CA2049937A1 - Glycosylated insulins - Google Patents
Glycosylated insulinsInfo
- Publication number
- CA2049937A1 CA2049937A1 CA002049937A CA2049937A CA2049937A1 CA 2049937 A1 CA2049937 A1 CA 2049937A1 CA 002049937 A CA002049937 A CA 002049937A CA 2049937 A CA2049937 A CA 2049937A CA 2049937 A1 CA2049937 A1 CA 2049937A1
- Authority
- CA
- Canada
- Prior art keywords
- insulin
- glycosylated
- phe
- gly
- human insulin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
Specifically glycosylated insulins and pharmaceutical preparations hereof are provided. The novel insulins will preferably be mono-, di- or triglycosylated and contain a monosaccharide group or an oligosaccharide group with two or three sugar units. The specifically glycosylated insulins will have a high solubility and a more rapid onset of insulin action than for instance human insulin.
Description
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 -~ .
'. ~ '.: , ~, .~;.- - - , : . : ~ - . . : :: , ; :
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 . ~ ' 5~ .
':~
`i ' .
~ . .
j' ' 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.
:., . : .
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 .:
~` :
.~, `' . : ~ .. :.. -......... , - .. .. ,, . :
'',:,,-, : , . : . . ' .. .: ': . ': ' :: . ' . , ~0 ~/1~5 2~ 7 PCT/DK~/~XK2 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)).
.. . .
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.
.. .~ ' .
~, ' ~'' .,,'.
. .
.. ~, . .:
~ ~/1~5 - PCT/D~/~X~2 '-'' ' ~':
~ .
' ; , ~.
- 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. `~.
`~ ,:
'.~ ; '' '. ' , ~;~ ` ' ' : ' "
~3 , - .., . ' . ;~ .
:: . - - , - ~
- ~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 -~ .
'. ~ '.: , ~, .~;.- - - , : . : ~ - . . : :: , ; :
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 . ~ ' 5~ .
':~
`i ' .
~ . .
j' ' 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.
:., . : .
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 .:
~` :
.~, `' . : ~ .. :.. -......... , - .. .. ,, . :
'',:,,-, : , . : . . ' .. .: ': . ': ' :: . ' . , ~0 ~/1~5 2~ 7 PCT/DK~/~XK2 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)).
.. . .
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.
.. .~ ' .
~, ' ~'' .,,'.
. .
.. ~, . .:
~ ~/1~5 - PCT/D~/~X~2 '-'' ' ~':
~ .
' ; , ~.
- 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. `~.
`~ ,:
'.~ ; '' '. ' , ~;~ ` ' ' : ' "
~3 , - .., . ' . ;~ .
:: . - - , - ~
Claims (15)
1. Specifically glycosylated insulins containing one or more monosaccharide groups or one or more oligosaccharide groups with up to three sugar units.
2. Glycosylated insulin according to claim 1 con-taining one monosaccharide group or one oligosaccharide group with up to three sugar units.
3. Glycosylated insulin according to claim 1 con-taining two monosaccharide groups or two oligosaccharide groups with up to three sugar units.
4. Glycosylated insulin according to claim 1 con-taining three monosaccharide groups or three oligosaccharide groups with up to three sugar units.
5. Glycosylated insulin according to claim 2 being monoglycosylated in position A1, B1 or B29.
6. Glycosylated insulin according to claim 3 being diglycosylated in position A1 and B1; A1 and B29; or B1 and B29.
7. Glycosylated insulin according to claim 4 being triglycosylated in position A1, B1 and B29.
8. Glycosylated insulin according to any one of the preceding claims containing Asp in position 310.
9. Glycosylated insulin according to any one of the preceding claims, characterized in that the parent insulin species are human.
10. Phe(B1) glucose human insulin.
11. Phe(B1),Gly(A1) diglucose human insulin.
12. A composition containing at least 90%, preferably at least 95%, most preferred at least 99%, of a specifically glycosylated insulin according to any one of the preceding claims.
13. Pharmaceutical preparations containing a speci-fically glycosylated insulin according to any of the previous claims or a pharmaceutically acceptable salt thereof optionally together with pharmaceutically acceptable adjuvants and additives and preservatives.
14. A process for the preparation of specifically glycosylated insulins according to any of the previous claims 1 - 11 wherein the appropriate insulin is reacted in an aqueous or organic medium with a monosaccharide with a free aldehyde group or an oligosaccharide with a free aldehyde group and with up to three sugar units whereupon the desired product is iso-lated from the reaction mixture.
15. Any novel feature or combination of features de-scribed herein.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DK111489A DK111489D0 (en) | 1989-03-08 | 1989-03-08 | PEPTIDES |
| DK1114/89 | 1989-03-08 |
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| Publication Number | Publication Date |
|---|---|
| CA2049937A1 true CA2049937A1 (en) | 1990-09-09 |
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ID=8101165
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002049937A Abandoned CA2049937A1 (en) | 1989-03-08 | 1990-03-06 | Glycosylated insulins |
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|---|---|
| EP (1) | EP0462192A1 (en) |
| JP (1) | JPH04504117A (en) |
| KR (1) | KR920701249A (en) |
| CN (1) | CN1045586A (en) |
| AU (1) | AU638701B2 (en) |
| CA (1) | CA2049937A1 (en) |
| CS (1) | CS114290A3 (en) |
| DD (1) | DD296933A5 (en) |
| DK (1) | DK111489D0 (en) |
| FI (1) | FI914226A0 (en) |
| GR (1) | GR1000604B (en) |
| HU (1) | HUT59942A (en) |
| IL (1) | IL93674A0 (en) |
| NO (1) | NO913517L (en) |
| NZ (1) | NZ232808A (en) |
| PT (1) | PT93366A (en) |
| WO (1) | WO1990010645A1 (en) |
| YU (1) | YU45490A (en) |
| ZA (1) | ZA901737B (en) |
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| CN103215328B (en) | 2004-01-21 | 2016-08-03 | 诺和诺德医疗保健公司 | The joint of transglutaminase mediated peptide |
| US7597884B2 (en) | 2004-08-09 | 2009-10-06 | Alios Biopharma, Inc. | Hyperglycosylated polypeptide variants and methods of use |
| RU2011135730A (en) | 2009-01-28 | 2013-03-10 | Смартселлз, Инк. | CRYSTAL INSULIN CONJUGATES |
| WO2010088294A1 (en) | 2009-01-28 | 2010-08-05 | Smartcells, Inc. | Conjugate based systems for controlled drug delivery |
| WO2010088268A1 (en) | 2009-01-28 | 2010-08-05 | Smartcells, Inc. | Exogenously triggered controlled release materials and uses thereof |
| US8623345B2 (en) | 2009-03-20 | 2014-01-07 | Smartcells | Terminally-functionalized conjugates and uses thereof |
| WO2010107520A1 (en) | 2009-03-20 | 2010-09-23 | Smartcells, Inc. | Soluble non-depot insulin conjugates and uses thereof |
| AU2011282988A1 (en) | 2010-07-28 | 2013-01-31 | Smartcells, Inc. | Recombinantly expressed insulin polypeptides and uses thereof |
| JP2013541500A (en) | 2010-07-28 | 2013-11-14 | スマートセルズ・インコーポレイテツド | Recombinant lectins, binding site modified lectins and their uses |
| CA2805739A1 (en) | 2010-07-28 | 2012-02-02 | Smartcells, Inc. | Drug-ligand conjugates, synthesis thereof, and intermediates thereto |
| US9624287B2 (en) | 2012-07-17 | 2017-04-18 | Case Western Reserve University | O-linked carbohydrate-modified insulin analogues |
| WO2014015078A1 (en) * | 2012-07-17 | 2014-01-23 | Michael Weiss | O-linked carbohydrate-modified insulin analogues |
| EP3332810B1 (en) | 2012-11-13 | 2021-01-13 | Adocia | Fast-acting insulin formulation comprising a substituted anionic compound |
| US9707276B2 (en) | 2012-12-03 | 2017-07-18 | Merck Sharp & Dohme Corp. | O-glycosylated carboxy terminal portion (CTP) peptide-based insulin and insulin analogues |
| CN105358531B (en) | 2013-03-14 | 2017-11-14 | 利兰-斯坦福大学初级学院的董事会 | Mitochondrial aldehyde dehydrogenase-2 modulators and its application method |
| BR112016007176A2 (en) | 2013-10-04 | 2018-01-23 | Merck Sharp & Dohme | conjugate, composition, uses of a conjugate and composition, and method for treating an individual having diabetes |
| US9795678B2 (en) | 2014-05-14 | 2017-10-24 | Adocia | Fast-acting insulin composition comprising a substituted anionic compound and a polyanionic compound |
| FR3020947B1 (en) | 2014-05-14 | 2018-08-31 | Adocia | AQUEOUS COMPOSITION COMPRISING AT LEAST ONE PROTEIN AND A SOLUBILIZING AGENT, ITS PREPARATION AND ITS USES |
| FR3043557B1 (en) | 2015-11-16 | 2019-05-31 | Adocia | RAPID ACID COMPOSITION OF INSULIN COMPRISING A SUBSTITUTED CITRATE |
| CN105535927A (en) * | 2016-01-29 | 2016-05-04 | 山东中海制药有限公司 | Medicine used for treating influenza, upper respiratory infection and viral pneumonia |
| CN105709207A (en) * | 2016-01-29 | 2016-06-29 | 徐宝贞 | Medicine for treating gout |
| CN105597080A (en) * | 2016-01-29 | 2016-05-25 | 程潜 | Medicine for treating uremia and urine protein |
| EP3463480A1 (en) | 2016-06-02 | 2019-04-10 | Sanofi | Conjugates of a pharmaceutical agent and a moiety capable of binding to a glucose sensing protein |
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| US3847890A (en) * | 1971-11-01 | 1974-11-12 | A Green | Acidic monosaccharide-substituted proteins |
| US4444683A (en) * | 1982-11-17 | 1984-04-24 | University Of Utah | Glycosylated insulin derivatives |
| EP0119650A3 (en) * | 1983-03-21 | 1987-09-30 | THE PROCTER & GAMBLE COMPANY | Galactosyl-insulin conjugates useful in treating diabetics |
| HU206890B (en) * | 1986-10-13 | 1993-01-28 | Sandoz Ag | Process for producing sugar-modified somatostatin peptide derivatives and pharmaceutical compositions containing them as active components |
-
1989
- 1989-03-08 DK DK111489A patent/DK111489D0/en not_active Application Discontinuation
-
1990
- 1990-03-06 JP JP2504720A patent/JPH04504117A/en active Pending
- 1990-03-06 EP EP19900904777 patent/EP0462192A1/en not_active Ceased
- 1990-03-06 NZ NZ232808A patent/NZ232808A/en unknown
- 1990-03-06 AU AU52807/90A patent/AU638701B2/en not_active Expired - Fee Related
- 1990-03-06 WO PCT/DK1990/000062 patent/WO1990010645A1/en not_active Application Discontinuation
- 1990-03-06 KR KR1019910701014A patent/KR920701249A/en not_active Application Discontinuation
- 1990-03-06 HU HU8727A patent/HUT59942A/en unknown
- 1990-03-06 CA CA002049937A patent/CA2049937A1/en not_active Abandoned
- 1990-03-07 YU YU00454/90A patent/YU45490A/en unknown
- 1990-03-07 PT PT93366A patent/PT93366A/en not_active Application Discontinuation
- 1990-03-07 GR GR900100159A patent/GR1000604B/en unknown
- 1990-03-07 DD DD90338488A patent/DD296933A5/en not_active IP Right Cessation
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- 1990-03-07 CN CN90101282A patent/CN1045586A/en active Pending
- 1990-03-08 CS CS901142A patent/CS114290A3/en unknown
-
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- 1991-09-06 FI FI914226A patent/FI914226A0/en not_active Application Discontinuation
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| AU5280790A (en) | 1990-10-09 |
| JPH04504117A (en) | 1992-07-23 |
| EP0462192A1 (en) | 1991-12-27 |
| GR900100159A (en) | 1990-07-31 |
| NZ232808A (en) | 1992-04-28 |
| DD296933A5 (en) | 1991-12-19 |
| IL93674A0 (en) | 1990-12-23 |
| WO1990010645A1 (en) | 1990-09-20 |
| HU902787D0 (en) | 1991-11-28 |
| FI914226A0 (en) | 1991-09-06 |
| GR1000604B (en) | 1992-08-26 |
| NO913517D0 (en) | 1991-09-06 |
| ZA901737B (en) | 1990-11-28 |
| NO913517L (en) | 1991-11-06 |
| PT93366A (en) | 1990-11-07 |
| AU638701B2 (en) | 1993-07-08 |
| CN1045586A (en) | 1990-09-26 |
| CS114290A3 (en) | 1992-02-19 |
| DK111489D0 (en) | 1989-03-08 |
| KR920701249A (en) | 1992-08-11 |
| YU45490A (en) | 1991-10-31 |
| HUT59942A (en) | 1992-07-28 |
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