, with some
clusters, in 80.6% yield.
EXAMPLE 13
pH 9.5 with 10% Ethanol and Zinc
[0066] A solution of V8-GLP-1 at 10 mg/ml in 50 mM glycine-150 mM
NaCl buffer at pH 10.5 was passed through a sterile 0.2 .mu.m
Acrodisc filter (Gelman Sciences, Ann Arbor, Mich.). To 500 .mu.l
of this solution was added 500 .mu.l of a 50 mM glycine-150 mM NaCl
buffer at pH 9.0. To this solution was then added 110 .mu.l of
absolute ethanol followed by 7.5 .mu.l of a 20.85 mg/ml zinc
chloride solution in water. Small additions of 1N HCl were used to
adjust the solution to pH 9.5. After gentle swirling the final
solution was enclosed in a 3-ml glass vial and stored at ambient
temperature for two days. Individual crystalline plates of V8-GLP-1
up to 150 .mu.m in length, approximately 25 .mu.m wide and less
than 5 .mu.m thick were generated in 72% yield.
EXAMPLE 14
pH 7.9 with 8.5% Ethanol and Zinc
[0067] V8-GLP-1 was prepared at 4 mg/ml in 50 mM glycine pH 9.5
buffer, followed by passage through a 0.2 .mu.m filter (Gelman
Sciences, Ann Arbor, Mich.). To 1-ml of this solution was added 100
.mu.l of absolute ethanol and then 60 .mu.l of 2.08 mg/ml zinc
chloride solution in water. Small additions of 0.1N HCl were used
to adjust the solution to pH 8.0. After gentle swirling the final
solution was enclosed in a 3-ml glass vial and stored at ambient
temperature for two hours. The pH of the clear solution was then
adjusted to pH 7.86 with small additions of 0.1N HCl and storage at
ambient temperature continued for two days. Microscopic examination
revealed modest-sized, individual tetragonal plates and some
clusters. The V8-GLP-1 content remaining in the clear supernatant
was quantitated by spectroscopic evaluation at a wavelength of 280
nm, using an extinction coefficient of 2.015 for a 1 mg/ml solution
of V8-GLP-1 in a 1 cm cell. The crystallization yield was
quantitated by subtracting the V8-GLP-1 level in the supernatant
from the V8-GLP-1 level in the starting solution. This sample
showed a crystallization yield of 92.2%.
EXAMPLE 15
pH 8.3 with 10% Ethanol and Zinc
[0068] V8-GLP-1 was prepared at 7 mg/ml in 100 mM glycine pH 10.5
buffer, followed by passage through a 0.2 .mu.m filter (Gelman
Sciences, Ann Arbor, Mich.). To 0.5 ml of this solution was added
0.4 ml of water. Then 100 .mu.l of absolute ethanol was added,
followed by about 6 .mu.l of a 20.86 mg/ml zinc chloride solution
in water. Small additions of 1N HCl were used to adjust the
solution to pH 8.33. After gentle swirling the final solution was
enclosed in a 3-ml glass vial and stored at ambient temperature for
one day. Microscopic examination revealed small, individual
tetragonal plates and some clusters. The V8-GLP-1 content remaining
in the clear supernatant was quantitated by spectroscopic
evaluation at a wavelength of 280 nm, using an extinction
coefficient of 2.015 for a 1 mg/ml solution of V8-GLP-1 in a 1 cm
cell. The crystallization yield was quantitated by subtracting the
V8-GLP-1 level in the supernatant from the V8-GLP-1 level in the
starting solution. This sample showed a crystallization yield of
92.4%.
EXAMPLE 16
pH 7.4 with 8.6% Ethanol and Zinc
[0069] V8-GLP-1 was prepared at 4 mg/ml in 50 mM glycine pH 9.0
buffer, followed by passage through a 0.2 .mu.m filter (Gelman
Sciences, Ann Arbor, Mich.). To 5 ml of this solution was added 500
.mu.l of absolute ethanol followed by 300 .mu.l of a 2.08 mg/ml
zinc chloride solution in water. Small additions of 1N HCl were
used to adjust the solution to pH 7.40. After gentle swirling the
final solution was enclosed in a 10-ml glass vial and stored at
ambient temperature for two days. Microscopic examination revealed
modest-sized, individual tetragonal crystals. The V8-GLP-1 content
remaining in the clear supernatant was quantitated by spectroscopic
evaluation at a wavelength of 280 nm, using an extinction
coefficient of 2.015 for a 1 mg/ml solution of V8-GLP-1 in a 1 cm
cell. The crystallization yield was quantitated by subtracting the
V8-GLP-1 level in the supernatant from the V8-GLP-1 level in the
starting solution. This sample showed a crystallization yield of
85.0%.
EXAMPLE 17
pH 6.4 with 5% Ethanol and 1.0% Ammonium Sulfate
[0070] V8-GLP-1 (12.5 mg) was weighed into a 20 ml glass vial. 2.0
ml of a 10 mM ammonium acetate buffer containing 150 mM NaCl at pH
6.4 was added. The pH of the turbid solution was clarified by
adjustment to pH 9.5 with 5N NaOH, then lowered to pH 6.4 with 2N
HCl. The clear solution was filtered through a 0.22 .mu.m Millex GV
13 syringe filter (Millipore, Bedford, Mass.) into a new 20 ml
glass vial. The concentration of the V8-GLP-1 stock solution was
determined from the absorbance at 280 nm using an extinction
coefficient of 2.015 for a 1.0 mg/ml solution of V8-GLP-1 in a 1 cm
cell. The protein concentration was adjusted to 5.0 mg/ml. A pH 6.4
precipitant solution containing 10 mM ammonium acetate, 150 mM
NaCl, 2% ammonium sulfate and 10% ethanol was prepared and filtered
through a 0.22 .mu.m Millex GV13 syringe filter. 2 ml of the
precipitant solution was slowly added to 2 ml of the V8-GLP-1 stock
solution in a glass vial. The vial was gently swirled and incubated
at room temperature for 2 days. Tetragonal plate-shaped crystals
were observed with a yield of 92%.
[0071] The pH of the crystal suspension was adjusted to pH 5.5 with
1N HCl and zinc chloride was added to a final concentration of 0.15
mg/ml. After zinc soaking overnight at room temperature, the pH of
the suspension was adjusted to pH 7.5 with 1N NaOH and the
preservative meta-cresol was added to a concentration of 3.16
mg/ml. This example shows that, if desired, preserved formulations
of GLP-1 crystals can be prepared directly for pharmaceutical use
without isolation of the crystals by centrifugation or filtration
in an intermediate step.
EXAMPLE 18
pH 7.6 with 8.5% Ethanol and Zinc
[0072] V8-GLP-1 was prepared at 4 mg/ml in 50 mM glycine pH 9.0
buffer, followed by passage through a 0.2 .mu.m filter (Acrodisc
from Gelman Sciences, Ann Arbor, Mich.). To 10 ml of this solution
was added 1 ml of absolute ethanol followed by 600 .mu.l of a 6.7
mg/ml zinc acetate (2-hydrate) solution in water. 100 .mu.l of 2%
acetic acid was added, resulting in a pH of about 7.6. After gentle
swirling the final solution was enclosed in a 20-ml glass vial and
stored at ambient temperature for 24 hours. Microscopic examination
revealed modest-sized, individual tetragonal crystals. To the
entire solution was then added 3.555 ml of a solution containing
3.5 ml of a 14 mg/ml solution of m-cresol in water and 55 .mu.l of
2% acetic acid, resulting in a suspension with a final pH of about
7.2. After gentle swirling the suspension was enclosed in a 20-ml
glass vial and stored at ambient temperature for 24 hours.
Microscopic examination again revealed modest-sized, individual
tetragonal crystals.
[0073] After centrifugation of an aliquot for 5 minutes at ambient
temperature, the V8-GLP-1 content remaining in the clear
supernatant was determined by HPLC analysis of a diluted sample
compared to HPLC analysis of V8-GLP-1 standard solutions. The
crystallization yield was quantitated by subtracting the V8-GLP-1
level in the supernatant from the V8-GLP-1 level in the starting
solution. This preserved V8-GLP-1 formulation showed a
crystallization yield of 97.7 %.
EXAMPLE 19
Crystal Stability
[0074] Single tetragonal crystals of V8-GLP-1 were prepared in 10
mM NH.sub.4OAc, 1% (NH.sub.4).sub.2SO.sub.4, 10% ethanol buffer at
pH 6.4 at 18.degree. C. as described in Example 8. The crystals
were removed from the mother liquor by low speed centrifugation and
resuspended in a 10 mM NH.sub.4OAc, 16 mg/ml glycerin, pH 5.5,
buffer to a concentration of about 4.9 mg/ml of V8-GLP-1.
[0075] 2 ml of this suspension was low speed centrifuged and the
supernatant was removed by pipette. The pellet was resuspended in 4
ml of a 10 mM ammonium acetate, 16 mg/ml glycerin pH 5.5 buffer
containing 0.1 mg/ml zinc. This crystal suspension was allowed to
soak in the zinc solution overnight at 4.degree. C.
[0076] The zinc-soaked crystal suspension was divided into four
1-ml aliquots. These suspensions were low speed centrifuged and
their supernatants were removed by pipette. Four crystal
suspensions were prepared in 10 mM ammonium acetate, 16 mg/ml
glycerin, 0.1 mg/ml zinc buffer at pH 6.0. Further pH adjustments
to pH 7.4 with 0.1N NaOH and/or additions of meta-cresol to a final
concentration of 3.16 mg/ml were made to selected samples as
illustrated in Table 2. Each suspension was further divided in half
for storage at both room temperature (about 22.degree. C.) and at
4.degree. C., providing a total of 8 test samples as shown in Table
2.
[0077] After 10 days, the crystal suspensions were examined under
the microscope. The suspension filtrates were then evaluated by
HPLC to quantitate the soluble V8-GLP-1 in the crystalline
suspensions. The HPLC results are reported in Table 2.
2TABLE 2 Soluble V8-GLP-1 in Crystal Suspensions after Storage for
10 days Storage mg/ml Soluble V8-GLP-1 Sample pH Temperature
meta-cresol by HPLC A 6.0 4.degree. C. 0 0.19% B 7.4 4.degree. C. 0
0.10% C 6.0 4.degree. C. 3.16 0.05% D 7.4 4.degree. C. 3.16 0.06% E
6.0 22.degree. C. 0 0.16% F 7.4 22.degree. C. 0 0.08% G 6.0
22.degree. C. 3.16 0.03% H 7.4 22.degree. C. 3.16 0.04%
[0078] This experiment showed that less than 0.2% of the V8-GLP-1
peptide became solublized in either the preserved or non-preserved
crystal formulations over a 10-day period.
[0079] Microscopically, the crystal suspensions showed less
agglomeration or clumping of the single, tetragonal crystals at pH
7.4 than at pH 6.0, and less at 4.degree. C. than at 22.degree. C.
The meta-cresol did not seem to have a significant effect on
crystal agglomeration. Additional testing showed the presence of
more than 3% ethanol in the crystal suspension, either from the
original crystallization mother liquor or from subsequent
additions, greatly reduced the clumping tendency of the crystals in
both preserved and non-preserved formulations. Further tests
revealed that, although the crystals are relatively stable in the
presence of meta-cresol, they are less stable in the presence of
0.5% phenol, which slowly leads to the formation of amorphous
material.
[0080] Additional crystal stability tests showed that the V8-GLP-1
crystals prepared at pH 6.4 are very stable chemically, with no
degradation peaks observed by HPLC analysis after storage at
5.degree. C. or room temperature for up to two months.
[0081] Stability tests of crystals prepared in glycine buffer as
described in Example 16 showed the V8-GLP-1 crystals stored in the
original mother liquor were not stable when meta-cresol was added
to the level of 3.16 mg/ml. This test resulted in dissolution of
the crystals after only 1 day. The crystal instability in this
composition could be effectively blocked by addition of zinc (via a
zinc chloride solution) prior to addition of the preservative.
Sequence CWU 1
1
29 1 31 PRT Homo sapiens 1 His Ala Glu Gly Thr Phe Thr Ser Asp Val
Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala
Trp Leu Val Lys Gly Arg Gly 20 25 30 2 31 PRT Artificial synthetic
construct 2 Xaa Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Xaa Gly 1 5 10 15 Gln Ala Ala Lys Xaa Phe Ile Ala Trp Leu Val Lys
Gly Arg Xaa 20 25 30 3 29 PRT Artificial synthetic construct 3 His
Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10
15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Xaa Xaa 20 25 4 31
PRT Artificial synthetic construct 4 Xaa Ala Glu Gly Thr Phe Thr
Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Xaa Glu
Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa 20 25 30 5 31 PRT
Artificial synthetic construct 5 His Xaa Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 6 29 PRT Artificial
synthetic construct 6 Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
Leu Xaa Gly Gln Ala 1 5 10 15 Ala Lys Xaa Phe Ile Ala Trp Leu Val
Lys Gly Arg Xaa 20 25 7 30 PRT Artificial synthetic construct 7 Ala
Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln 1 5 10
15 Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Xaa 20 25 30
8 28 PRT Artificial synthetic construct 8 His Ala Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu Val Lys 20 25 9 29 PRT Artificial synthetic
construct 9 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly 20 25 10 30 PRT Artificial synthetic construct 10 His Ala Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 11 31
PRT Artificial synthetic construct 11 His Ala Gln Gly Thr Phe Thr
Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu
Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 12 31 PRT
Artificial synthetic construct 12 His Ala Xaa Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 13 31 PRT Artificial
synthetic construct 13 His Ala Glu Gly Thr Phe Thr Ser Asp Thr Ser
Lys Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp
Leu Val Lys Gly Arg Gly 20 25 30 14 31 PRT Artificial synthetic
construct 14 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Lys Tyr
Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val
Lys Gly Arg Gly 20 25 30 15 30 PRT Artificial synthetic construct
15 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30 16 31 PRT Artificial synthetic construct 16 His Gly Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 17
31 PRT Artificial synthetic construct 17 His Val Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 18 31 PRT
Artificial synthetic construct 18 His Met Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 19 31 PRT Artificial
synthetic construct 19 His Ala Xaa Gly Thr Phe Thr Ser Asp Val Ser
Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp
Leu Val Lys Gly Arg Gly 20 25 30 20 31 PRT Artificial synthetic
construct 20 His Ala Thr Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val
Lys Gly Arg Gly 20 25 30 21 31 PRT Artificial synthetic construct
21 His Ala Xaa Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
Gly 20 25 30 22 31 PRT Artificial synthetic construct 22 His Ala
Asn Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15
Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25
30 23 31 PRT Artificial synthetic construct 23 His Ala Xaa Gly Thr
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala
Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 24 31 PRT
Artificial synthetic construct 24 His Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Ser 1 5 10 15 Arg Arg Ala Gln Glu Phe
Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 25 31 PRT Artificial
synthetic construct 25 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser
Ser Tyr Leu Glu Gly 1 5 10 15 Arg Ala Ala Lys Glu Phe Ile Ala Trp
Leu Val Lys Gly Arg Gly 20 25 30 26 31 PRT Artificial synthetic
construct 26 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
Leu Glu Gly 1 5 10 15 Gln Arg Ala Lys Glu Phe Ile Ala Trp Leu Val
Lys Gly Arg Gly 20 25 30 27 30 PRT Artificial synthetic construct
27 His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg
20 25 30 28 31 PRT Artificial synthetic construct 28 His Gly Glu
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Gln Gly 1 5 10 15 Gln
Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 29
31 PRT Artificial synthetic construct 29 His Thr Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30
* * * * *