United States Patent
`
`[191
`
`Kornfelt et al.
`
`[11] Patent Number:
`
`5,652,216
`
`[451 Date of Patent:
`
`Jul. 29, 199'?
`
`USOOSGSZZIEA
`
`[54] PHARMACEUTICAL PREPARATION
`
`5,059,537 1011991 Yamamoto .............................. 514112
`
`[75]
`
`Inventors: Trocls Komfclt, Virum; Hmrik
`Raslnusaen, Copenhagen; Flemming
`Steel: Jensen. Allemacd. all of Denmark
`
`[73] Assigncc: Novo Nordisk AIS. Bagsvacrd.
`Dcmnarlc
`
`[21] App]. No; 275.855
`[22] Filed:
`Jul. 15, 1994
`
`FOREIGN P.P.TENT DOCUMEPWS
`WD93fl22Hl
`"H1993 WIFO .
`
`OTHER PUBLICATIONS
`
`Thu Marc!-1 Index.
`(1983).
`
`lfllh Edifiun. Monograph No. 4307.
`
`Primary Etam:'r|er——Pct:.l‘ O'SuIlivan
`Artomegg Agem‘, or F.I‘rm«—+Steve T. Zelson. Esq.; Valun A.
`Grcgg. Esq.
`
`[30]
`
`Foreign Applicafiun Priority Data
`
`[57]
`
`ABSTRACT
`
`May 25, 1994
`
`[DR]
`
`Denmfirk .....
`
`A6‘.ll( 33:25
`Int. cl.‘
`[51]
`51I=U12
` [52] US. Cl.
`[53] Field nfsearch .......................................... 514112
`
`0590194
`
`Thr: prcscnt i.uv¢ntion relates to a phanmwnical prcpara-
`Lion (:Dm1:n'Si.I:Ig glucagon and a stabilizing amount of a
`phannaccutically acceptable: arnpholyta. especially an
`antdnc acid or dipczptidc or a m.i:-trun: thcrncuf and optionally
`an cxcipient.
`
`The prcparaxion is stable for cxumded pcfiods of time in
`snlnlien at room tcrnpcranmr.
`
`I3 Claims, 9 Drawing Shuts
`
`[56]
`
`Refemnces Cited
`U.S. I'.PII'EN'l‘ DOCUMENTS
`
`435ili8
`.'5fI9E9 Nmxisct :11.
`4.826.763
`4111473
`.
`5.023.083 M991 Wong _....~..
`
`EXHIBIT
`
`/673
`
`CFAD Exhibit 1090
`CFAD Exhibit 1090
`CFAD v. NPS
`CFAD V. NPS
`IPR2015-01093
`IPR2015—O1093
`
`CFAD Exhibit 1027
`
`

`
`FUI-93-063. Decomposition determined by RP-HPLC. Al-A3 +Ref.
`5 mM Aminoacid
`
`100
`
`~
`
`~
`
`so I
`
`~ 60 I
`
`\.-(
`
`;:j 40
`~
`
`20
`
`0
`
`0
`
`2
`Time (weeks)
`-.-- Glycylglycine ~ Histidine
`____ Glycine
`Accelerated stability (60°C).
`Figure 1
`
`1
`
`3
`
`4
`
`-a- Reference
`
`2
`
`I
`
`Cj
`
`• \fJ. •
`~
`[
`
`~
`~
`'""' ~
`
`-...J
`
`~ a
`'""' s,
`"°
`
`tll
`-..
`Q'\
`tll
`N
`-..
`N
`~
`Q'\
`
`

`
`FUl-93-063. Decomposition determined by RP-HPLC. A4-A6 + Ref.
`5 mM Aminoacid
`100
`
`80
`
`I
`
`""~ ~
`
`~ 60 I ~ ----
`
`H
`:::1
`P-<
`
`40
`
`20
`
`0
`
`0
`
`z
`Titne (weeks)
`--+- Glutamic acid -+-- Leucine
`- 11- Aspartic acid
`Accelerated stability (60°C).
`Figure2
`
`1
`
`3
`
`I
`
`3
`
`4
`
`-a- Reference
`
`e • r:J1 • i
`
`~
`="""
`--~
`......
`'° \C
`
`"'-l
`
`~
`!
`N
`~
`'°
`
`01
`
`"' "" ~
`"' N
`~ ""'
`
`

`
`FUl-93-063. Decomposition determined by RP-HPLC. A7-A9 + Ref.
`5 mM Alninoacid
`
`100
`
`80
`
`I
`
`•
`
`,-.....
`~ 60
`
`'-" . e ~
`
`~ 40
`
`20
`
`0
`
`0
`
`___ Alanine
`Accelerated stability (60°C).
`Figure 3
`
`1
`
`2
`Time (weeks)
`-+- Asparagine --...- Valine
`
`3
`
`4
`
`-a- Reference
`
`4
`
`~ • 00
`•
`~
`[
`
`~
`"~
`.....
`~
`
`~
`!l
`w
`~
`\C
`
`...
`tll
`Q\
`~ ...
`N
`~
`Q\
`
`

`
`FUI-93-063. Decomposition determined by RP-HPLC. Al - A3 +Ref.
`10 mM Aminoacid
`
`100
`
`~
`~
`
`so I
`
`* 60 I
`
`>-.
`.......
`• .-<
`H
`~ 40
`
`20
`
`0
`
`0
`
`2
`Time (weeks)
`_._ Glycylglycine ~ Histidine
`-11- Glycine
`Accelerated stability (60°C).
`Figure 4
`
`1
`
`3
`
`4
`
`-a- Reference
`
`I
`
`cj
`• rri. •
`
`~ ....... a
`
`~
`
`~
`~
`.....
`~
`"-..!
`
`g3 a .a;:.
`
`~
`\Cl
`
`th
`"' ~
`th
`N
`"' N
`li-6
`~
`
`5
`
`

`
`FU! -93- 063. Decomposition determined by RP - HPLC. A 4 - A6 + Ref.
`10 mM Aminoacid
`
`100
`
`80
`
`i 60 r
`
`~ 40
`
`20
`
`d
`00
`•
`
`~ ~ a
`
`~ = ~
`~~
`.....
`::g
`
`--.J
`
`gl
`!
`s,
`\C
`
`tit
`
`0
`
`0
`
`2
`Time (weeks)
`-+- Glutamic acid -+- Leucine
`___ Aspartic acid
`Accelerated stability ( 60°C).
`Figure 5
`
`1
`
`6
`
`3
`
`4
`
`-a-- Ref ere nee
`
`tll
`~
`
`~
`
`~ ~ N
`
`~
`~
`
`

`
`FUl-93-063. Decomposition determined by RP-HPLC. A? - A9 + Ref.
`10 mM Aminoacid
`
`100
`
`80
`
`~ 60 1
`.£' H
`~ 40
`
`20
`
`0
`
`0
`
`-11- Alanine
`Accelerated stability (60°C).
`Figure 6
`
`•
`
`•
`
`1
`
`2
`Time (weeks)
`__.__ Asparagine ~Valine
`
`3
`
`4
`
`-a- Reference
`
`7
`
`Cj
`•
`'CFJ.
`•
`
`~ ~ a
`
`~
`r""'
`N
`
`~ ,....
`~
`
`ga
`!'tl a
`s,
`
`Q'>,
`
`\0
`
`Ol
`
`"' °" ~
`"' N
`~ °"
`
`

`
`FUI-93-063. Decomposition determined by RP-HPLC. A1-A1 +Ref.
`20 mM Aminoacid
`
`100
`
`,,,.-..
`~ ...._,
`:>--,
`-;...>
`•...-4
`H
`;::i
`~
`
`80
`
`60
`
`40
`
`20
`
`•
`~
`
`d • 00
`~ ;-a
`
`~
`=-'
`--~
`~
`'I
`
`1--1
`
`gJ
`"' ll
`
`-..J
`~
`\C
`
`0
`
`0
`
`2
`Time (weeks)
`-+- Glycylglycine --..-- Histidine
`--11- Glycine
`Accelerated stability (60°C).
`Figure 7
`
`1
`
`8
`
`3
`
`4
`
`-a- Ref ere nee
`
`Ol
`
`"' °" ~
`~ °"
`
`~
`
`

`
`d
`FUl-93-063. Decomposition determined by RP-HPLC. A4-A6 + Ref. ~
`20 mM Aminoacid
`~
`[
`
`100
`
`~
`
`I
`
`~
`
`~
`~
`"
`~
`-..:i
`
`~
`
`80
`
`I
`
`~ 60 I
`
`C>
`• >-i
`;...,
`;::l
`~
`
`40
`
`20
`
`0
`
`0
`
`1
`
`2
`Time (weeks)
`-+- Glutamic acid -+- Leucine
`____ Aspartic acid
`Accelerated stability (60°C).
`Figure 8
`
`9
`
`3
`
`4
`
`-a- Ref ere nee
`
`~
`!
`00
`s,
`""
`
`...
`01
`~
`01
`...
`N
`N
`ii(cid:173)
`~
`
`

`
`FUl-93-063. Decomposition determined by RP-HPLC. A7-A9 + Ref.
`20 mM Aminoacid
`
`100
`
`'-....
`
`I
`
`,-,.
`~ ,,.._...
`.Q
`.......
`H &!
`
`80
`
`I
`
`60 I
`
`40
`
`20
`
`0
`
`0
`
`___ Alanine
`Accelerated stability (60°C).
`Figure 9
`
`1
`
`2
`Time (weeks)
`-+-Asparagine ~Valine
`
`3
`
`4
`
`-a- Reference
`
`10
`
`•
`~
`~
`
`d • \./').
`"""'" a
`
`~ = =-
`~
`'""" ~ ...;i
`
`~
`m.
`
`l,c
`~
`l,c
`
`Ol
`"' 0-..
`Ol
`N
`'N
`~
`0-..
`
`

`
`5,652,216
`
`5
`
`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, Glutarnic 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
`15 with 20 mM Alanine, Asparagine or Valine as compared
`with the corresponding formulation without addition of
`amine acid as a function of time.
`
`DErAIIBD DESCRIPTION OF THE
`INVENITON
`
`1
`PHARMACEUTICAL PREPARATION
`
`FIELD OF THE INVENTION
`
`The present invention relates to a stabilized pharmaceu-
`tical preparation comprising glucagon.
`
`BACKGROUND OF THE INVENTION
`
`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
`
`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 25
`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 35
`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
`
`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
`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, ~alanine, valine, leucine, nor-leucine,
`isoleucine, serine. threonine, aspartic acid, glutamic acid,
`hydroxyglutamic acid, lysine, hydroxylysine, omithine,
`arginine, histidine, methionine, asparagine and glutarnine;
`dipeptides such as glycylglycine; pharmaceutically accept-
`able sulfonic acids or derivatives thereof such as taurine;
`creatinine, and ethylenediaminetetraacetic 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 micromoles per mg glucagon giving an optimum
`60 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
`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
`
`Preparation of formulations comprising glucagon, lactose
`and an amino acid or a dipeptide.
`In an analogous manner as descn"bed above formulations
`were prepared from glucagon, lactose and an amino acid or
`15 a dipeptide.
`As reference was used a formulation comprising glucagon
`and lactose prepared as described above.
`Formulations having the following compositions per vial
`20 were prepared:
`
`Reference:
`
`Glucagon
`Lactose USP/Ph Eur
`INHCI
`Test funnulations:
`
`Glucagon
`Lactose USP/Ph Eur
`Ampholyte
`HCl/NaOH
`
`1.1 mg
`I!J1 mg
`ad pH 2.8
`
`1.1 mg
`I!Jl mg
`5, 10, 20mM
`ad pH 2.8
`
`The ampholytes tested were:
`
`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
`1-7. Preferably, the pH is adjusted to the interval 2-4, and
`most preferred to about 2.8.
`The invention also relates to a method for the preparation
`of a pharmaceutical preparation comprising glucagon and a
`stabilizing amount of a pharmaceutically acceptable
`ampholyte wherein glucagon is dissolved in a solution of the
`ampholyte and optional excipient and lyophilized, option(cid:173)
`ally after sterile filtration.
`The dissolution of the glucagon is preferably carried out
`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.
`Formulation
`The pharmaceutical preparations of the invention may be
`Al
`Glycine
`Glycylglycine
`A2
`formulated for administration in any suitable way, e.g. by
`A3
`Histidine
`parenteral or oral administration or administration to a
`Aspartic acid
`A4
`mucosa! membrane, e.g. nasal administration. The pharma- 40
`Glntamic acid
`A5
`ceutical preparation may be preserved in the form of a dose
`A6
`Leucine
`A7
`Alanine
`comprised in a vial or cartridge or any other suitable
`Asparagine
`A8
`container.
`Valine
`A9
`The invention is explained more in detail in the below 45 - - - - - - - - - - - - - - - - - - - - (cid:173)
`Examples which illustrate the invention. They are not to be
`The test formulations were incubated at 60° C., for a total
`considered as limiting the scope of the invention being
`period of 4 weeks. Tue degradation of the formulations were
`defined by the appended claims.
`measured weekly by reverse phase HPLC. The results are
`shown in Table 1 and FIGS. 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.
`
`MATERIALS AND METHODS
`
`50
`
`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 1N 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:
`
`55
`
`60
`
`12
`
`TABLE 1
`
`Week
`
`0
`
`2
`
`Cone.AA
`(mM)
`
`Glycine Glycylglycinc Histidine Reference
`RPC
`RPC
`RPC
`RPC
`%
`%
`%
`%
`
`5
`10
`20
`5
`10
`20
`5
`10
`20
`
`98.00
`97.90
`97.80
`97.10
`92.40
`93.40
`91.60
`9450
`91.60
`
`9790
`9790
`9790
`96.40
`97.00
`96.10
`95.20
`93.70
`90.80
`
`97SXJ
`98.00
`97.90
`94.70
`97.20
`94.00
`91.20
`94.40
`90.90
`
`97.f!J
`
`55.80
`
`2930
`
`

`
`5,652,216
`
`6
`
`5
`
`TABLE !-continued
`
`Cone.AA
`(mM)
`
`Week
`
`Glycine Glycylglycine Histidine
`RPC
`RPC
`RPC
`%
`%
`%
`
`Reference
`RPC
`%
`
`3
`
`4
`
`5
`IO
`20
`5
`10
`20
`
`94.80
`93.20
`91.80
`84.60
`93.30
`85.40
`
`9530
`9430
`93.60
`91.70
`87.50
`90.10
`
`88.IO
`87.60
`94.IO
`90.90
`93.30
`94.20
`
`25.30
`
`26.90
`
`TABLE2
`
`Aspartic
`acid
`RPC
`%
`
`Glutamic
`acid
`RPC
`%
`
`97.70
`97.80
`97.70
`72.70
`95.60
`94.80
`65.80
`93.30
`91.90
`
`97.10
`97.70
`97.70
`60.30
`95.80
`94.90
`48.10
`94.10
`93.30
`
`Leucine Reference
`RFC
`RPC
`%
`%
`
`97.80
`
`45.80
`
`33.50
`
`97.70
`97.flJ
`97.flJ
`81.00
`95.90
`95.40
`74.70
`94.20
`93.flJ
`
`57.80
`88.60
`8650
`
`32.70
`90.50
`88.20
`
`67.00
`91.00
`89.10
`
`I9.20
`
`Cone.AA
`(mM)
`
`5
`10
`20
`5
`IO
`20
`5
`10
`20
`5
`IO
`20
`5
`IO
`20
`
`TABLE3
`
`Cone.AA
`(mM)
`
`Alanine
`RPC
`%
`
`Asparagine
`RPC
`%
`
`Valine Reference
`RPC
`RPC
`%
`%
`
`5
`IO
`20
`5
`10
`20
`5
`IO
`20
`5
`10
`20
`5
`10
`20
`
`97.30
`97.50
`97.30
`93.60
`96.30
`95.90
`
`90.90
`9430
`93.20
`89.90
`93.40
`92.30
`
`97.30
`97.60
`97.60
`93.90
`96.20
`96.00
`
`91.90
`94.40
`93.80
`91.40
`93.60
`93.00
`
`97.00
`
`68.IO
`
`44.00
`
`35.90
`
`97.40
`97.60
`97.30
`85.90
`96.IO
`95.80
`
`80.00
`94.00
`92.80
`75.40
`93.50
`92.10
`
`Weck
`
`0
`
`2
`
`3
`
`4
`
`Week
`
`0
`
`2
`
`3
`
`4
`
`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. [3-alanine, valine, leucine, nor-
`leucine, isoleucine, serine, threonine, aspartic acid. glutamic
`acid, hydroxyglutamic acid, lysine, hydroxylysine.
`IO ornithine, arginine. methionine. asparagine, glutamine,
`taurine, creatinine, and ethylenediaminetetraacetic acid.
`2. The pharmaceutical preparation according to claim 1.
`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 l,
`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 1,
`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 micromoles per mg glucagon.
`12. The pharmaceutical preparation according to claim 8,
`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 ..
`
`40
`
`45
`
`50
`
`20
`
`25
`
`30
`
`35
`
`* * * * *
`
`13