Note: Descriptions are shown in the official language in which they were submitted.
17~3
-- 2 --
The invention relates -to insu:l ins of the formula I
.
Gly (A1
Ile Yal ( B2 )
Yal Asn
~lu Gln
Gln Hi.s
, ~--Cys - IJeu
, Cys--S_ S_ Cys
10 Thr Giy
Se r . Se r
Ile His
: i--Cys Leu
Ser Yal
15I,eu Glu
Tyr Ala
G;n Leu
I.eu Tyr -
Glu Leu
~0.Asn ~al
.
Tyr / S Cys .
- Cys - - S Gly
Y ~A2 1 ) . Glu
Arg
. C;ly
Phe
Phe
Tyr
Thr
3 Pro
Lys ~B29)
'
~.
~ :~ 7 3 ~3 ~3
-- 3
~ part from ~ small modification, the new compounds
have the am~noacid sequence of human insulin
Insulins in which the Bl-phenylalanine is missing
are already known, but desBl-phenylalanine-human insulin
(des-PheBl-human insulin) has not hitherto been described.
Since human insulin can now be prepared in several
ways as a starting material for experimental purposes,
it has for the first time been possible to recognize the
advantageous properties ofdes-PheBl-human insulin
(formula I, in which X = Thr and Y = Asn).
The solubility of the compounds accor~ing to the
invention in which X = Thr is/ quite generally, con$ider-
ably greater than that of compounds which still contain
PheBl, so that highly concentrated solutions such as are
15 required for insulin pumps can be prepared
Moreover, these compounds have advantages in the
immunological field inasmuch as they glve rise to a lesser
degree of formation of antibodies. As is known, the
production of antibodies, which can even be induced by
20 homologous insulin, plays a critical role in long-term
treatment with insulins. Thus, for example, it has
been observed that human insulin is al)so capable of pro-
ducing antibodies in humans.
A sensitive test model for the production of anti-
25 bodies is the goat, which very rapidly forms antibodiesagainst swine insulin and against human insulin in the
presence of Freund t S adjuvan-t. According to Diabetes
27 (197~), page 14, figure 4, the bonding capacity of
antibodies against the heterologous des-PheBl-swine
1 ~338
4 _
insulin is lower and antibody formation takes place more
slowly than in the case of non-modified swine insulin or
even in the case of homologous sheep insulin. In
this test model, -the compounds according to the invention,
above all those in which X = OH, cause an even slower
antibody formation, and the bonding capacity is reduced
by a further 15 %, compared with that of des-PheBl-swine
insulin
In a similar manner to human insulin, the compounds
10 according to the invention also exhibit the advantage of
a better glucose tolerance, compared with swine insulin,
in the treatment of humans. This property is evidently
unchanged by spli~ting off PheBl.
- MoreoverJ des-Phe31-human insulin (I in which
15 X = Thr), in particular, has an action which starts very
rapidly and is long-lasting.
If ThrB~O ~I in which X - OH~ is also split off
enzymatically from the human insulin, in addition -to
PheBl, all the advantageous properties mentioned for
20 des-PheBl-human insulin remain. The same applies to
the compound which carries, instead of AsnA21, an addi-
tional carboxyl group with AspA21 (I in which X = OH and
Y = Asp). In this case, an even further increase in
the solubility, which manifests i-tself, above all, in
25 the neutral and weakly alkaline range, occurs
The particular economic advantage of the
compoun~ I in which X _ OH is that human insulin, which
at present is still accessible only with grea-t difficulty,
does no-t have to be used in the preparation, but -that
, .~
3388
swine insulin can be used. Swine insulin differs from human
insulin only in the aminoacid B30 (Ala instead oE Thr), and,
after this aminoacid has been split off, becomes identical to des-
Thr -human insulin.
The splitting off of Ala 30 from swine insulin has
already been described in U.S. Patent Specification
3,364,116. However, in respect of its immunological properties,
des-Ala 30-swine insulin differs only slightly from swine
insulin. In contrast, the additional removal of PheBl causes
structural changes in the insulin molecule which are responsible
for the advantageous biological properties.
The invention also relates to a process for the pre-
paration of the insulins of the formula I. This comprises a) con-
verting human insulin, desamidoA21-human insulin, des-ThrB30-
human insulin, desamidoA21-des-ThrB30-human insulin, des-Ala 30-
swine insulin or desamidoA21-des-AlaB30-swine insulin into the
N Al-NEB29-bis-Boc compound and subjecting this to Edman
deyradation, b) treating des-PheBl-human insulin or des-PheB30
swine insulin with acid, or c) treating a compound of the formula
I in which X denotes Ala with carboxypeptidase A.
To prepare the compounds according to the invention
in which X = Thr and Y = Asn, human insulin, which is converted
into the corresponding des-PheBl-compound by a procedure analo-
gous to that in German Patent 2,005,658, is used as the starting
material.
To prepare the compound I in which ;~ =OH and
-- 6 --
Y = Asn, des-Ala~0-swine insulin, which is prepared,
for example, according to Hoppe-Seyler's Z. Physiol.
Chem 359 {1978), page 799t by enzymatic splitting off
of AlaB30, is used as the starting ma-terial, and PheBl
is split off by a procedure analogous to that in German
Patent 2,005,658. It is also possible to reverse the
sequence of the steps and first to split off PheBl
chemically and then to split off AlaB30 enzymatically.
If Y is to be Asp, the starting compounds are
first treated with aqueous acid at pH 2 - 3 and purified
by ion exchange chromatography, in a known manner,
PheBl or AlaB30 are then re~oved as described above.
It is also possible to carr~ out the acid treat-
ment after PheBl and, if appropriate, AlaB30 have been
split o~f. This has the advantage that a very uni-
form product appears in the final purification by ion
exchangerchromatography. 1 - 2 purification operations
are thus su ficient, whilst a further purification step
is usually required in the reverse process.
The new human insulin analbgues are used for the
treatment of diabetes mellitus and, because of their
very good solubility at pH 7 - 7.5, can also be used in
the insulin pump in a concentration of about 10 %.
The dose depends on the condition of the pa-tient and
approximately corresponds to tha-t of swine insulin, but
can be considerably lower in the case of patients with
an increased level of antibodies.
The invention also relates to a pharmaceutical
formulation of the compounds of the formula I which
,
~ 1 ~33~3~
contains a compound of the formula I in dissolved or
amorphous form, in addi-tion to a compound of the formula
I, in crystalline form, which howeve~, addltionally con-
tains phenylalanine ln Bl.
The new human insulin analogues can, of course,
also be used together with compounds which still
contain PheBl. They are then in the dissolved or
amorphous form, whilst the compounds containing PheBl
are employed in crystalline form. Two-phase insulins
in which a rapid onset of action is combined with a
long period of action are known per se. They are des-
cribed, for.example, in German Offenle~gungsschrift
2,4~9,515 (HOE 74/F 377) and German Offenlegungsschrift
2,256,215 ~HOE 72/F 348). The new ~ormulations accor-
ding to the invention are prepared analogously to the~nstructions given for swine insulin in these specifica-
tions. The invention also relates to another formula~
tion form in which a compound o~ the ~ormula I in which
Y = Asp is present in solution in addition to the corres-
po~ding compound in which Y = Asn, in crystalline form.
This formulation form can be prepared by a procedure
analogGus to that in German Offenlegungsschrift
2,803,9~6.
EXAMPLES
The intermediate and end produc-ts were characteri-
zed by paper electrophoresis and gel electrophoresis a-t
pH 2 and 8.3, and by aminoacid analysis, thin layer chroma-
tography and HPLC, in the known systems.
Example 1:
~ ~ 73388
_ ~ _
Des-PheBl-h~lman insulin
a) 1 g of human insulin is taken up in 70 ml of
80 % streng-th aqueous dimethylacetarnide. 1.45 g of
tert.-butoxycarbonyl azide and 3.3 ml of lN sodium bi-
carbonate are added and the mixture is stirred at 3~ Cfor 5 hours. The solution is then concentrated at a
bath temperature of at most 50C. ,The residue is
triturated with ether and digested with 10 rnl of 2 /0
strength acetic acid. Yield: 942 mg of NaAl,
N~B29-di-tert.-butoxycarbonyl-hùman insulin.
b) The resulting compound is dissolved in 4 cc of
95 % strength pyridine, 0.03 cc of phenyl isothio-
cyanate are added and mixture is stirred at room tempera-
ture for 4 hours~ It is then concentrated to a small
volume in vacuo at a bath temperature of at mast 50C,
and the compound is precipitated with ether. Yield:
'830 of NaAl ~ B29-di-tert,-butoxycarbonyl-M -
phenylthiocarbamoyl-human insulin.
c) 820 mg of the compound prepared according to b)
are kep-t in 8.5 cc of trifluoroacetic acid at room
temperature for 1 hour, When 100 cc of ether are
added, 725 mg of des-phenylalanineBl-human insulin pre-
cipita-te; the product can be made to crystallize at
pH 5.0 -to 5,5 in the known manner,
Phe calculated 2.00, found 2.02
Example 2:
Des-PheBl-des-ThrB30-human insu].in
a) 1,0 g of des-AlaB30-swine insulin, prepared accor-
' ding to Hoppe-Seyler's Z. Physiol. Chem. 359 (1978),
733~3~
_ g _
page 799, are reacted by a procedure analogous to that
in Example la c. 710 mg of the title compound are
obtained and can be purified by chromatography in 1 %
strength acetic acid on a Sephadex G 50 superfine 1 x
5 100 cm column. Yield: 623 mg. Crystallization
at pH 5.4.
Phe calcula-ted 2.00, *ound 1.99
b) 50b mg of des-PheBl-swine insulin, prepared from
swine insulin by a procedure analogous to tha-t in
10 Example 1, are dissolved in 100 ml of 0,lM ammonium
bicarbonate buffer at pH 8.2. 3 mg of carboxypeptid-
ase A are added and the mixture is kept at room tempera-
ture for 15 hours and then lyophilized. To remove
salts, the product is chromatographed in 1 % streng-th
acetic acid over a Sephadex G 15 1 x 50 cm column.
For further purification, the product was chroma-
tographed in 30 % strength isopropanol containing 0.05M
of tris-buffer a-t pH 8 and with a NaCl gradient of 0 to
0.25M in a DEAE-Sephadex A-25 2.5 x 5~ cm column.
The eluate was deszlinated by dialysis against distilled
water and lyophilized. Yield: 276 mg,
Phe calcula-ted 2, found 1.98; Ala calculated 1,
found 1.01
_ample ~:
Des-Phe31-rAspA21]-human insulin
a~ 0.5 g of [AspA21]-human insulin, prepared by
keeping human insulin in aqueous trifluoroacetic acid
at pH 2 for 3 days and freeze-drying the product and
purifying it by chromatography on DEAE-Sephadex A-25
~d~n~1les ~r~ rk
.~ .
7~33~
-- 10 --
by a procedure analogous to that in Example 2b), is
reacted by a procedure analogous to that in Example la -c).
A~ter chromatography by a procedure analogous to that
in Example 2a), 324 mg of the title compound are obtained.
Phe calculated 2, found 2.02
The product is characterized by gel electrophoresis
- at pH 8. The distance -travelled corresponds to that
in the case of desamido-insulin (slight deviation as a
- result of the absence of phenylalanine).
b) 005 g f des-PheBl-human insulin, prepared accor-
ding to Example 1, are kept in aqueous trifluoroacetic
acid as described under a) and then purified on DEAE-
Sephadex A-25. Crystalliza-tion is carried out in the
known manner at pH 5.0 to gi.ve 365 mg of the ti-tle com-
1~ pound, which is identical to that prepared according toa)
Exam~le 4-
t
Des-PheBl-des-ThrB30-LAspA21]-human insulin
0.5 g of des-PheBl-des-Thr330-human insulin,
prepared according to Example 2, is treated with acid
and purified, according to Example 3b). Yield:
344 mg
Phe calcula-ted 2, found 1.99; Ala calculated 1,
~ound 1.02
The product is fur-ther characterized by gel electro-
phoresis at pH 8, by a procedure analogous to that in
Example 3a).
