`Kornf elt et al.
`
`11•1111111111111
`
`US005652216A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,652,216
`Jul. 29, 1997
`
`[54] PHARMACEUTICAL PREPARATION
`
`5,059,587 10/1991 Yamamoto ................................ 514/12
`
`[75]
`
`Inventors: Troels Komfelt, Virum.; Henrik
`Rasmussen, Copenhagen; Flemming
`Steen Jensen, Alleroed, all of Denmark
`
`[73] Assignee: Novo Nordisk A/S. Bagsvaerd,
`Denmark
`
`[30]
`
`[22] Filed:
`
`[21] Appl. No.: 275,855
`Jul. 15, 1994
`Foreign Application Priority Data
`May 26, 1994
`[DK] Denmark ................................. 0590194
`Int. Cl.6
`•••••••••••••••••••••.•••.•..••••••••••••••.•••••.••• A61K 38/26
`[51]
`[52] U.S. Cl. ................................................................ 514/12
`[58] Field of Search ................................................. 514/12
`
`[56]
`
`References Cited
`
`U.S. PIUENT DOCUMENI'S
`
`FOREIGN PIUENT DOCUMENTS
`
`W093/12811
`
`7/1993 WIPO.
`
`ITTHER PUBLICATIONS
`
`The Merck Index, 10th Edition, Monograph No. 4307,
`(1983).
`
`Primary Examiner-Peter O'Sullivan
`Attorney, Agent, or Firm-Steve T. Zelson, Esq.; Valeta A.
`Gregg, Esq.
`
`[57]
`
`ABSTRACT
`
`The present invention relates to a pharmaceutical prepara(cid:173)
`tion comprising glucagon and a stabilizing amount of a
`pharmaceutically acceptable ampholyte, especially an
`amino acid or dipeptide or a mixture thereof and optionally
`an excipient.
`
`The preparation is stable for extended periods of time in
`solution at room temperature.
`
`4,826,763
`5,023,088
`
`5/1989 Norris et al .................•............. 435/68
`6/1991 Wong ...................................... 424/473
`
`13 Claims, 9 Drawing Sheets
`
`CFAD Exhibit 1027
`
`1
`
`
`
`FUI-93-063. Decomposition determined by RP- HPLC. Al-A3 + Ref.
`5 mM Aminoacid
`
`~
`
`I
`
`I
`
`100
`
`80
`
`,-......
`~ 60
`...._,
`c •l""'(
`:::l 40
`~
`
`~
`
`20
`
`0
`
`0
`
`2
`Time (weeks)
`--+- Glycylglycine __..___ Histidine
`-11- Glycine
`Accelerated stability (60°C).
`Figure 1
`
`1
`
`3
`
`4
`
`-a- Reference
`
`I
`
`0 • rJJ. •
`~ = """'" a
`
`~ = t""'
`
`N
`\C
`"'
`""""'
`\C
`\C
`~
`
`g3
`m.
`""""' s,
`
`\C
`
`~
`
`Ol
`~
`Ol
`N
`~ N
`~
`~
`
`2
`
`
`
`FUl-93-063. Decomposition determined by RP-HPLC. A4-A6 + Ref.
`5 mM Aminoacid
`
`100
`
`~ • 00
`
`•
`
`'"'C i
`
`80
`
`I
`
`~~ ~
`
`~ 60 I ~ ----
`
`l-4
`::i
`P-l
`
`40
`
`20
`
`0
`
`0
`
`2
`Titne (weeks)
`---+- Glutamic acid __.,___ Leucine
`____ Aspartic acid
`Accelerated stability (60°C).
`Figure2
`
`1
`
`I
`
`----
`
`~ = :""'
`
`N
`--~
`lo-'
`~
`~
`~
`
`ga
`!!
`N
`~
`~
`
`3
`
`4
`
`-a- Reference
`
`0-.
`,_.
`
`N
`,_.
`N
`
`°" 0-.
`~ °"
`
`3
`
`
`
`Cj
`•
`rJJ. •
`FUl-93-063. Decomposition determined by RP-HPLC. A7-A9 +Ref. ~
`5 mM Aminoacid
`~
`i
`
`•
`
`..___ ___ _
`
`a
`..
`
`~ F-
`'"~ ....
`"° "° ""
`
`~ ('!) a
`
`w
`~
`"°
`
`~
`
`Ot
`~
`Ot
`N
`~ N
`~
`~
`
`100
`
`80
`
`~
`
`~ 60
`....._.,,,
`p
`
`• r-i
`J-1
`~ 40
`
`20
`
`0
`
`0
`
`-11- Alanine
`Accelerated stability (60°C).
`Figure 3
`
`1
`
`2
`Time (weeks)
`-+- Asparagine ~Valine
`
`3
`
`4
`
`-a- Reference
`
`4
`
`
`
`FUI-93-063. Decomposition determined by RP- HPLC. Al - A3 + Ref.
`10 mM Aminoacid
`
`~
`
`I
`
`I
`
`~ •
`00
`•
`
`~ ~ a
`
`~
`[:.
`N
`... 1.e
`
`""'""
`\C
`\C
`-...I
`
`I
`
`100
`
`80
`
`~ 60
`:>...
`~
`•,.....(
`$-1
`::::s
`~ 40
`
`20
`
`0
`
`0
`
`2
`Time (weeks)
`--+- Glycylglycine --.-- Histidine
`___ Glycine
`Accelerated stability (60°C).
`Figure 4
`
`1
`
`g2 a
`
`,&:;...
`
`s,
`\C
`
`3
`
`4
`
`-a- Reference
`
`01
`,.,.
`=".
`01
`N
`,.,.
`N
`~
`=".
`
`5
`
`
`
`FUI-93-063. Decomposition determined by RP-HPLC. A4 - A6 +Ref.
`10 mM Aminoacid
`
`100
`
`80
`
`* 60 I
`
`c ........
`H
`~
`~ 40
`
`20
`
`0
`
`0
`
`2
`Time (weeks)
`---+-- Glutamic acid _..._ Leucine
`-11- Aspartic acid
`Accelerated stability ( 60°C).
`Figure 5
`
`1
`
`3
`
`4
`
`--a--- Reference
`
`~ •
`00
`•
`
`~ = """'" a
`
`~ = ~
`~
`.......
`~
`~
`'I
`
`g: a
`
`(II
`
`~
`~
`
`...
`Ol
`~
`~ ...
`N
`~
`~
`
`6
`
`
`
`FUl-93-063. Decomposition determined by RP-HPLC. A7 - A9 +Ref.
`10 mM Aminoacid
`
`100
`
`80
`
`I
`
`..............
`
`I
`
`~ 60 r
`
`c . .-;
`~ 40
`
`H
`
`20
`
`0
`
`0
`
`___ Alanine
`Accelerated stability (60°C).
`Figure 6
`
`1
`
`2
`Time (weeks)
`_.__ Asparagine ~Valine
`
`3
`
`4
`
`-e- Reference
`
`d •
`rJJ
`•
`'"'O
`~
`
`"""'" a
`
`~
`:""
`N
`~
`......
`\C
`\C
`'I
`
`~
`~ a
`a-..
`s,
`
`\C
`
`Ot
`
`N
`~ N
`
`~ °' Ot
`~ °'
`
`7
`
`
`
`FUI-93-063. Decomposition determined by RP-HPLC. Al-A3 +Ref.
`20 mM Aminoacid
`
`100
`
`~
`
`so I
`
`~ 60 I
`
`H
`::1
`~
`
`40
`
`I
`
`20
`
`f-
`
`~
`
`I
`
`I
`
`I
`
`I
`
`~ •
`rJl
`•
`~ = ;-
`a
`
`~ = =-
`~
`--....
`
`~
`~
`-...!
`
`rri
`t:T'
`('t)
`i:a.
`-...!
`~
`~
`
`...
`tit
`Q'..
`tit
`...
`N
`N
`~
`Q'..
`
`3
`
`4
`
`-a- Ref ere nee
`
`I
`
`0
`
`0
`
`1
`
`2
`Time (weeks)
`--+-- Glycylglycine --..-- Histidine
`-11- Glycine
`Accelerated stability (60°C).
`Figure 7
`
`8
`
`
`
`FUl-93-063. Decomposition determined by RP-HPLC. A4-A6 + Ref.
`20 mM Amino acid
`
`100
`
`~
`
`I
`
`,..-....
`~
`'..._./ c .,....,
`H
`::l
`p.,
`
`80
`
`I
`
`60 I
`
`40
`
`20
`
`0
`
`0
`
`2
`Time (weeks)
`---+-- Glutamic acid --+--- Leucine
`--11- Aspartic acid
`Accelerated stability (60°C).
`Figure 8
`
`1
`
`3
`
`4
`
`-a- Reference
`
`0 •
`rJl
`•
`
`~ ;-a
`
`~ = t"""
`
`N
`... 'C
`
`~
`\C
`\C
`""-l
`
`~
`m.
`
`00
`~
`\C
`
`Ol
`,_.
`~
`Ol
`N
`,_.
`N
`i-(cid:173)
`~
`
`9
`
`
`
`FUl-93-063. Decomposition determined by RP-HPLC. A7-A9 +Ref.
`20 mM Aminoacid
`
`100
`
`80
`
`I
`
`............_
`
`I
`
`~ I
`
`~ 60
`'.._/ c • ....-!
`~ 40
`
`H
`
`20
`
`0
`
`0
`
`___ Alanine
`Accelerated stability (60°C).
`Figure 9
`
`1
`
`2
`Time (weeks)
`-+- Asparagine _,__ Valine
`
`3
`
`4
`
`-a- Reference
`
`Cj
`• 00
`•
`~
`~
`
`"""'" a
`
`~ = =--
`
`N
`"'\Cl
`!--'-
`\Cl
`\Cl
`-....J
`
`ga
`a
`
`\Cl
`~
`\Cl
`
`Ol
`-..
`~
`Ol
`N
`-..
`N
`~
`~
`
`10
`
`
`
`5,652,216
`
`1
`PHARMACEUTICAL PREPARATION
`
`FIELD OF THE INVENTION
`
`The present invention relates to a stabilized pharmaceu-
`tical preparation comprising glucagon.
`
`5
`
`BACKGROUND OF THE INVENTION
`
`2
`FIG. 6 shows the decomposition of glucagon stabilized
`with 10 mM Alanine, Asparagine or Valine as compared
`with the corresponding formulation without addition of
`amine acid as a function of the time,
`FIG. 7 shows the decomposition of glucagon stabilized
`with 20 mM Glycine, Glycylglycine or Histidine as com(cid:173)
`pared with the corresponding formulation without addition
`of amine acid as a function of the time,
`FIG. 8 shows the decomposition of glucagon stabilized
`with 20 mM Aspartic Acid, Glutamic Acid or Leucine as
`compared with the corresponding formulation without addi(cid:173)
`tion of amine acid as a function of the time, and
`FIG. 9 shows the decomposition of glucagon stabilized
`with 20 mM Alanine, Asparagine or Valine as compared
`with the corresponding formulation without addition of
`amine acid as a function of time.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`15
`
`Human glucagon is a polypeptide hormone secreted by
`a-cells of the pancreatic islets of Langerhans. It is a single- 10
`chain polypeptide consisting of 29 amino acid residues the
`sequence of which is published inter alia in The Merck
`Index, 10th Edition (1983), Monograph No.4307.
`Glucagon is used for the treatment of hypoglycemia in
`diabetics due to its glycogenolytic effect on the liver. Glu(cid:173)
`cagon also exerts a spasmolytic effect on smooth muscles
`which is used clinically in connection with several imaging
`procedures, especially radiology.
`Glucagon is at present marketed in the form of a lyo(cid:173)
`philized product for injection comprising lactose as the sole 20
`excipient. The lyophilisate is to be reconstituted using a
`suitable diluent.
`In the production of those conventional pharmaceutical
`preparations comprising glucagon, utmost care must be
`taken in order to avoid undesired decomposition of the
`glucagon during preparation, dispensing and lyophilization.
`Furthermore, glucagon undergoes decomposition during
`storage of the finished product at room temperature signifi(cid:173)
`cantly limiting the shelf-time of the preparation.
`Thus, there is a need for a more stable formulation of
`glucagon retaining its activity for extended periods of time
`at room temperature. Such stabilized preparations compris(cid:173)
`ing glucagon are desirable for emergency treatment of acute
`hypoglycemia rendering it possible for diabetic patients to
`carry a dose of glucagon in their hand bag enabling them(cid:173)
`selves or another person present to treat an incidence of
`hypoglycemia immediately.
`
`30
`
`BRIEF DESCRIPTION OF THE INVENTION
`
`The present invention relates to a stabilized pharmaceu(cid:173)
`tical parenteral preparation comprising glucagon.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`The invention relates to a stabilized pharmaceutical
`preparation comprising glucagon and a stabilizing amount of
`a pharmaceutically acceptable ampholyte, especially an
`amino acid or dipeptide or a mixture thereof and optionally
`25 an excipient. Such preparations retain the glucagon activity
`at room temperature, e.g. 25° C .. for extended periods of
`time.
`A pharmaceutically acceptable ampholyte to be used in
`accordance with the invention may be selected from the
`group consisting of amino acids or derivations thereof such
`as glycine, ethylglycine (sarcosine), trimethylglycine
`(betaine), alanine, [3-alanine, valine, leucine, nor-leucine,
`isoleucine, serine, threonine, aspartic acid. glutamic acid,
`hydroxyglutamic acid, lysine, hydroxylysine, ornithine,
`35 arginine, histidine, methionine, asparagine and glutarnine;
`dipeptides such as glycylglycine; pharmaceutically accept(cid:173)
`able sulfonic acids or derivatives thereof such as taurine;
`creatinine, and ethylenediarninetetraacetic acid (EDTA).
`An amino acid to be used in accordance with the present
`40 invention is preferably a naturally occurring alpha amino
`acid. Such amino acids may be 1 or d amino acids or a
`mixture thereof.
`Preferably glycine, glycylglycine, histidine or a mixture
`of two or more of these is used.
`A pharmaceutical preparation of the invention in lyo(cid:173)
`philized form preferably also comprises an excipient, e.g. for
`facilitating the lyophilization and rapid and complete redis(cid:173)
`solution thereof when reconstituting the preparation before
`use.
`An excipient may be selected from disaccharides such as
`lactose, trehalose, and sucrose, sugar alcohols such as sor(cid:173)
`bitol or mannitol, polysaccharides such as the polymers
`commercialized as Dextran® products such as Dextran® 40,
`Dextran® 70 or Dextran® 75, and Ficoll® and polyvalent
`alcohols such as polyethylene glycol or polyvinyl alcohol or
`a combination of two or more of these.
`The excipient is preferably present in an amount of from
`10 to 600 rnicromoles per mg glucagon giving an optimum
`stabilization. The excipient preferred according to the inven(cid:173)
`tion is lactose.
`In a further aspect of the invention, the pharmaceutical
`preparation of the invention is in the form of a stabilized
`solution of glucagon.
`In order to obtain the desired stabilization, the stabilizing
`amino acid or dipeptide may be present in an amount from
`0.01 to 50 micromoles per mg glucagon. The amount of
`
`50
`
`The invention is further explained with reference to the 45
`drawings in which
`FIG. 1 shows the decomposition of glucagon stabilized
`with 5 mM Glycine, Glycylglycine or Histidine as compared
`with the corresponding formulation without addition of
`amine acid as a function of the time,
`FIG. 2 shows the decomposition of glucagon stabilized
`with 5 mM Aspartic Acid, Glutamic Acid or Leucine as
`compared with the corresponding formulation without addi(cid:173)
`tion of amine acid as a function of the time,
`FIG. 3 shows the decomposition of glucagon stabilized
`with 5 mM Alanine, Asparagine or Valine as compared with
`the corresponding formulation without addition of amine
`acid as a function of the time,
`FIG. 4 shows the decomposition of glucagon stabilized 60
`with 10 mM Glycine, Glycylglycine or Histidine as com(cid:173)
`pared with the corresponding formulation without addition
`of amine acid as a function of the time,
`FIG. 5 shows the decomposition of glucagon stabilized
`with 10 mM Aspartic Acid, Glutamic Acid or Leucine as 65
`compared with the corresponding formulation without addi(cid:173)
`tion of amine acid as a function of the time,
`
`55
`
`11
`
`
`
`5,652,216
`
`4
`Prefreezing at -45° C. for 3-5 hours,
`primary drying from -45° C. to 20° C. and a pressure of
`0.1 hPa for 29 hours, and
`Secondary drying at 20° C. (and full vacuum) for 4 hours
`using a Heto CDS apparatus.
`
`EXAMPLES
`
`EXAMPLE 1
`
`Reference:
`
`Glucagon
`Lactose USP/Ph Eur
`lNHCl
`Test formulations:
`
`Glucagon
`Lactose USP/Ph Eur
`Ampholyte
`HCl/NaOH
`
`1.1 mg
`107 mg
`adpH2.8
`
`1.1 mg
`107 mg
`5, 10, 20mM
`ad pH 2.8
`
`The ampholytes tested were:
`
`F=ulation
`
`Glycine
`Glycylglycine
`Histidine
`Aspartic acid
`Glutamic acid
`Leucine
`Alanine
`Asparagine
`Valine
`
`Al
`A2
`A3
`A4
`A5
`A6
`A7
`AB
`A9
`
`45
`
`3
`stabilizing amino acid or dipeptide present per dose of
`pharmaceutical preparation comprising glucagon is accord(cid:173)
`ing to the invention from 0.1 to 50 micromoles.
`A preferred preparation of the invention comprises
`glycine, histidine or glycylglycine in an amount about 10 5
`micromoles per mg glucagon. The amount of glycine, his(cid:173)
`tidine or glycylglycine is preferably about 10 micromoles
`per dose.
`The pH of the preparations according to the invention in
`the form of a solution is preferably adjusted to the interval 10
`Preparation of formulations comprising glucagon, lactose
`1-7. Preferably, the pH is adjusted to the interval 2-4, and
`and an amino acid or a dipeptide.
`most preferred to about 2.8.
`In an analogous manner as described above formulations
`The invention also relates to a method for the preparation
`were prepared from glucagon, lactose and an amino acid or
`of a pharmaceutical preparation comprising glucagon and a
`15 a dipeptide.
`stabilizing amount of a pharmaceutically acceptable
`ampholyte wherein glucagon is dissolved in a solution of the
`As reference was used a formulation comprising glucagon
`and lactose prepared as described above.
`ampholyte and optional excipient and lyophilized, option-
`Formulations having the following compositions per vial
`ally after sterile filtration.
`The dissolution of the glucagon is preferably carried out 20 were prepared:
`at a temperature of from 4 ° to 8° C.
`A most preferred preparation according to the invention
`comprises glucagon, lactose or mannitol as excipient and
`glycine, histidine or glycylglycine or a mixture of two or
`more of these as a stabilizing agent. Such a preparation 25
`shows a buffer effect at pH about 2.8 giving a minimum for
`the rate of decomposition of glucagon. Thus, the invention
`enables the formulation of a preparation in which glucagon
`is stable at room temperature.
`The amino acid sequence of human glucagon is identical 30
`to the amino acid sequence of porcine and bovine glucagon.
`Hence, glucagon may be isolated by conventional extraction
`form porcine or bovine pancreatic glands. In the alternative,
`glucagon may be prepared by fully or partially chemical
`synthesis or by recombinant techniques, e.g. as disclosed in 35
`U.S. Pat. No. 4,826,763.
`The pharmaceutical preparations of the invention may be
`formulated for administration in any suitable way, e.g. by
`parenteral or oral administration or administration to a
`mucosal membrane, e.g. nasal administration. The pharma- 40
`ceutical preparation may be preserved in the form of a dose
`comprised in a vial or cartridge or any other suitable
`container.
`The invention is explained more in detail in the below
`Examples which illustrate the invention. They are not to be
`considered as limiting the scope of the invention being
`defined by the appended claims.
`
`MATERIALS AND METHODS
`
`50
`
`55
`
`Preparation and lyophilization of formulations compris(cid:173)
`ing glucagon was carried out using the following procedure:
`Glucagon: Recombinant glucagon prepared in saccharo(cid:173)
`myces cerivisiae as disclosed in U.S. Pat. No. 4.826,763.
`10.7 g lactose and the amino acid or dipeptide was
`dissolved in approximately 75 ml of distilled water and the
`pH was adjusted to 2.6-3.0 using lN hydrochloric acid/
`sodium hydroxide. The volume was adjusted to 100 ml and
`the solution was cooled to 4°-8° C. in a refrigerator.
`110 mg glucagon was dissolved in the solution and the pH
`adjusted to pH 2.6-3.0 using lN hydrochloric acid
`(maintaining the solution at 4°-8° C.).
`The solution was sterile filtered through a 0.2 µm filter
`(using a 50 cc syringe) and divided into vials, 1 ml in each. 65
`The solution was lyophilized in total 36 hours, using the
`following procedure:
`
`60
`
`The test formulations were incubated at 60° C .• for a total
`period of 4 weeks. The degradation of the formulations were
`measured weekly by reverse phase HPLC. The results are
`shown in Table 1 and F1GS. 1-9.
`The results show that a very pronounced stabilization of
`glucagon is obtained by adding stabilizing agent in accor(cid:173)
`dance with the invention.
`
`TABLE 1
`
`Week
`
`0
`
`2
`
`Cone.AA
`(mM)
`
`Glycine Glycylglycine Histidine
`RPC
`RPC
`RPC
`%
`%
`%
`
`Reference
`RPC
`%
`
`5
`10
`20
`5
`10
`20
`5
`10
`20
`
`98.00
`g].90
`g].80
`g].10
`92.40
`93.40
`91.60
`9450
`91.60
`
`g].90
`g]9Q
`g]9Q
`96.40
`g].OO
`96.10
`95.20
`93.70
`90.80
`
`g].60
`
`55.80
`
`29.30
`
`g].90
`98.00
`g].90
`94.70
`g].20
`94.00
`91.20
`94.40
`90.90
`
`12
`
`
`
`5
`
`TABLE 1-continued
`
`5,652,216
`
`6
`
`Week
`
`Cone. AA
`(mM)
`
`Glycine Glycylglycine Histidine
`RFC
`RFC
`RPC
`%
`%
`%
`
`Reference
`RPC
`%
`
`3
`
`4
`
`5
`10
`20
`5
`10
`20
`
`94.80
`93.20
`91.80
`84.60
`93.30
`85.40
`
`95.30
`94.30
`93.60
`91.70
`87.50
`90.10
`
`88.10
`87.60
`94.10
`90.90
`93.30
`94.20
`
`25.30
`
`26.90
`
`TABLE2
`
`Aspartic
`acid
`RFC
`%
`
`Glutamic
`acid
`RFC
`%
`
`Cone.AA
`(mM)
`
`Leucine Reference
`RFC
`RPC
`%
`%
`
`5
`10
`20
`5
`10
`20
`5
`10
`20
`5
`10
`20
`5
`10
`20
`
`97.70
`97.80
`97.70
`72.70
`95.60
`94.80
`65.80
`93.30
`91.90
`
`57.80
`88.60
`86.50
`
`97.10
`97.70
`97.70
`60.30
`95.80
`94.90
`48.10
`94.10
`93.30
`
`97.70
`97.60
`97.60
`81.00
`95.90
`95.40
`74.70
`94.20
`93.60
`
`97.80
`
`45.80
`
`33.50
`
`32.70
`90.50
`88.20
`
`67.00
`91.00
`89.10
`
`19.20
`
`Week
`
`0
`
`2
`
`3
`
`4
`
`TABLE3
`
`20
`
`25
`
`30
`
`35
`
`Valine Reference
`Alanine
`Asparagine
`RPC
`RFC
`RPC
`RFC
`Cone.AA
`%
`%
`(mM)
`%
`%
`Week
`~~~~~~~~~~~~~~~~~~~~~- 40
`97.40
`97.00
`97.30
`97.30
`5
`0
`97.60
`97.50
`97.60
`10
`97.30
`97.30
`97.60
`20
`85.90
`93.60
`5
`93.90
`96.10
`96.30
`96.20
`10
`95.80
`95.90
`96.00
`20
`5
`10
`20
`5
`10
`20
`5
`10
`20
`
`2
`
`3
`
`4
`
`68.10
`
`44.00
`
`35.90
`
`90.90
`94.30
`93.20
`89.90
`93.40
`92.30
`
`91.90
`94.40
`93.80
`91.40
`93.60
`93.00
`
`80.00
`94.00
`92.80
`75.40
`93.50
`92.10
`
`We claim:
`1. A pharmaceutical preparation comprising glucagon and
`a stabilizing amount of a pharmaceutically acceptable
`ampholyte, wherein the ampholyte is selected from the
`5 group consisting of glycine. ethylglycine, glycylglycine.
`trimethylglycine, alanine, ~-alanine. valine, leucine, nor(cid:173)
`leucine, isoleucine, serine. threonine. aspartic acid. glutamic
`acid, hydroxyglutamic acid. lysine, hydroxylysine,
`10 ornithine, arginine, methionine, asparagine, glutamine,
`taurine. creatinine, and ethylenediaminetetraacetic acid.
`2. The pharmaceutical preparation according to claim l,
`wherein the ampholyte is a pharmaceutically acceptable
`naturally occurring amino acid or a dipeptide or a mixture
`15 thereof.
`3. The pharmaceutical preparation according to claim 2,
`wherein the amino acid or dipeptide is an amino acid
`selected from the group consisting of glycine, glycylglycine,
`or a mixture thereof.
`4. The pharmaceutical preparation according to claim 1.
`further comprising a pharmaceutical acceptable excipient.
`5. The pharmaceutical preparation according to claim 4,
`wherein the excipient is one or more compounds selected
`from the group consisting of disaccharides, sugar alcohols,
`polysaccharides, and polyvalent alcohols.
`6. The pharmaceutical preparation according to claim 4,
`wherein the excipient is present in an amount 10 to 600
`micromoles per mg glucagon.
`7. The pharmaceutical preparation according to claim 3,
`wherein the glycine or glycylglycine is present in an mount
`from 0.01 to 50 micromoles per mg glucagon.
`8. The pharmaceutical preparation according to claim l,
`wherein the pH is adjusted to a value in the interval 2-7.
`9. A method for the preparation of a pharmaceutical
`preparation according to claim 1, comprising (a) dissolving
`glucagon in a solution which comprises the ampholyte and
`optionally an excipient, and (b) lyophilizing the pharmaceu-
`tical preparation.
`10. The pharmaceutical preparation according to claim 5,
`wherein the disaccharide one of lactose, trehalose, or
`sucrose, the sugar alcohol is sorbitol or mannitol, the
`polysaccharide is Dextran or Ficoll, and the polyvalent
`alcohol is polyethylene glycol or polyvinyl alcohol.
`11. The pharmaceutical preparation according to claim 7,
`wherein the glycine. or glycylglycine is present in an amount
`about 10 rnicromoles per mg glucagon.
`12. The pharmaceutical preparation according to claim 8,
`50 wherein the pH is adjusted to between 2.3 to 3.8.
`13. The pharmaceutical preparation according to claim
`12, wherein the pH is adjusted to 2.8 ..
`
`45
`
`* * * * *
`
`13