jc891 U.S. PTO
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`111111111111111111111111111111111\111111
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`12/29/00
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`Atty. Dkt. No. 016777/0454
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`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
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`Applicant:
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`lndu J. Isaacs
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`Title:
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`GLP-2 FORMULATIONS
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`Appl. No.: Unknown
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`Filing Date: Herewith
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`Examiner:
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`Unknown
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`Art Unit:
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`Unknown
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`..
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`UTILITY PATENT APPLICATION
`TRANSMITTAL
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`Commissioner for Patents
`Box PATENT APPLICATION
`Washington, D.C. 20231
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`Sir:
`
`Transmitted herewith for filing under 37 C.F.R. § 1.53{b) is the nonprovisional
`utility patent application of:
`
`lndu J. Isaacs
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`Enclosed are:
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`IX] Specification, Claim(s), and Abstract (27 pages including a cover page}.
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`[ X ]
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`Informal drawings (6 sheets, Figures 1-6).
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`[ X] Unexecuted Declaration and Power of Attorney {4 pages}.
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`[ ]
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`[ ]
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`[
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`]
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`[ ]
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`[ ]
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`[
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`]
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`Assignment of the invention to NPS ALLELIX CORP ..
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`Assignment Recordation Cover Sheet.
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`Check in the amount of $40.00 for Assignment recordation.
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`Small Entity statement.
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`Information Disclosure Statement.
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`Form PT0-1449 with copies of _
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`listed reference(s).
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`002.430797. 1
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`-1-
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`CFAD Exhibit 1004
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`1
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`Atty. Dkt. No. 016777/0454
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`The filing fee is calculated below:
`
`Claims
`as Filed
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`Included in
`Basic Fee
`
`Extra
`Claims
`
`Basic Fee
`
`20
`54
`Total Claims:
`3
`5
`Independents:
`If any Multiple Dependent Claim(s) present:
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`34
`2
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`x
`x
`+
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`Rate
`$710.00
`$18.00
`$80.00
`$270.00
`
`[
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`]
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`SUBTOTAL:
`Small Entity Fees Apply (subtract Y2 of above):
`TOTAL FILING FEE:
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`Fee
`Totals
`$710.00
`$612.00
`$160.00
`$0.00
`$1482.00
`$0.00
`$1482.00
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`[
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`]
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`A check in the amount of $1482.00 to cover the filing fee is enclosed.
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`[ X ] The required filing fees are not enclosed but will be submitted in response to the
`Notice to File Missing Parts of Application.
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`[
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`]
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`The Commissioner is hereby authorized to charge any additional fees which may
`be required regarding this application under 37 C.F.R. § § 1.16-1.17, or credit
`any overpayment, to Deposit Account No. 19-0741. Should no proper payment
`be enclosed herewith, as by a check being in the wrong amount, unsigned, post(cid:173)
`dated, otherwise improper or informal or even entirely missing, the Commissioner
`is authorized to charge the unpaid amount to Deposit Account No. 19-0741.
`
`Please direct all correspondence to the undersigned attorney or agent at the
`address indicated below.
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`Respectfully submitted,
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`_.r(}thJ /YJ ;j~{V\
`By~-~~~~N_ol_s_~_,_7_17~~~~/'--
`
`stephen A. Bent
`Attorney for Applicant
`Registration No. 29, 768
`
`()
`
`Date
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`FOLEY & LARDNER
`Washington Harbour
`3000 K Street, N. W., Suite 500
`Washington, D.C. 20007-5109
`Telephone:
`(202) 672-5404
`Facsimile:
`(202) 672-5399
`
`002.430797 .1
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`-2-
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`2
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`

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`U.S. PATENT APPLICATION FOR
`
`GLP-2 FORMULATIONS
`
`BY
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`Indu J. Isaacs
`
`3
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`

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`)_
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`GLP-2 FORMULATIONS
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`FIELD OF INVENTION
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`The present invention provides formulations for GLP-2 peptides and analogs thereof
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`5
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`In particular, the invention provides formulations of GLP-2 peptides and GLP-2 analogs with
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`improved stability.
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`BACKGROUND OF THE INVENTION
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`Administration of therapeutic peptides requires peptide formulations that remain
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`stable during storage. In general, parenteral administration is used with peptides because of
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`10
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`their increased size and subsequent difficulty in crossing biological membranes. Peptides can
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`be particularly difficult to formulate because of their tendency to degrade over time and/or
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`undergo aggregation and precipitation. Degradation, aggregation, and precipitation are all
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`indicative of an unstable formulation. Such an unstable formulation is not commercially
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`viable, as it cannot pass U.S. Food and Drug Administration approval.
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`Formulation variables which affect the degradation of peptides during storage include,
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`but are not limited to, pH, the quantity of salts present, and the type and quantity of
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`excipients. In addition, temperatures, pressures, and time for freezing and drying cycles can
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`affect the stability of a lyophilized peptide formulation. The role of most of these variables
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`has been studied; however, the synergistic effect of the variables is still poorly understood.
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`'zb
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`Glucagon-like peptide-2 (GLP-2) is a 33 amino acid peptide having therapeutic
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`applications in the treatment of diseases of the gastrointestinal tract . In particular, it has been
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`determined that GLP-2 and analogs thereof act as trophic agents to enhance and maintain the
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`functioning of the gastrointestinal tract and to promote growth of intestinal tissue. See e.g.,
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`U.S. Patent Nos. 5,834,428; 5,789,379; and 5,990,077; and International Publication No. WO
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`25
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`98/52600.
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`Commercial exploitation ofGLP-2 or an analog thereofrequires a stable GLP-2
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`formulation that can be readily prepared using a commercially acceptable process. Because
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`GLP-2 is a protein, and thus far more labile than traditional small molecular weight drugs, the
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`formulation ofGLP-2 or an analog thereof presents challenges not commonly encountered by
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`30
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`the pharmaceutical industry. For example, methionine oxidation at position 10 and aspargine
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`deamination at position 11, 16, and/or 24 of GLP-2 are potential routes of degradation.
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`1
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`4
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`

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`Furthermore, GLP-2 or an analog thereof may also be adsorbed to surfaces to form
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`aggregates and/or precipitate, which would then render the formulation unstable.
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`There is a need in the art for stable formulations of GLP-2 peptides and analogs
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`thereof which can be prepared using a commercially acceptable process. The present
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`s
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`invention satisfies these needs.
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`SUMMARY OF THE INVENTION
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`The present invention provides stable formulations of GLP-2 and analogs thereof,
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`which can be prepared using a commercially acceptable process.
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`10
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`It has been discovered that relatively high concentrations of GLP-2 can be used in
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`pharmaceutically acceptable formulations. Moreover, it has been discovered that a pH of
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`greater than about 5.5, more preferably greater than about 6, even more preferably from about
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`6.9 to about 7.9, and most preferably about 7.3 to about 7.4, is suitable for a stable
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`formulation.
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`It has also been discovered that the GLP-2 analog h[Gly2]GLP-2 undergoes a phase
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`transition between 40-55°C, depending upon the salt concentration, and becomes hydrophobic
`in the presence of salt. It has also been discovered that Tween so®, salt, and arginine are not
`suitable materials for producing a stable formulation for h[Gly2]GLP-2.
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`According to one aspect of the present invention, there is provided a GLP-2
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`formulation comprising: (1) a medically useful amount of GLP-2; (2) a phosphate buffer
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`sufficient to adjust the pH of the formulation to a pharmaceutically acceptable level, and in
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`particular above about 6.0; (3) a stabilizing amount of the amino acid L-histidine; and (4) a
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`bulking agent selected from sucrose and mannitol.
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`More particularly, there is provided a GLP-2 formulation comprising: (1) a medically
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`25
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`useful amount ofGLP-2 comprising from about 0.1 to about 50 mg/ml ofGLP-2, preferably
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`about 5 to about 40 mg/ml, more preferably about 7 to about 30 mg/ml, even more preferably
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`about 10 to about 20 mg/ml, and most preferably about 20 mg/ml; (2) a phosphate buffer to
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`maintain the pH at a physiologically tolerable level, i.e., above 6; (3) a stabilizing amino acid,
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`particularly L-Histidine; and ( 4) a bulking agent, particularly mannitol. All percentages
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`30
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`described herein (except for percentages for water) are weight/volume of formulated product
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`prior to lyophilization in gms/ml (xlOO). Percentages for water content are weight/weight of
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`lyophilized product (xlOO).
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`2
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`5
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`

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`In one embodiment of the present invention, the GLP-2 formulation is a h[Gly2]GLP-
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`2 lyophilized formulation comprising in the reconstituted product: (1) phosphate buffer in an
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`amount necessary to maintain the pH of the reconstituted product between about 6.9-7.9, and
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`preferably in an amount to maintain a pH of about 7.3 to about 7.4; (2) about 0.5 to about 1 %
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`5
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`L-histidine; (3) about 2 to about 5% mannitol, preferably about 2.5 to about 3.5% mannitol,
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`and most preferably about 3% mannitol; and (4) from about 0.1 to about 50 mg/ml ofGLP-2
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`or an analog thereof, preferably about 5 to about 40 mg/ml, more preferably about 7 to about
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`30 mg/ml, even more preferably about 10 to about 20 mg/ml, and most preferably about 20
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`mg/ml.
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`10
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`In a more preferred embodiment of the invention, a h[Gly2]GLP-2 lyophilized
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`formulation is provided comprising in the reconstituted product: (1) about 7 to about 30
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`mg/ml, preferably about 10 to about 20 mg/ml, and most preferably about 20 mg/ml of
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`h[Gly2]GLP-2; (2) a phosphate buffer sufficient to maintain the pH at about 7.3 to about 7.4;
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`(3) about 0.5 to about 1 % L-histidine; and (4) about 3% mannitol.
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`;i5
`-
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`In another aspect of the present invention there is provided a process for making the
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`lyophilized formulation of GLP-2. Such a process comprises the following steps:
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`(a) preparing the GLP-2 formulation comprising GLP-2 or an analog thereof, a
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`phosphate buffer, L-histidine, and mannitol;
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`(b) freezing the formulation to about-4Q°c;
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`(c) performing a first drying step at about -2D°c; and
`(d) performing a second drying step at +20°C.
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`In a preferred embodiment the liquid formulation subjected to the lyophilization
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`process comprises:
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`(1) the h[Gly2]GLP-2 analog; (2) 35 mM phosphate buffer to maintain the reconstituted
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`25
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`product at a pH of about 6.9 to about 7.9, and more preferably at a pH of about 7.3 to about
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`7.4; (3) about 0.5 to about 1 % L-histidine; and (4) about 3% mannitol.
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`According to another aspect of the present invention, there is provided a method for
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`preparing a GLP-2 pharmaceutically acceptable formulation for parenteral administration,
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`comprising the step ofreconstituting the lyophilized GLP-2 formulation.
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`30
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`There is further provided in accordance with the present invention a therapeutically
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`useful kit comprising: (1) a sterile vial comprising a lyophilized GLP-2 formulation of the
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`invention, (2) a vehicle suitable for reconstitution thereof, preferably sterile water, (3)
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`instructions for reconstitution; and ( 4) optionally instructions for administration. The kit may
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`:further comprise a device suitable for injection of the reconstituted preparation.
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`10
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`Uis
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`Both the foregoing general description and the following detailed description are
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`exemplary and explanatory and are intended to provide further explanation of the invention
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`as claimed. Other objects, advantages, and novel features will be readily apparent to those
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`skilled in the art from the following detailed description of the invention.
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`Brief Description of the Figures
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`Figure 1:
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`Shows a bar graph of the effect of certain amino acid stabilizers on a
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`formulation ofh[Gly2]GLP-2 using a heat stress test. The precent (%)purity
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`is plotted for three different amino acid formulations, both before and after the
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`application of heat;
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`Figure 2:
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`Shows a bar graph of the effect ofL-histidine on a phosphate buffered
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`formulation ofh[Gly2]GLP-2. The% purity is plotted for three different
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`formulations at 0 and at 4 hours;
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`Figure 3:
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`Shows a bar graph of the screening of bulking agents analyzed by
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`reverse-phase high performance liquid chromatography (RP-HPLC) at room
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`temperature and 60°C. The % purity is plotted for seven
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`different amino acid formulations;
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`Figure 4:
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`Shows a bar graph of the screening ofbulking agents analyzed by
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`size exclusion high performance liquid chromatography (SE-HPLC). "HMW"
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`represents a high molecular weight peak. The % purity is plotted for seven
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`different formulations;
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`Figure 5:
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`Shows a bar graph of the stability of mannitol and sucrose
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`formulations of h[Gly2]GLP-2 in a liquid state, prior to lyophilization, which
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`25
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`have been stored at 4°C. The% purity is plotted for four different
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`formulations at 0 min. through 49 min., at 7 min. intervals; and
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`Figure 6:
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`Shows a bar graph of the stability oflyophilized mannitol and sucrose
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`formulations of h[Gly2]GLP-2 which have been stored at 60°C. The% purity
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`is plotted for four different amino acid formulations.
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`30
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`4
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`7
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`Detailed Description of the Invention
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`The invention relates to GLP-2 formulations which exhibit superior storage stability.
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`The term "GLP-2," as used herein, means a naturally occurring GLP-2 peptide or a GLP-2
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`analog thereof (unless specifically indicated otherwise).
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`5
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`The present GLP-2 formulations can be provided as liquid formulations suitable for
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`administration, such as by injection, in unit or multi-dose amounts. The liquid formulations
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`can also serve as stock solution from which lyophilized dosage forms can be prepared.
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`Accordingly, the present GLP-2 formulations can also be provided in lyophilized form, e.g.,
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`as freeze-dried powders suitable for reconstitution and subsequent administration as
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`10
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`injectable liquid formulations.
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`Lyophilized formulations of the present invention exhibit storage stability of six
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`months at ambient temperature, and eighteen months at 4 °C. Storage stability is exhibited by
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`minimal peptide degradation, preferably less than about 5% peptide degradation, more
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`preferably less than about 3 to about 4% peptide degradation, and even more preferably less
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`than about 1 to about 2% peptide degradation. Peptide degradation can be measured using
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`standard reverse-phase HPLC (RP-HPLC) techniques.
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`The naturally occurring GLP-2 peptides are highly conserved peptides. Accordingly,
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`GLP-2 peptides for use in the present invention include the various naturally produced forms
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`of GLP-2, particularly vertebrate species (including piscine and avian species), more
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`'~o
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`particularly mammalian (such as primate, rodent (including rat, mouse, degu, hamster, and
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`guinea pig), porcine, and bovine, ), and more particularly the human form. Desirably, but not
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`essentially, the naturally occurring GLP-2 peptide selected for use is of the same species as
`the subject identified for treatment.
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`GLP-2 analogs potentially useful in the present invention include agonists and
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`25
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`antagonists of the GLP-2 receptor. GLP-2 agonists activate the GLP-2 receptor by first
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`binding to the receptor, followed by stimulating an intracellular second messenger system
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`coupled to the receptor. In one embodiment of the invention, the GLP-2 agonists act
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`selectively at the GLP-2 receptor. Selectively-acting GLP-2 agonists are compounds that, in
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`the context of a suitable GLP-2 receptor binding or functional assay, bind to the GLP-2
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`30
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`receptor with greater affinity. Such greater affinity is preferably at least an order of
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`magnitude greater relative to different receptor types, such as the GLP-1 receptor. In other
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`embodiments, the GLP-2 analogs bind to the GLP-2 receptor with an affinity at least
`equivalent to the affinity of naturally occurring GLP-2.
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`In other embodiments of the invention, the GLP-2 peptide is an analog of natural
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`GLP-2 that incorporates one or more amino acid substitutions, additions, deletions, or
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`modifications and retains biological acitivity.
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`The agonist activity of human GLP-2 and rat GLP-2 is believed to require an intact N-
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`5
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`terminus, but various deletions of up to several residues at the C-terminus are tolerated
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`without loss of agonist activity. Substitutions are tolerated at sites outside regions conserved
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`across the various GLP-2 species homologs. Similarly, substitutions are also tolerated at sites
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`within regions conserved across GLP-2 species. In preferred embodiments, the amino acid
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`substitutions are conservative substitutions. For example, one member of an amino acid class
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`10
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`can be substituted by another member, e.g., the substitution of alanine by glycine, the
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`substitution of asparagine by glutamine, the substitution of methionine by leucine or
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`isoleucine, and the like.
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`Antagonist activity of GLP-2 analogs in humans and rats is exhibited when the
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`naturally occurring GLP-2 peptide is mutated in any one or more of the first four N-terminal
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`residues, in particular by deleting any one or more of these N-terminal residues. In addition,
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`antagonist activity is exhibited when naturally occurring hGLP-2 is substituted: (1) with an
`amino acid which does not naturally occur at any of the following positions: Asp15
`, Phe22
`,
`Thr29
`; .(2) and when Ala2 is replaced by anyone of the following amino
`, Thr32 and/or Asp33
`acids: Leu, Cys, Glu, Arg, Trp and P03-Tyr2. In addition, antagonists ofGLP-2 analogs
`include any mutation or variation of the naturally occurring GLP-2 peptide which results in
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`;[.10
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`the inhibition of intestinotrophic activity of naturally occurring GLP-2 or GLP-2 analogs
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`which exhibit agonist acitivity. Structural analogs of GLP-2 which act as antagonists are
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`specifically described in WO 98/03547.
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`The GLP-2 receptor analogs can be identified by screening peptides against cells
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`25
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`genetically engineered to produce the GLP-2 receptor. The GLP-2 receptor has been cloned.
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`See Munroe et. al., Proc. Natl. Acad. Sci. USA, 96(4):1569 (1999). Cells functionally
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`incorporating the GLP-2 receptor, and their use to screen GLP-2 analogs, are also described
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`in International Publication No. WO 98/25955, published on June 18, 1998.
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`In a preferred embodiment, the GLP-2 analog with agonist activity has been altered
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`30
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`to confer resistance to degradation by endogenous enzymes, such as DPP-N. Such analogs
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`suitably incorporate a replacement of the alanine residue at position 2. In specific
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`embodiments, the Ala2 residue is replaced by glycine or serine, or by other residues as
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`described for example in U.S. Patent No. 5,789,379. In a preferred embodiment, the GLP-2
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`receptor agonist is [Gly2]GLP-2. For use in treating humans, the GLP-2 analog is desirably
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`but not essentially a human GLP-2 peptide or analog, particularly including the Gly2 analog
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`of human GLP-2.
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`It was discovered that the h[Gly2]GLP-2 analog precipitated at a pH ofless than 5.5,
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`and that temperature profiles suggested a heat-induced and salt-dependent transition
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`5
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`temperature of about 40°C. Based on pH solubility profiles, it was determined that a
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`phosphate buffer provides optimal buffering capacity for GLP-2 peptides. Furthermore, the
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`addition ofL-histidine to the phosphate buffer was found to effectively stabilize GLP-2
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`peptides, whereas the addition of arginine citrate or lysine did not effectively stabilize GLP-2
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`compositions. L-histidine acts as a stabilizing amino acid that increases the length of time
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`10
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`that the GLP-2 peptide remains intact prior to degradation.
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`The lyophilized formulations of the present invention are preferably provided in a
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`powder form comprising not more than about 5% water by weight, preferably not more than
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`2% water by weight, and more preferably not more than about 1 % water by weight.
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`The bulking agent incorporated in the preparation produces a non-crystalline
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`amorphous cake. It was found that lactose, trehalose, and maltose sugars did not effectively
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`stabilize the GLP-2 formulation as well as mannitol and sucrose. Mannitol was found to be
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`the preferred excipient for the GLP-2 formulations.
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`The buffering agent incorporated in the formulation of the present invention is
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`selected from those capable of buffering the preparation to a pH within a physiologically
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`tolerable range for administration to a patient. "Physiologically tolerable" formulations are
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`those that elicit reactions, in a recipient, that are not so extreme as to preclude further
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`administration of the formulation. acceptable range for administration to a patient. More
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`particularly, it was found that the pH of the formulation should by greater than about 5.5,
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`more preferably greater than about 6, even more preferably of about 6.9 to about 7.9, and
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`25 most preferably about 7.3 to about 7.4. Preferably, the buffering agent is phosphate based,
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`and most preferably a 35 mM phosphate buffer is used.
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`The formulations of the present invention incorporate GLP-2 in a medically effective
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`amount, namely an amount which is useful either therapeutically or diagnostically. Such an
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`amount can be determined based on the type ofGLP-2 peptide or analog selected and on the
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`30
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`intended end-use of the preparation. Therapeutically useful amounts ofGLP-2 include those
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`unit dosage amounts useful in a regimen to treat a subject that would benefit from GLP-2
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`administration, as described more fully in U.S. Patent Nos. 5,834,428; 5,789,379; 5,990,077;
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`and 5,952,301, and in International Publication No. WO 98/52600.
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`7
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`10
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`In one application, the formulation maybe exploited for the treatment of
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`gastrointestinal disease, particularly diseases, disorders or conditions of the intestine.
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`Therapeutically useful amounts also include multi-dose amounts of GLP-2, which can be
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`delivered to an intended subject. Diagnostically useful amounts of GLP-2 include those
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`5
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`amounts useful as a calibrant when assessing endogenous levels of GLP-2 or levels of GLP-2
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`drug in a subject, for instance as a prelude to GLP-2 therapy, or during the course of GLP-2
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`treatment. Medically useful amounts of GLP-2 thus can range widely from a few
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`micrograms to many milligrams. The formulations of the present invention preferably
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`provide about 0.1 to about 50 mg/ml of GLP-2, preferably about 5 to about 40 mg/ml, more
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`10
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`preferably about 7 to about 30 mg/ml, even more preferably about 10 to about 20 mg/ml, and
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`most preferably about 20 mg/ml of GLP-2.
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`In an embodiment of the invention, a liquid formulation ofh[Gly2]GLP-2 suitable for
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`lyophilization comprises: (1) preferably about 7 to about 30 mg/ml, even more preferably
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`about 10 to about 20 mg/ml, and most preferably about 20 mg/ml of h[Gly2]GLP-2; (2)
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`1~15
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`about 2 to about 5% ofmannitol, preferably about 2.5 to about 3.5%, most preferably about
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`3%; (3) about 0.5 to about 1 % of an amino acid stabilizer, which is preferably L-histidine;
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`and (4) a phosphate buffer in an amount capable of buffering the reconstituted product to a
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`pH of about 6.9-7.9, and preferably a pH of about 7.3 to about 7.4.
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`-·
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`The GLP-2 formulations of the present invention are preferably filled in individual
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`20
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`vials to the desired volume and the vials are subjected to a lyophilization process. The
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`lyophilization process includes a temperature cycling process that is carefully controlled to
`
`ensure that drying proceeds uniformly. The drying process is continued until there is less
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`than about 5% of water, preferably less than about 2% of water, and more preferably no more
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`than about 1 % of water, in the GLP-2 formulation.
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`25
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`A lyophilization process suitable for the present invention involves a freezing step and
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`a two-step drying process. In an exemplary freezing process: (1) the formulation vials are
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`first cooled from ambient temperature to about -1° C at about 2 ° C/minute, and then held at
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`about -1° C for about 15 minutes, (2) next the vials are cooled from about -1 ° C to about -
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`40°C at about 2°C/minute, and then held at about -40°C for about 4 hours.
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`30
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`In an exemplary first drying cycle, the temperature is increased from about -40 ° C to
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`about -20 ° C at about 2 ° C/minute, and then held at about -20 ° C for about 14 hours under a
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`vacuum of about 150 mT with a condenser temperature of about- 80°C. In an exemplary
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`second drying cycle, the vials are warmed from about-20°C to about +20°C at about
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`2°C/minute, and then held at about +20°C for about 14 hours at a vacuum of about 150 mT
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`8
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`11
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`and a condenser temperature of about-80°C until there is less than about 5% of water,
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`preferably less than about 2% of water, and more preferably no more than about 1 % of water.
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`The vials are then preferably stored at about 4° C.
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`The present invention also provides a medically useful kit comprising: (1) at least one
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`5
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`vial containing the lyophilized freeze-dried GLP-2 formulation of the invention; (2) at least
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`one vial of sterile water for reconstitution; (3) instructions directing reconstitution; and ( 4)
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`optionally an injection device for administration. To use the kit, the user mixes the water
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`with the formulation vial, preferably by transferring the water to the formulation vial. The
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`lyophilized formulation of the present invention rapidly dissolves upon reconstitution and,
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`10 when reconstituted, is stable for at least about 12 hours, preferably up to about 24 hours, at 4
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`°C. In a preferred embodiment, reconstitution of the lyophilized formulation is carried out
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`using sterile water, preferably no more than about 1 mL of sterile water per dose of GLP-2.
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`To reconstitute, the sterile water may be drawn into a syringe and then transferred to the vial
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`containing the lyophilized GLP-2 formulation.
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`The following examples are given to illustrate the present invention. It should be
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`understood, however, that the invention is not to be limited to the specific conditions or
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`details described in these examples. Throughout the specification, any and all references to a
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`-·
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`publicly available document, including a U.S. patent, are specifically incorporated by
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`reference.
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`1d.20
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`Example 1: Formulation and Lyophilization ofh[Gly2]GLP-2
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`The purpose of this example was to prepare a lyophilized formulation of the GLP-2
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`peptide h[Gly2]GLP-2.
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`A base formulation buffer, comprising 35 mM sodium phosphate at pH 7.4, was
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`prepared as follows: (1) purified water was added to a sterile, depyrogenated flask; (2)
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`25
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`sodium heptahydrate was added to the flask; and (3) monobasic sodium phosphate
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`monohydrate was added to the flask. The buffer was mixed and the pH was verified to be
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`7.4±0.2. The base formulation buffer was then used to dilute the GLP-2 peptide
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`h[Gly2]GLP-2 liquid bulk drug substance to a concentration of 10 mg/ml. L-histidine was
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`then added to a final concentration of7.76 gm.IL, and mannitol was added to a final
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`30
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`concentration of 30 gm/L.
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`The preparation was carefully mixed, followed by filtering the preparation through a
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`0.22 µm filter into a sterile filling tank. The GLP-2 preparation was then aseptically filled, in
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`9
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`12
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`

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`1 ml aliquots, from the tank into 3 cc sterile USP Type I glass vials, which were then partially
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`capped with sterile rubber stoppers and placed into lyophilization trays.
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`The vials were then loaded into the lyophilizer, and the lyophilization cycle was
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`commenced by pre-freezing the formulation to a temperature of -40±i' C for about 4 hours.
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`5
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`In the freezing step, the formulation vials were first cooled from ambient temperature to
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`-1° C at 2 ° C/minute and then held at -1° C for approximately 15 minutes. This first freezing
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`step was followed by cooling the vials from -1° C to -40 ° C at 2 ° C/minute, and the vials were
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`then maintained at -40 ° C for 4 hours.
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`In the first and primary drying cycle, the temperature was increased from-40°C to -
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`10
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`20°C at 2°C/minute and then held at-20°C for about 14 hours under a vacuum of 150 mT
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`with a condenser temperature of - 80° C. In the second drying cycle, the vials were warmed
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`from -20°C to +20°C at 2°C/minute and then held at +20°C for about 14 hours at a vacuum of
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`150 mT and a condenser temperature of-80°C. The second drying cycle was continued until
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`there is less than about 5% of water, preferably less than about 2% of water, and more
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`preferably no more than about 1 % of water, remaining in the GLP-2 formulation. The vials
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`were then stored at 4°C.
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`At the end of the lyophilization cycle, the vials were purged with filtered nitrogen and
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`the rubber stoppers were fully depressed into the vials. The stoppered vials were removed
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`from the lyophilizer and permanently sealed with a crimped aluminum seal and capped with a
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`;1)20
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`polypropylene flip-off button.
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`Example 2: Screening of Amino Acid to Stabilize the Formulation
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`The prnpose ofthis example was to determine the effect of various amino acid
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`additives on the stability of GLP-2 following exposure to elevated temperatures.
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`25
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`The h[Gly2]GLP-2 formulation was tested with several amino acids as set out below.
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`The tested formulations comprised: (1) h[Gly2]GLP-2 at a concentration of 10 mg/ml; and
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`(2) the additives listed below. The pH of the composition was maintained between 7.1-7.5.
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`30
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`1.
`2.
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`3.
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`4.
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`5.
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`6.
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`10 mM phosphate, 10 mM Glutamate
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`10 mM phosphate, 10 mM Glutamate, 50 mM Arginine
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`10 mM phosphate, 10 mM Citrate
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`10 mM phosphate, 10 mM Citrate, 50 mM Arginine
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`I 0 mM phosphate, 100 mM Citrate
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`10 mM phosphate, 100 mM Citrate, 50 mM Arginine
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`10
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`13
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`

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`7.
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`8.
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`9.
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`10.
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`11.
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`12.
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`13.
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`14.
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`15.
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`16.
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`10 mM phosphate, 10 mM Serine
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`10 mM phosphate, 10 mM Serine, 50 mM Arginine
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`10 mM phosphate, 10 mM Pro line
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`10 mM phosphate, 10 mM Pro line, 50 mM Arginine
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`10 mM phosphate, 10 mM Histidine
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`10 mM phosphate, 10 mM Histidine, 50 mM Arginine
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`10 mM phosphate, 10 mM Glycine
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`10 mM phosphate, 10 mM Glycine, 50 mM Arginine
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`10 mM His, 10 mM Glycine
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`10 mM His, 10 mM Glycine, 50 mM Arginine
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`5
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`10
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`Following preparation, the samples were lyophilized according to the protocol of
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`Example l, stored at 40 °C for 14 days, diluted to 0.4 mg/ml, and then heated at 60 °C for 4
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`hours.
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`All of the formulations containing arginine precipitated upon heating (Formulations 2,
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`4, 6, 8, 10, 12, 14, and 16). Formulation 5 (100 mM citrate) and Formulation 15 (L-histidine
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`and glycine) also precipitated. Formulations comprising L-histidine, 10 mM citrate, serine,
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`proline, glutamate, and glycine (Formulations 1, 3, 7, 9, 11, and 13) showed similar stability
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`when these compounds were used without the addition of other amino acids. (See Figure 1.)
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`20
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`As shown in Figure 2, when L-histidine was used as a stabilizer in combination with a
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`phosphate buffer, the GLP-2 peptide remained stable following heat stress for 4 hours at 60
`oc.
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`Example 3: Screening Bulk Agents
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`25
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`The purpose of this example was to determine the effect of various bulk agent
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`additives on the stability of a GLP-2 peptide following exposure to elevated temperatures.
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`The following formulations of the GLP-2 peptide h[Gly2]GLP-2, at a concentration of
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`0.4 mg/ml, were lyophilized according to lyophilization process of Example 1. The
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`compositions were then reconstituted and heated to 60 °C.
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`30
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`1. 25 mM histidine, 35 mM phosphate, 3% mannitol
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`2. 50 mM histidine, 35 mM phosphate, 3% mannitol
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`3. 75 mM histidine, 35 mM phosphate, 3% mannitol
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`4. 25 mM histidine, 25 mM phosphate, 3% sucrose
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`11
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`14
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`

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`5. 25 mM histidine, 25 mM phosphate, 3% trehalose
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`6. 25 mM histidine, 25 mM phosphate, 3% maltose
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`7. 25 mM histidine, 25 mM phosphate, 3% lactose
`
`s
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`As shown in Figures 3 and 4, the reverse phase HPLC data (Fig. 3) demonstrate that
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`the mannitol samples (Formulations 1, 2, and 3) exhibited the least amount of GLP-2
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`degradation. In addition, all three L-histidine concentrations (25 mM, 50 mM, and 75 mM)
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`showed comparable stability. The SE-HPLC analysis (Fig. 4) also showed that, except for
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`maltose and lactose (Formulations 6 and 7), the GLP-2 analog in all of the formulations
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`10
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`eluted as a single peak without aggregation. Formulations 6 and 7 gave an additional high
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`molecular weight (HMW) impurity peak that accounted for approximately 6%. However
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`when these samples were heat stressed at 60 °C, the high molecular weight impurity
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`aggregates increased to approximately 20% in Formulations 6 and 7.
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`Accordingly, mannitol and sucrose were determined to be acceptable candidates for
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`addition to the GLP-2 formulations of the invention.
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`Example 4: Screening Bulk Agents
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`The purpose of this example was to compare the effectiveness of the bulk agent
`additives sucrose and mannitol on the stability ofGLP-2 following exposure to elevated
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`temperatures.
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`The following formulations ofh[Gly2]GLP-2, at 10 mg/ml, were prepared and the
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`stability of GLP-2 in each formulation was analyzed. The concentration of sucrose in
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`Formulation 2 was increased to 5% to satisfy physiological osmolarity.
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`25
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`1.
`2.
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`3.
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`4.
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`35 mM phosphate, 50 mM histidine, 3% mannitol, pH 7.4
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`35 mM phosphate, 50 mM histidine, 5% sucrose, pH 7.4
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`35 mM phosphate, 25 mM lysine, 3% mannitol, pH 7.4
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`35 mM phosphate, 25 mM lysine, 5% mannitol, pH 7.4
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`The formulations were then lyophilized according to lyoph