Note: Descriptions are shown in the official language in which they were submitted.
UO ~/1~5 PCT/DK~/~XK2z~ 7
Glvcosvlated insullns.
- ~he presen~ invention relates to specifically glyco-
sylated insulins a~d cor~inations thereoC pharmaceutical ~re^-
arations containing such glycosylated co~pounds and a method
~- for their p eparatio~
- 5 ~n the recent years, several insulin analogues have
been suggested for the treatment of diabetes mellitus. ~he pur-
pose of developing such insulin analogues has been to improve
- the insulin replace~ent therapy by making available insulin
analogues ~ith eithe~ a more rapid or a more protracted insulin
action compared to especially human insulin.
A pro~le~ in the development of insulin analogues b.
suDstitutir.g one or ~o-e Oc the amino a~.d residues ir. na- ~e
` insulin is tne poten:iai iru~unogenicity of such compounds. Also
unforeseeable solubility and stability problems ~ay arise fror~
such substicutions.
Although insulin has a very short half life time in
circulating blood, it can not be excluded that a small amount
of insulin is glycosylated in vivo not only in diabetic
patients as postulated by Nakayama et al. (Nonenzymatic glyco-
sylation of insulin in "Current topics in clinical and experi-
mental aspects of diabetes mellitus" (1985), 201 - 204, Saka-
moto, Min and Baba, Eds., Elsevier Science Publishers 8.~.) bu~
also in non-diabetics. I~ i5 therefore pos~ible that t;~e orga~.-
;ism has developed mechanisms to suppress th-e formation of an~i-
-~25 bodies against glycosylated insulin. It is furthermore posslble
that the conformational changes of the saccharide part will be
able to camouflage the antigen.
. ,~ . .
The binding of glucose, mannose and certain oligo-
saccharides to insulin has been the subject of nu~erous in
vitro studies in the past with the purpose of investigating
whether in vivo fornation of glycosylated insulin might be re-
3~sponsible for late complications in diabetic patients Anzen-
bacher et al. (Biochimica et Biophysica Acta 386 (1975), 603-
607) studied the binding of D-glucose to insulin by equilibrium
3S dialysis. The binding was not found to be very the specific and
-~ .
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WO 90/10645 PCT/D~i90/00062
2~a ~937 2
,
. .
the average number of glucose molecules bound to the insulin
molecule was found to be eight.
Interaction of insulin with glucose and mannose ~as
studied by Dolhofer et al. (Febs Letters loo (15,9), 133~
136). The results indicated that both hexoses were covalently
incorporated into the insulin molecule upon incubation in vitro
at 37 C. Under the chosen reaction conditions, in average 3.6 _
0.39 glucose and 5.0 2 0.43 mannase residues ~ere found to be
' taXen up per molecule of insulin.
; 10 A glucose controlled insùlin delivery svste- ~as sug-
gested by Bro~nlee & Cerami (Science 206 (1979), 1190 - 1191,
and Diabetes 32 (1933), 499 - 505) by synthesl~ing gl"~^,;la e~
insulin derivatives which are able to compete wit;~ gluc3se for
binding to lectins. In this study maltose and othe- oligo-
~ saccharides were reacted with insulin.
,~Nakayama et al. (supra) investigated non-enzymatic
glycosylation of insulin in vitro and in vivo (diabetic
~'patients) and concluded that glucose was incorporated into the
insulin molecule in vivo under pathological conditions. By the
20 in vitro studies, 3 ~olecules of glucose were found to be in-
q~corporated per molecule of insulin.
, In 1988, Lapolla et al. lDiabetes 37 (1~o8), 7~/-
~j'791) reported a reduced in vivo biological activi~y o' in vitro
-glycosylated insulin. Insulin was glycosylated in ambien_ high
25 glucose concentration according to Dolhofer (supra) in aqueous
solution at 37'C for 17 hours at a pH value o- 7.~ e incor-
'poration of glucose was found to be in average 2 mcl glucose
7residue/mol insulin.
Taking into account that native insulin has three
30 free primary amino groups viz. at position Bl (Phe), Al (Gly)
'~' and B29 (Lys), respectively, it is apparent that the abo~e de-
.
scribed glycosylated insulin preparations will all be inhomo-
~,geneous mixtures of glycosylated insulin molecules. ' '
~Nobody has so far taken any steps to fractionate the
-,~35 above mixture into the individual components.
,1,
?
'I ,
~0 90/106~5 PCr/DI;90/00062
C~3~
r
Glycosylated insulin derivatives for self-regulating
- insulin delivery systems have been described by ~i~ et al.
- (Journal of Controlled Release 1 (1984) ~ 57 ~ 66~ and US patent
- specification Nos. 4~483~792 4~478~830; 41C178,7'.6 an-'
4~489~063)~ In these glycosylated insulins, glucose or mannose
is coupled with insulin via a spacer group derived fro~ di-
carboxylic acids, acid anhydrides or p~enyl amines or a combi-
nation thereof.
European patent application No. 8~20032~3 h~ving oub-
lication No. 119,650 relates to galactosyl insulir.s h~i~h like
-' the glycosylated insulins described by Kim (sup.a) con'ain a
spacer group.
It is the purpose of this invention to pre~are insu-
5~;lin derivatives having improved properties. More soecifically,
.115 it is the purpose of the present invention to develop non-im-
munogenic insulin derivatives. It is furthermore the purpose
of the present invention to develop insulin derivatives with a
faster onset of insulin action than native insulin and to im-
prove the solubility of less soluble insulins in order to allo~
the use of highly concentrated solutions, for exa~ple in insu-
.lin pumps. A still further purpose of this invention is to pre-
pare insulin derivatives with an improved stability a~inst fi-
-~3brillation.
-~The present invention provides specifically glyco-
~25 sylated insulins. Hereinafter the term specifically glycosy-
Ilated insulins designates insulins having the carbohydrate sub-
stituent in a specific pos,tion in the insulin ~.olecule. S~
"prisingly, such specifically glycosylated insulir.s offer cer-
..tain therapeutical advantages as will be apparent from the fol-
:.~30 lowing description and examples.
IIn its broadest aspect the present invention provides
specifically glycosylated insuiins. In a narro~er aspect the
~,present invention provides insulin derivatives being either
monoglycosylated in position Al, Bl or B29; diglycosylated in
position A1 and Bl; Al and B29; or Bl and B29 or triglycosy-
?; . .
^s'~ ' ' . '
:
PCT/DK~/~XK2
- ' " ~; ' '
lated in position Al, Bl and B29. -
The glycosylated insulins described by Ki~ et al. and
dealt with above are remote from the insulins of the present
invention, which utilizes the aldehyde function of the sugar
itself to for~ a covalent bond to insulin, without the use of
artificial spacer groups. A further advantage of the present
invention over the insulins of Klm et al. is the retention of
the natural cha-ge distribution of the insulin molecule ~hus,
; the amino grou~is invol~ed which by the glycosylation reaction
(see diagra~) are converted into secondary amino groups are
still capable of ~eing protonated, as in nor~al insuli.,.
~ he p-esent ir.sulin derivatives may ln eac~. o' t~.e
th ee positions contain a monosaccharide or an oligosaccharide
- with up to three sugar residues. Suitable monosaccha-ides are
15 glucose, mannose and galactose. Suitable oligosaccha-ides are ;
maltose, isomaltose, lactose, maltotriose, melibiose and cello-
' biose.
The specifically glycosylated insulin derivatives of
this invention may be used as such for the treatnent of dia-
~ 20 betes mellitus. With the purpose of monitoring the insulin
Y therapy, selected mixtures of the individual specifically gly-
~ cosylated compGund~ may however also be used.
-~ As used herein, the expression insulins is ~ean to
cover native for~s of insulin such as human, bovine and porcine
25 insulin, but also derivatives thereof wherein one or F,^re a~ino
acid residues have been substituted, added or deleted, compared
with native insulin, for example as described in European
patent appiications having publication Nos. 0194864A and
0214826A.
~,i 30As mentioned above, native insulins have three poten-
~ tial glycosylation sites, namely the two N-terminal a.ino acid
3 residues in the A- and B-chain and the lysine residue in posi-
j tion a29. It is apparent that the number of potential glycosy- ~ -
~ lation sites in insulin analogues of the above described type
`j - 3 may be from two (the two N-terminal residues) and upwards de-
ii :
. .
~0 ~/1~5 PCT/DK~/~XK'
2~ $'~
pending on ho~ many lysine residues are present in the modified
insulin molecule, l~sine being the only naturally occurring
amino acid with a free primary amino group in the side chain.
The glycosylation schematically proceeds according to
5 the following diagramme using D-glucose: ~.
, . .
:: CH2OH CH2OH
OH _ ~ ' ~ O ~ :
HO
OH OH
t'` 1 ~ D-glucose D-glucose
pyranose structu~e chain structure
The chain structure is the reactive component
i :
~, 20
CH2O~ CH2OH
H~o + H2N-"insulin~ rH2O HO ~ N~-"insulin"
` 25 OH OH
H0 ~ CH2-NH-"insulin" .
. HO
.. ~ 1 deoxy-D-fructosyl insulin (glucose insulin)
.¦ "insulin" designates desamino insulin.
. 3~
The above reaction will proceed in an analogous
manner with other monosaccharides or oligosacchar_des having a
free aldehyde group.
~:~3~ - Specific examples of the p-esent glycosylated insu-
lins are:
Phe(Bl) glucose human lnsulin,
Phe(Bl) mannose human insulin,
~ .
.~ . ' .
. ~ . - . .
UO 90/10645 PCT/DI~90/00062
i ~ 6
Gly(Al) mannose human insulin, :
Lys~B29) mannose human insulin, :.
~ Phe(Bl) galactose human insulin,
. Gly(Al) galactose human insulin,:: -
5 Lys(B29) galactose human insulin, ..
Phe(Bl) maltose human insulin, : .
Phe(B1) lactose human insulin,
' 3 GlytAl) glucose human insulin, : .
.~ Gly(A1) maltose human insulin, .
10 Gly(A1) lactose human insulin, .
: Lys(B29) glucose human insulin, -
Lys(B29) maltose human insulin,
:1 Lys(B29) lactose human insulin,
Gly(Al),Phe(Bl) diglucose human insulin, ~.
~i 15 Gly(Al),Lys(B29) diglucose human insulin,
Phe(Bl),Lys(B29) diglucose human insulin, ..
~I Phe(B1) isomaltose hunan insulin,
~ Gly(A1) isomaltose human insulin,
.~ Lys(B29) isomaltose human insulin,
Phe(B1) maltotriose human insulin,
Gly(A1) maltotriose hu~an insulin, ;.
Lys (B29) maltotriose human insulin,
.~ Gly~Al),Phe(B1) di~altoce human insulin,
. - Gly(Al),Lys(829) dimaltose human insulin,
Phe(Bl),Lys(B29) dimaltose hu~an insulin,
~! Gly(Al),Phe(B1) dilactose human insulin,
i Gly(Al),Lys(B29) dilactose human insulin,
Phe(Bl),Lys(B29) dilactose human insulin,
. Gly(Al),Phe(B1) dimaltotriose human insulin,
, 30 Gly(Al),Lys(B29) dimaltotriose human insulin,
~ Phe(Bl),Lys(B29) dimaltotriose human insulin,
-x~ Phe(Bl),Gly(Alj dimannose human insulin,
Phe(Bl),Lys(B29) dimannose human insulin,
Gly(Al),Lys(B29) dimannose human insulin,
Phe(Bl),Gly(Al) digalactose human insulin,
, .
,~ .
WO ~/1~5 2~ 3~ PC~/D~/~XK2
.~ .
.
- Phe(Bl),Lys(B29) digalactose human insulin,
Gly(Al),Lys(B29) digalactose human insulin,
Phe(Bl),Gly(Al) diisomaltose human insulin,
Phe(Bl),Lys(B29) diisomaltose human insulin,
Gly(Al),Lys(B29) diisomaltose human insulin,
Gly(Al),Phe(Bl),Lys(~29) triglucose human insulin,
Gly(Al),Phe(Bl),Lys(B29) trimaltose human insulin,
Gly(Al),Phe(Bl),Lys(B29) trilactose human insulin,
Gly(Al),Phe(Bl),Lys(~29) trimaltotriose human insulin.
Gly(Al),Phe(Bl),Lys(829) trimannose human insulin,
Gly(Al),Phe(Bl),Lys(B29) trig~alactose human insulin,
~; Gly(Al),Phe(Bl),Lys(B29) triisomaltose hu~an insulin,
Phe(Bl) glucose ~Asp310] human insulin,
Gly(Al),Phe(~l) diglucose [Asp310] human insulin.
~ 15
; Also, specifically glycosylated insulins from other
. species such as porcine are interesting.
~; The present glyco5ylated insulins may be prepared by
.~ reacting insulin or an insulin analogue with an excess of the
selectéd monosaccharide or oligosaccharide in a suitable or-
ganic or aqueous medium. The temperature may vary from 20 to
~, 60'C. As organic solvents lower carboxylic acids, for exa~pls
acetic acid and propionic acid, lower aliphati~ alcohols, for
~ example methanol, ethanol and 2-propanol, ethylene glycol and
propylene glycol may be used. However, phenols may also ~e
` used.
The duration of the reaction and the composition of
~ the reaction mixture will depend on whether a mono-, di- or
.~ triglycosylated end product is desired. The reaction may con-
veniently be followed by reversed phase high pressure liquid
chromatography (hereinafter designated RP HPLC) to deter~ine
~ the point of maximum for~ation of each of the individual glyco-
:~ sylated products.
~? The reaction is stopped by cooling, for example to
-20-C, and the reaction mixture is concentrated to dryness
. ~ '
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~ . .
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WO ~/1~5 z ~C ~ 3-~ PCT/DK~/~XK2
..
whereupon the major components of the reaction mixture are iso-
lated and purified by preparative RP HPLC. After desalting, the
products are characterized by fast atom bombardment mass spec-
trometry (hereinafter designated FAB-MS), quantitative amino
; 5 acid analysis after borohydride reduction and bioassays.
The present glycosylated insulins and mixtures there-
of may be substituted for the human or porcine insulin in the
insulin preparations heretofore known in the art to prepare
novel insulin preparations. Such novel insulin preparations
will contain the glycosylated insulin or a phar~aceuticall~
acceptable salt thereof in an aqueous solution, preferably at a
neutral pH value. Preferably, the aaueous medium is ~de iso~c-
; nic, for example with sodium chloride, sodium acetate or gly-
cerol. Furthermore, the aaueous medium may contain zinc ions,
15 buffer components such as acetate or phosphate and a preserva-
.' tive such as m-cresol, methylparaben or phenol. The pH vaiue of
~ the preparation may be adjusted to the desired value and the
3 preparation can be sterilized by filtration.
The insulin preparation of the present invention can
20 be used similarly to the use of the ~nown insulin preparations.
's EXPERIMENTAL PART
.~ .
Example 1
25 Phe(B11 alucose human insulin
~1
Human insulin 10.1 m~ol) was suspended in methanol
(30 ml) and glacial acetic acid (5 ml) was added at ambient
temperature. ~he mixture was gently stirred until the insulin
30 had dissolved. Then, a further quantity of methanol (3~ ml) was
~ added and after addition of D-glucose t2.2 mmol), the mixture
was gently stirred at 40 C for 8 hours, by which the title com-
pound became tXe main component.
The solution was concentrated almost to dryness on a
35 rotatory evaporator. The residue was dissolved in water and
., .
~,
'` :
..... .,. . ... ,; . . .. . . . .. , ., ~ ~
wo ~/1~ ~5 ~ ~J ~ 3~ PCT/DK~/~WK2
9 ,
fractionated by preparative RP HPLc. Column: 16 x 250 ~m with 7
. ~m 100 A C18 particles. Temperature 30'C. Mobile phase. A: 0.04
M phosphoric acid, 0.2 M sodium sulphate, 10% acetonitrile, pH
value adjusted to 2.5 with ethanol amine. B: 50~ acetonitrile.
- 5 The fraction corresponding to the central part of the
major peak was desalted and lyophilized. The yield was 0.02
; mmol. The product was characterized by FAB-MS and quantitative
amino acid analysis after borohydride reduction. The ar,ino acid
analysis sho~ed the prese~ce of two phenylalanlne residues
(i.e. one less phenylalanine residue compa~ed to hu-,an insulin)
proving substitution in Phe(B1). The molecular weigh' ~as found
i' to be 5g70 (calc~lated: 5570). ~ :
, :.
...
15 Example 2
PhetBl~ Gly(Al ! dialucose human insulin
The above compound was prepared as described in
Example 1 with the exception that the reaction time was 16
20 hours instead of 8 hours, by which the title compound became
the main component.
The yield was 0.06 mmol.
The amino acid analysis sho-~ed the presence of one
3 : less Phe and one less Gly residue proving a substit~tion in po-
~ 25 sition Al and Bl.
~ . .
j The molecular weight measured ~as 6132 (calculated:
6132). ~-
i~ The subcutaneous absorption was measured in pigs by
-~ injection of 125I Iabelled PhetB1),~ly(Al) diglucose insulin
30 prepared using the iodate method essentially as described (J0r-
gensen et al., Diabetologia 19 (1980), 545 - 554). The absorp-
tion rate after subcutaneous injection into pigs of 12'I human
insulin and 125I Phe(Bl),Gly(Al) diglucose human insulin is
shown in Table 1, below. The T7s, T50 and T25 values given in
35 Table 1 are the time (in hours) elapsed from the moment of in-
.. ~ - ,
.
WO ~/I~5 PCT/D~/~XK2
2~
.. ~.:
. jection of the sample until the radioactivity measured at the
site of injection has decreased to 75%, 50% and 25%, respecti-
vely, of the initial value. It appears from Table 1 that the
glycosylated insulin has a significantly faster absorption than :
~ 5 human insulin.
:' ' ' '
TA8LE 1 ~
~75 T50 ~25 ';
10 human insulin 1.12 2.33 3.6~
; diglucose human insulin 0.65 1.47 2.76
. .
. . .
The blood glucose lo~ering effect of hu~an insulin
,; 15 (ActrapidTM) and Gly(Al),Phe(B1) diglucose human insulin by
~ su~cutaneous injection in pigs (mean of 5 animals) in an amount
:~; of 0.1 U/kg appears from Table 2, below. Table 2 gives the
;:~ values for glucose in mmol/1.
:., . : .
TABLE 2
Time, ~uman Diqluco~e ~u~an
ho~ insulLn insulln
-0.33 5.30 5.24
. 25 0 5.36 5.32
~ 0.33 4.~8 4.76
.q~ 0.67 4.72 4.06
1 4.12 3.34
1.5 3.60 2.98
2 3.24 2.84
s 2.5 3.28 2.96
q 3 3.18 3.14
:~ 4 3.34 3.96
~ 35
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~0 ~/1~5 2~ 7 PCT/DK~/~XK2
11
Table 2 shows the fast action of diglucose insulin
compared with human insulin.
The i~une responses in rabbits (mean values for 10
animals) of human insulin, bovine insulin and Gly(Al~,Phe(Bl)
diglucose hu~an insulin appear from Table 3, below, giving
values for percentage binding in rabbit serum. (Method:
Schlichtkrull et al. (Horm. Me~tab- Res. Suppl. ser. 5 (1974),
134 - 143)).
TABLE 3
.. . .
Time, Human Bovine Di~lucose human
d days insulin insulin insulin
15 0 1.9 -0.4 1.2
13 2.3 0.6 1.3
27 3.0 28.5 1.5
41 3.2 29.7 1.4 -
s 5S 4.3 30.7 1.1
20 69 3.9 32.7 2.2
83 2.4 30.1 1.1
.
97 2.0 32.6 1.5
~ It appears ~rom Table 3 that diglucose human insulin
. 25 has a surprisingly low immune response which corresponds to
¦ ~ that of human insulin.
` Example 3
. 30 The following compounds were prepared analogously.
The molecular weight measured by FAB-MS or plasma desorption
mass spectroscopy, together with the calculated molecular
weight, is given for each of the compounds.
.. .~ ' .
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~ ~/1~5 - PCT/D~/~X~2
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- Molecular_weiqht :
Compound Measured Calcu-
.; lated
5 Phe(Bl) galactose hu~an insulin5956 5970
Phe(Bl) ~al'ose hu.. ,an insulin 6119 6132
. Phe(Bl) lactc,e hu~an insulin 6125 6132
Phe~Bl) maltc~riose h~man insulin 6288 62C~
~ Gly(Al),Phe(B;) di~,a;tose human insulin 6444 64,$
.~ 10 Gly(Al),Phe(31) dilactose hur.,an insulin ~6 6~6
; Gly(Al),Phe(B;) dimaltotriose h~ran i.~sulin 6771 6780
Gly(Al),Phe(B'), Lys(B29) triglu~ose
hu~an insulir. 629~ 62~ ;
,~ Gly(Al),Phe(B ) dlglucose ~Asp310~
15 human insulin 6112 6110
. . .
.,~; -
;- The location of the bound sugar residues was confirmed by boro-
~ hydride reduction followed by quantitative amino acid analysis. `~.
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