AU745983B2 - Acylated insulin - Google Patents

Description

I I.

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Novo Nordisk A/S Actual Inventor(s): SVEND HAVELUND, JOHN BROBERG HALSTROM, IB JONASSEN, ASSER SLOTH ANDERSEN, JAN MARKUSSEN Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: ACYLATED INSULIN Our Ref: 623355 POF Code: 375902/119285 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 1

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A ACYLATED INSULIN The present application is a divisional application from Australian patent application number 48461/97, the entire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates to novel human insulin derivatives which are soluble and have a protracted profile of action, to a method of providing such derivatives, to pharmaceutical compositions containing them, and to the use of such insulin derivatives in the treatment of diabetes.

BACKGROUND OF THE INVENTION Many diabetic patients are treated with multiple daily insulin injections in a regimen comprising one or two daily injections of a protracted insulin to cover the basal requirement supplemented by bolus injections of a rapid acting insulin to cover the requirement related to meals.

Protracted insulin compositions are well known in the art.

Thus, one main type of protracted insulin compositions comprises injectable aqueous suspensions of insulin crystals or amorphous insulin. In these compositions, the insulin compounds utilized typically are protamine insulin, zinc insulin or protamine zinc insulin.

Certain drawbacks are associated with the use of insulin suspensions. Thus, in order to secure an accurate dosing, the insulin particles must be suspended homogeneously by gentle shaking before a defined volume of the suspension is withdrawn from a vial or expelled from a cartridge. Also, for the storage of insulin suspensions, the temperature must be "kept within more narrow limits than for insulin solutions in order to avoid lump formation or coagulation.

ooeo While it was earlier believed that protamines were nonimmunogenic, it has now turned out that protamines can be oooo \VARIOUS lsbta.

oeooe It (I 2 immunogenic in man and that their use for medical purposes may lead to formation of antibodies (Samuel et al., Studies on the immunogenecity of protamines in humans and experimental animals by means of a micro-complement fixation test, Clin. Exp.

Immunol. 33, pp. 252-260 (1978)).

Also, evidence has been found that the protamine-insulin complex is itself immunogenic (Kurtz et al., Circulating IgG antibody to protamine in patients treated with protamineinsulins. Diabetologica 25, pp. 322-324 (1983)). Therefore, with some patients the use of protracted insulin compositions containing protamines must be avoided.

Another type of protracted insulin compositions are solutions having a pH value below physiological pH from which the insulin will precipitate because of the rise in the pH value when the solution is injected. A drawback with these solutions is that the particle size distribution of the precipitate formed in the tissue on injection, and thus the timing of the medication, depends on the blood flow at the injection site and other parameters in a somewhat unpredictable manner. A further drawback is that the solid particles of the insulin may act as a local irritant causing inflammation of the tissue at the site of injection.

WO 91/12817 (Novo Nordisk A/S) discloses protracted, soluble insulin compositions comprising insulin complexes of cobalt(III). The protraction of these complexes is only intermediate and the bioavailability is reduced.

*Human insulin has three primary amino groups: the N-terminal group of the A-chain and of the B-chain and the e-amino group of Lys 9 Several insulin derivatives which are substituted in one or more of these groups are known in the prior art. Thus, US Patent No. 3,528,960 (Eli Lilly) relates to N-carboxyaroyl insulins in which one, two or three primary amino groups of the insulin molecule has a carboxyaroyl group. No specifically Ne 1 29 substituted insulins are disclosed.

According to GB Patent No. 1.492.997 (Nat. Res. Dev. Corp.), it has been found that insulin with a carbamyl substitution at N" 29 has an improved profile of hypoglycaemic effect.

JP laid-open patent application No. 1-254699 (Kodama Co., Ltd.) discloses insulin wherein a fatty acid is bound to the amino group of PheB" or to the e-amino group of LysB 29 or to both of these. The stated purpose of the derivatisation is to obtain a O 10 pharmacologically acceptable, stable insulin preparation.

Insulins, which in the B30 position have an amino acid having at least five carbon atoms which cannot necessarily be coded for by a triplet of nucleotides, are described in JP laid-open patent application No. 57-067548 (Shionogi). The insulin analogues are claimed to be useful in the treatment of diabetes mellitus, particularly in patients who are insulin resistant due to generation of bovine or swine insulin antibodies.

By "insulin derivative" as used herein is meant a compound having a molecular structure similar to that of human insulin including the disulfide bridges between CysA 7 and Cys B 7 and 0 between CysA 20 and Cys 819 and an internal disulfide bridge between CysA 6 and CysA 11 and which have insulin activity.

However, there still is a need for protracted injectable insulin compositions which are solutions and contain insulins 25 which stay in solution after injection and possess minimal inflammatory and immunogenic properties.

One object of the present invention is to provide human insulin derivatives, with a protracted profile of action, which are soluble at physiological pH values.

Another object of the present invention is to provide a pharmaceutical composition comprising the human insulin derivatives according to the invention.

It is a further object of the invention to provide a method of making the human insulin derivatives of the invention.

SUMMARY OF THE INVENTION Surprisingly, it has turned out that certain human insulin derivatives, wherein the E-amino group of LysB 29 has a lipophilic substituent, have a protracted profile of action and are soluble at physiological pH values.

Accordingly, in its broadest aspect, the present invention relates to an insulin derivative having the following sequence: A-Chain S

S

1 7 1 Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser- 1 2 3 4 5 6 8 9 10 11 12

S

B-Chain

S

Xaa-Val-Xaa-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val- 1 2 3 4 5 6 7 8 9 10 11 12 A-Chain (contd.) 25 Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cs-Xaa (SEQ ID NO:1) 13 14 15 16 17 18 19 j 21

S

B-Chain (contd.)

S

S 30 Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe- 13 14 15 16 17 18 19 20 21 22 23 24 B-Chain (contd.) Phe-Tyr-Thr-Pro-Lys-Xaa (SEQ IDNO:2) 35 25 26 27 28 29 oooo• wherein Xaa at positions A21 and B3 are, independently, any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; Xaa at position B1 is Phe or is deleted; Xaa at position B30 is a non-codable, lipophilic amino acid having from 10 to 24 carbon atoms, in which case an acyl group of a carboxylic acid with up to 5 carbon atoms is bound to the e-amino group of LysB 29 any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys, in Swhich case the E-amino group of Lys" 29 has a lipophilic substituent or deleted, in which case the E-amino group of LySB 29 has a lipophilic substituent; and any Zn 2 complexes thereof,provided that when Xaa at position B30 is Thr or Ala, Xaa at positions A21 and B3 are both Asn, and Xaa at position Bl is Phe, then the insulin derivative is a Zn 2 complex.

In one preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid residue is deleted or is any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; the A21 and the B3 amino acid residues are, independently, any amino acid residues which can be coded for by the genetic code except Lys, Arg and Cys; Phe 1 may be deleted; the E-amino group of LysB 2 has a lipophilic substituent which comprises at least 6 carbon atoms; and 2-4 Zn 2 ions may be bound to each insulin hexamer with the S: proviso that when B30 is Thr or Ala and A21 and B3 are both Asn, and PheB' is not deleted, then 2-4 Zn 2 ions are bound to each hexamer of the insulin derivative.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid residue is deleted or is any amino acid residue which can be coded for by 35 the genetic code except Lys, Arg and Cys; the A21 and the B3 •oooe amino acid residues are, independently, any amino acid residues which can be coded for by the genetic code except Lys, Arg and Cys, with the proviso that if the B30 amino acid residue is Ala or Thr, then at least one of the residues A21 and B3 is different from Asn; Phe

B

may be deleted; and the e-amino group of Lys B 29 has a lipophilic substituent which comprises at least 6 carbon atoms.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid residue is deleted or is any amino acid residue which can be coded for by the genetic code except Lys, Arg and Cys; the A21 and the B3 amino acid residues are, independently, any amino acid residues which can be coded for by the genetic code except Lys, Arg and Cys; Phe B1 may be deleted; the e-amino group of LysB 29 has a lipophilic substituent which comprises at least 6 carbon atoms; and 2-4 Zn 2 ions are bound to each insulin hexamer.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid residue is deleted.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid residue is O Asp.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid residue is 25 Glu.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid residue is Thr.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid is a lipophilic amino acid having at least 10 carbon atoms.

o o e ooooo oooo• In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid is a lipophilic a-amino acid having from 10 to 24 carbon atoms.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid is a straight chain, saturated, aliphatic a-amino acid having from to 24 carbon atoms.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid is D- or L-N'-dodecanoyllysine.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid is a-amino decanoic acid.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid is a-amino undecanoic acid.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid is a-amino dodecanoic acid.

20 In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid is a-amino tridecanoic acid.

S. In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid is a-amino 25 tetradecanoic acid.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid is a-amino pentadecanoic acid.

8 In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid is a-amino hexadecanoic acid.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B30 amino acid is an aamino acid.

In another preferred embodiment, the invention relates to a human insulin derivative in which the A21 amino acid residue is Ala.

In another preferred embodiment, the invention relates to a human insulin derivative in which the A21 amino acid residue is Gln.

In another preferred embodiment, the invention relates to a human insulin derivative in which the A21 amino acid residue is Gly.

In another preferred embodiment, the invention relates to a human insulin derivative in which the A21 amino acid residue is Ser.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B3 amino acid residue is Asp.

In another preferred embodiment, the invention relates to a human insulin derivative in which the B3 amino acid residue is .Gln.

25 In another preferred embodiment, the invention relates to a human insulin derivative in which the B3 amino acid residue is Thr.

*o In another preferred embodiment, the invention relates to a human insulin derivative in which the e-amino group of Lys" 29 has a lipophilic substituent which is an acyl group corresponding to a carboxylic acid having at least 6 carbon atoms.

s In another preferred embodiment, the invention relates to a human insulin derivative in which the E-amino group of LysB 29 has a lipophilic substituent which is an acyl group, branched or unbranched, which corresponds to a carboxylic acid having a chain of carbon atoms 8 to 24 atoms long.

S0lo In another preferred embodiment, the invention relates to a human insulin derivative in which the e-amino group of LysB 29 has a lipophilic substituent which is an acyl group corresponding to a fatty acid having at least 6 carbon atoms.

In another preferred embodiment, the invention relates to a human insulin derivative in which the E-amino group of Lys" 2 9 has a lipophilic substituent which is an acyl group corresponding to a linear, saturated carboxylic acid having from 6 to 24 carbon atoms.

In another preferred embodiment, the invention relates to a human insulin derivative in which the E-amino group of Lys B29 has O a lipophilic substituent which is an acyl group corresponding to a linear, saturated carboxylic acid having from 8 to 12 carbon atoms.

o In another preferred embodiment, the invention relates to a 25 human insulin derivative in which the e-amino group of LysB 2 has 16 0 a lipophilic substituent which is an acyl group corresponding to a linear, saturated carboxylic acid having from 10 to 16 carbon atoms.

*oo In another preferred embodiment, the invention relates to a 30 human insulin derivative in which the e-amino group of LysB 29 has a lipophilic substituent which is an oligo oxyethylene group comprising up to 10, preferably up to 5, oxyethylene units.

In another preferred embodiment, the invention relates to a human insulin derivative in which the e-amino group of Lys" 29 has a lipophilic substituent which is an oligo oxypropylene group comprising up to 10, preferably up to 5, oxypropylene units.

In another preferred embodiment, the invention relates to a human insulin derivative in which each insulin hexamer binds 2 Zn 2 ions.

o0 In another preferred embodiment, the invention relates to a human insulin derivative in which each insulin hexamer binds 3 Zn 2 ions.

In another preferred embodiment, the invention relates to a human insulin derivative in which each insulin hexamer binds 4 Zn 2 ions.

In another preferred embodiment, the invention relates to the use of a human insulin derivative according to the invention for the preparation of a medicament for treating diabetes.

SIn another preferred embodiment, the invention relates to a pharmaceutical composition for the treatment of diabetes in a patient in need of such a treatment comprising a therapeutically effective amount of a human insulin derivative according to the invention together with a pharmaceutically acceptable carrier.

In another preferred embodiment, the invention relates to a pharmaceutical composition for the treatment of diabetes in a patient in need of such a treatment comprising a therapeutically effective amount of a human insulin derivative according to the invention, in mixture with an insulin or an *O insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier.

In another preferred embodiment, the invention relates to a pharmaceutical composition comprising a human insulin derivative according to the invention which is soluble at physiological pH values.

In another preferred embodiment, the invention relates to a pharmaceutical composition comprising a human insulin derivative according to the invention which is soluble at pH values in the interval from about 6.5 to about In another preferred embodiment, the invention relates to a protracted pharmaceutical composition comprising a human insulin derivative according to the invention.

In another preferred embodiment, the invention relates to a pharmaceutical composition which is a solution containing from about 120 nmol/ml to about 1200 nmol/ml, preferably about 600 nmol/ml of a human insulin derivative according to the invention.

In another preferred embodiment, the invention relates to a method of treating diabetes in a patient in need of such a treatment comprising administering to the patient a therapeutically effective amount of an insulin derivative according to this invention together with a pharmaceutically acceptable carrier.

0@ S 25s In another preferred embodiment, the invention relates to a method of treating diabetes in a patient in need of such a treatment comprising administering to the patient a therapeutically effective amount of an insulin derivative according to this invention, in mixture with an insulin or an 3o insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier.

oVoooo ooooo Examples of preferred human insulin derivatives according to the present invention in which no Zn 2 ions are bound are the following:

N'

29 trideciy des(B30) human insulin, N18 29 -tetradecanoyl des(B30) human insulin, N tB 2 9 -decanoyl des(B30) human insulin, N IB 2 9 -dodecanoyl e(3)hmnisl, Nt29trdeanylGl"des (B3 0) human insulin, N t1 2 9 -terdecanoyl ly2 e(3)hmnisi, lo NB29 decaoyl Gl yA des (B30) human insulin, N B 2 9 -tetdecanoyl GyA 2 l des(B30) human insulin,

N'

92 9 -decanoyl G lyA 2 1G8 des(B 0) human insulin, N c8 2 9 tdordecanoyl Gl yA2 B des (B30) human insulin, N ,8 2 9 -tdecanoyl GlyAi Gn 3 des(B30) human insulin, 1N6B 2 9 -tetdecanoyl Gl yA21 Gin 3 des(B) human insulin, N cB 29 -trdecanoyl GlA2 Gi 9 des (B30) human insulin,

N'

29 -dordecanoyl lA2 1 Gi 9 des(B30) human insulin,

N'

2 9 -tdecanoy Aa A des(B30) human insulin, N B 2 9 -ttdecanoyl Aa A des(B0) human insulin, 2N I 2 9 -trdecanoyl Ala A 'G B des(B30) human insulin, N eB 2 9 -dotrdecanoy Ala A 1G 1 des (B30) human insulin, 13 2 9 -tdecanoyi l AlaA' Gin 3 des(B30) human insulin, -tetra-d decanoyl Aa A Gin 3 des(B30) human insulin, NB29 -tdecanoy ll Gin 3 des(B30) human insulin, 25£9(29-dotdecanoy ll Gin 3 des(B30 human insulin, N IB 2 9 -tdecanoyl Gn 3 des(B30) human insulin, N"829 -ddecanoyl Gin 3 des(B30) human insulin,

N'

0 'r-ddecanoyl Gl 3 dsB) human insulin, N B£29 -terdecanoyl Gl yA2 human insulin, IB2

A

3o N 9 2 -trdecanoyl G l1 human insulin 680 2 9 -dcny GyA 2 hmnisi,

N'

2 -dodecanoyl GlyA1 human insulin, N B29-tridecanoyl G lyA2i Gln B 3 human insulin, N ,8 2 9 -tetradecanoyl GlyAll Gin 9

B

3 human insulin, N 8 2 9 -decanoyl Gl yA 2 l Gin 9

B

3 human insulin, 35 N 8 2 9 -dodecanoyi Gl yA2i Gln 1 3 human insulin, N'8 2 9 tridecanoyl Ala A21 human insulin, Nc' 29 -tetradecanoyl AlaA 2 1 human insulin, N18 29 decanoyi AlaA21 human insulin, N"8 2 9 -dodecanoyl Ala A 2 human insulin, N" 2 9 -tridecanoyi Ala A2 Gln1 3 human insulin, N B 2 9 -tetradecanoyl AlaA 2 1 GlnB 3 human insulin, N" 2 9 -decanoyl Ala A 2 GinB 3 human insulin,

N"B

2 9 -dodecanoyi Ala A 2 1 Gln 9 3 human insulin, N" 9 -tridecanoyi GlnB 3 human insulin, N"8 2 9 -tetradecanoyl Gln 9 3 human insulin, l0 NB 29 -decanoyl Gin 3 human insulin, N 8 2 9 -dodecanoyl Gin' 3 human insulin, N 2 9 -tridecanoyl Gu 3 0 human insulin, N'8 2 9 -tetradecanoyl GlUB 3 0 human insulin, N8 2 9 -decanoyl

GUB

3 0 human insulin, N I 2 9 -dodecanoyi GluB 3 0 human insulin,

N

2 9 -tridecanoyl GlyA 2 l GlU1 30 human insulin,

NB

29 -tetradecanoyl GlyA 2 1 Glu 30 human insulin, N"8 2 9 -decanoyi GlA 2 GlUB 30 human insulin, Nc8 2 9 -dodecanoyl Gly 1 2' Glu1 30 human insulin,

NB

2 9 -tridecanoyl GlyA 2 l Gln8 3 GlUB 30 human insulin, N 2 9 -tetradecanoyl Gly2i GlnB 3 Glu 30 human insulin,

N'

2 9 -decanoyl GlyA 2 l Gln' 3 G1UB 30 human insulin, N 1 29 -dodecanoyl GlyA 21 Gin' 3 Glu1 30 human insulin, N 2 9 -tridecanoyl AlaA 2 1 Glu 3 0 human insulin,

NB

2 9 -tetradecanoyl Ala A 2 GluB 3 0 human insulin, N B 2 9 -decanoyl Ala A2 GlUB 30 human insulin,

NB

2 9 -dodecanoyl AlaA2 Gu1 30 human insulin,

N

2 9 -tridecanoyi AlaA21 Gln1 3 Gl' 3 0 human insulin, N B 2 9 -tetradecanoyl AlaA 1 Gln 93 Gl u 3 0 human insulin,

NB

2 9 -decanoyl Ala A 2 Gin' 3

GUB

3 0 human insulin, Nt*N 2 9 -dodecanoyl AlaA2z Gin 3 GuB 30 human insulin, N 8 2 9 -tridecanoyl Gin' 3 Glu 3 0 human insulin, N B 2 9 -tetradecanoyi Gin' 3 GlU1 30 human insulin, N 8 2 9 -decanoyl GlnB 3 Glu 3 0 human insulin and

NB

29 -dodecanoyi Gln 93

GUB

3 0 human insulin.

Examples of preferred human insulin derivatives according to the present invention in which two Zn 2 ions are bound per insulin hexamer are the following: (N B 9 tridecanoyl des(B30) human insulin) 6 2Zn 2

NB

29 ttaeao1dsBJ)hmnisln 6 2n 2 tB 2 adecanoyl des(B30) human insuln) 6 2Zn

(N'

29 -decanoyl des(B30) human isln6, 2Zn 2 (NtB 2 9 -trdecanoyl GlYAB30 deuman0 hnuan)6 2Zn2Zn (NtB 2 9 trdecanoy GlyA 2 l des(B30) human insulin) 6 2Zn 2 1(NB 9 ttdecanoyl Gl des(B30) human insulin) 6 2Zn 2 l(NIB 29 decanoyl GyA 2 l des(B30) human insulin) 6 2Zn 2 2 9 trdecanoy1 G1/A 21 Gn des(B) human insulin) 6 2Zn 2

(NB

29 trdecanoy Gy 21 Gln' des(B30) human insulin) 6 2Zn 2 (NtB 29 -ttdecanoyl G1/ 2 1 GlnB I des(B30) human insulin) 6 2Zn 2 1(NtB 2 9 decanoyl GlyA 2 Gln 3 des(B30) human insulin) 6 2Zn 2 1(N,,B 2 9 -trdecanoyl laA 2 ln des(B30) human insulin) 6 2Zn 2

(N'B

2 9 .trdecanoyl AlaA 2 l des(B30) human insulin) 6 2Zn 2

I

(B

29 ~ddcny A A 2 e(3)hmnisln 6 2 8 2 -trdecanoyl Ala~ 1l 9 des(B30) human insulin) 6 2Zn

N

6 2 9 ttrdcny Aa~ 2l+ e(3)hmn nui),2n~

(NED

2 9 decanoyl AlaA~ 1l des(B30) human insulin) 6 2Zn 2

(B

29 ~ddcny

AA

2 l l 9 e(3)hmnisln 6 2Zn 2 -trdecanoyl AlnB1 des(B30) human insulin) 6 2Zn 2 2(NB 29 ttrdcny A2n 6 1e(30)hmnisln 6 2 2(N" 2 -trdecanoyl A1 Gln des(B30) human insulin) 6 2Zn

:(NE

2 9 dotrdecanoyl A2 GlnB 3 des(B30) human insulin) 6 2Zn 2 ',1 t8' 2 9 -tidcAo l huma inuin 6 2Z *(NEB trdecanoyl a1GndB0 human insulin) 6 2Zn 0 B' 29 -dcny huA inuin 6 2+~ (N 9 ~dodecanoyl Aa'GnsB0 human insulin) 6 2Zn (N 9 2 -tridecanoyl Gln ds(0 human insulin) 6 2Zn (N -tetradecanoyl GlyA 2 human insulin) 6 2Zn

(NEB

2 9 -decanoyl GlA 2 (0 human. insulin) 6 2Zn 35(N" 2 -dodecanoyl Gln ds(0 human insulin) 6 2Zn (NtB 2 9 tridecanoyl Gy 2 l 9 human insulin) 6 2Zn 2 (N 8 2 9 -tetradecanoyl G lyA2l Gin 9 8 3 human insulin) 6 2Zn 2 (N 29 -decanoyl Gl1yA2 1 Gijn 9 3 human insulin) 6 2Zn 2 (N 9 2 9 -dodecanoyl GlyAll Gin 9 1 3 human insulin) 6 2Zn 2

(N'B

29 -tridecanoyl Ala A 2 1 human insulin) 6 2 Zn 2

NB

29 terdcny A2~ ua isln 6 2n 2 9 2 -edecanoyl Ala human insulin) 6 2Zn (N8 2 9 -dd ny Aa 2 lhmnisi) 6 2 2 (N 9 2 -decanoyl AlaA~ Gi1 human insulin) 6 2Zn 2 (t 92 9 -eadcny Aa 2 l Gi 9 2uanisln),2n+ 6 2 -decanoyl Ala Gi1 human insulin) 6' 2Zn 2 18' 2 9 -ddcny

AA

2 3 hmnisl) 6 n 2

(N'

9 2 -tridecanoylAl Gin human insulin) 6 2Zn ,,8 2 9 -t~rdcny Gin2 huma inuin 6 2 (N tetr-decanoya Gin human insulin) 6 2Zn

N'

29 -d cny i 9 ua inuin3 2Z 2 l(N 9 2 -decanoyl Aa2 Gin human insulin) 6 2 Zn

BN~

2 9 -erdcny A2 6 3 hua 3 2Z 2 2 -ddecanoyl l1 3

G

0 human insulin) 6 2Zn 2(N" 2 9 tridecanoyl Gyi Gu human insulin) 6 2Zn

(N'

9 2 -tetradecanoyl GlyinGu human insulin) 6 2Zn 2

(NB

29 ~dcny BiA~ 3i 0 hmn nui)6 2Zn+

(N'

9 2 -ddecanoyi Al G l 3 human insulin) 6 2Zn 2 (N -dottdecanoyl G~ Gin 9 Gi 3 human insulin) 6 2Zn 2 2 -trdecanoyl Gy 2 Gin 9 i 9 0 human insulin) 6 2Zn 2 (N18 29 -dodecanoyl Glyl GU8 Gu 3 0 human insulin) 6 2Zn 2+, 2(NIB 29 tridecanoyl GAlaA21 Gl UB 30 human insulin) 6 2 Zn 2 (N iB 2 9 tetradecanoyl GlI 1GilU 30 human insulin) 6 2Zn 2 3(NCB 2 9 -decanoyl Al A 2 GlU 30 human insulin) 6' 2Zn 2 8 29 -dodecanoyl GAlA 2 G lUB 3 0 human insulin 6 2Zn 2

IB

29 tridecanoyl AlA~l 1Gin 9

B

3 GlU 30 human insulin) 6 2Zn 2 6B2-tezaecanoyl AlA 2 Gin 3 G 1U 9 30 human insulin 6 2Zn 2 *(N,8 2 9 -decanoyl Al A 2 l Gi1n 3 G1iU 30 human insulin) 6 2Zn 2 3(N18 2 9 -dodecanoyl Al A 2 1 GinB 3 Glu' 30 human insulin) 6 2Zn 2

*(NIB

2 9 -tridecanoyl Gin 9 3 2 G lU 3 0 human insulin) 6 2Zn 2

B

2 9 -tetradecanoyi Gin 9 3 2 Gi 9 31U 30 human insulin 2 n 2 Zn2 N*2 e a o l A a A G n B G 1 0 h m n i s l n 6 z (N 8 29 -decanoyl Gin 6 8 3 GlUB 30 human insulin 6 2Zn 2 and (N B 2 9 -dodecanoyl. Gin 6

B

3 G1lU8 30 human insulin 6 2Zn 2 Examples of preferred human insulin derivatives according to the present invention in which three Zn 2 ions are bound per insulin hexamer are the following:

(N

6 9 tridecanoyl. des (B3 0) human isln6, 3Zn, (N8 2 9 ttrdcnyde(B0)hmnisln 6 3 n 2 (N tetr-decanoyl des(B30) human insulin) 6 3Zn 29 -ddecanoyl des(B30) human isln6, 3Zn 2 1(N" B 2 9 -trdecanoyl Al des(B30) human insulin) 6 3Zn l(N B 2 9 trdecanoyl GlyA1 des (B30) human insulin 6' 3Zn 2 (N 'E 29 -ttdecanoyl G 1yA des(B30) human insulin) 6 3Zn 2% (629 -eaolG A2 1 dsB0 ua nui),3n2 (N 2-dodecanoyj. G lyA1 des(B30) human insulin) 6 3Zn 2 (N'8 2 9 -tridecanoyl G1lyA 2 Gin 6

B

3 des (B3 0) human insulin 6' 3Zn 2 (N'8 29 -tetradecanoyl G lyA2 Gin 6

B

3 des(B30) human insulin) 6 3Zn 2 (N tB 29 -decanoyl Gl1yA 2 1 Gin 6

B

3 des(B30) human insulin) 6 3Zn 2 (N B 2 9 -dodecanoyi G1lyA 2 l Gin 6 8 3 des (B3 0) human insulin) 6' 3Zn 2

(N'

2 9 tidcny AlA 2 l e( )hmn6 3Z 2 (N 6 2 -trdecanoyl AlaA 1 des(B30) human insulin) 6 3Zn 2 t0E1~ 2 9 -dcny A2A~ 2e(3)hmninui),3n+ (N tet-dodecanoyl Aa 1 des(B30) human insulin 6 3Zn 2(N ,1 29 -tdecanoy Ala A 2 6 des(B30) human insulin) 6 3Zn 2

,N'B

29 -trdcny

A

2 Gi 6 2e(30 ua nuln 6

Z

2 (N 2 -ddecanoyl Ala~ Gi 6 des(B30) human insulin) 6 3Zn 2 25 (N 29 _ddc~j lA21 Gin 3 de(3)hmnisln 6

Z

2 6 9 tridecanoyl Gin 63 l des(B30) human insulin) 6 3Zn (Nt29 tetacaloyl Gin 6 1l 3 des(B30) human insulin) 6 3n 2 2+

(N'

62 -decanoyl Alin 63 l des(B30) human insulin 3n, Z (N -dotrdecanoylAl ln'sB0 human insulin) 6 3Zn

(N'

2 9 -terdcny huma 2+3Z 629 -decano humn de(3hmninsulin) 6 )6,n 2

Z

(N -te-dodecanoyl.GnsB0 human insulin) 6 3Zn (N 2 -trdecanoyi GiYA 2 (0 human insulin) 6 3Zn 3~(N 6 9 tetradecanoyi Al human insulin) 6 3Zn 2 c62 2 (N d c n yl h m n n ul n) Z

(N'B

2 9 -decanoyi GlyA 2 l human insulin) 6 3Zn 2

(N'

2 9 dodecaniy GlyA 2 l human insulin) 6 3Zn 2 (N'8 2 9 -tridecanoyl GlyA21 Gin" 3 human insulin) 6 3 zn 2 (Nt 2 9 -tetradecanoyl GlyA 2 l Gin 93 human insulin) 6 3Zn 2

(N"

29 -decanoyi GlyA 2 l Gin 9 3 human insulin) 6 3Zn 2 (NlB 29 -dodecanoyl GlyA 2 l Gin 9 1 3 human insulin) 6 3Zn 2

(N'

2 9 tridecanoyl AlaA 1 human insulin) 6 3Zn, 8N~ 2 9 -erdcny A2? umn6 3Z 2 tet-decanoyl Ala human insulin) 6 3Zn 1(N'8 29 -ddecanoyl AlaA~ human insulin) 6 3Zn 2 l(N 9 9 -tetdecanoyl AlaA~ Gi1 human insulin) 6 3Zn

BN'

29 -d~cny A A 2 Gin 3 hua nui)6 Z 2 (Nt triddecanoyl Ala 1 Gin 3 human insulin) 6 3Zn~ 15~~A B3 9 9 tidcny 2lB uanisln),3n (NtB 2 9 -tetradecanoylAl1 Gin human insulin) 6 3Zn (N6 2 9 -dcanoyl A A GinB 3 human insulin) 6 3Zn 2

B

2 9 -dodecanoyl Gin 3 humn iunin 6 3n)6, Z2 (N -tridecanoyl Gin 93 human insulin) 6 3Zn, 2(N 9 2 -tetradecanoyl Glun 3 human insulin) 6 3Zn 2 (NtB 2 9 -decanoyl Glun 3 human insulin) 6 3Zn 2

(NEB

2 9 dodecanoyl Glun 3 human insulin) 6 3Zn 2 (N B 2 9 -tridecanoyl GlUBl u 30 human insulin) 6 3Zn 2(N 8 2 9 tetradecanoyl GlyUB u 30 human insulin) 6 3Zn 2 2(N'8 29 -decanoyl G lyUB u 30 human insulin) 6' 3Zn 2 (NcB 9 dodecanoyl Gll u 30 human insulin) 6 3Zn 2 (N,8 29 -tridecanoyl GlyA2 1 Gi 9 Giu 9 human insulin) 6 3Zn 2 (N 8 2 9 tetradecanoyl. GlyA 2 I i' Glu 30 human insulin) 6 3Zn 2 2(N' 9 2 9 -decanoyl GlyA 2 l i Gl 30 human insulin) 6 3Zn 2 0(N B 2 9 -dodecanoyJ. Gl yA21Gi Glu 9 3 0 human insulin) 6 3Zn tB 2 9 -tridecanoyl AiaA 2 l in GluB 30 human insulin) 6 3Zn 2+, N t 2 9 -tetradecanoy. AlaA2 lnB3 93 0 3 human insul i) 6 3Z 2 n2* (Nt 9 2 9 -decanoyl AlA 2 Glu'

U

3 0 human insulin) 6 3Zn 2 (N tB 2 9 -doidecanoyl Ala A2' G lU 9 30 human insulin) 6 3Zn 2 3(N"8 2 9 -tdecanoyJ. Ala A 2 9 Glu 3 0 human insulin) 6 3Zn (N"8 2 9 -trdecanoyl Ala A1 Gin 3 GlU 30 human insulin) 6 3Zn 2

(N"

29 -tedecanoy Ala A2 GinB 3 Gl 30 human insulin) 6 3Zn 2+ N B 9 d c n y *l A 1 G nB U 3 u a n u i 3 n

(N'

62 9 -dodecanoyl AlaA' Gin 6 3 Glu 3 human insulin 6 3Zn 2

(N

6 -tiecnylGn 3 Gu 30 hmnnsl), 3Z 2

(N'B

29 -trdecanoyl Gn 6 Glu 0 human insulin) 6 3Zn (N B 2 9 -decanoyl Gin 6

B

3 G lUB 30 human insulin) 6 3Zn 2 and (N 18 2 9 -dodecanoyl Gin 6

B

3 GlU11 3 0 human insulin) 6 3Zn 2 Examples of preferred human insulin derivatives according to the present invention in which four Zn 2 ions are bound per insulin hexamer are the following: io(N 62 -trdecanoyl des(B30) human insulin) 6 4Zn 2

BN'

29 -eany de2+)hmninui),4n~ l(NI tetdodecanoyl des (B30) human insulin) 6 4n t8' 29 -rdcny 2lAidsB0+uanisln 6

Z

2 (N 6 9 -trdecanoyl y~ des(B30) human insulin 6 4Zn 2 t5 N3 29 -dcny.Gy~ e(3)hmnisln 6 4 2 (Ne 6 2 -dodecanoyl Gy~ des(B30) human insulin) 6 4Zn 2 (N 18 2 9 tridecanoyi GlyA2iGi des(B30) human insulin 6 4Zn (NE8 2 9 -tetradecanoyl GlyA2iGi des(B30) human insulin) 6 4Zn 2 1(N 8 2 9 -decanoyl GlyA2iGi des(B30) human insulin) 6 4Zn 2(N' 6

B

2 9 -dodecanoyl GlyA2iGi des(B human insulin) 6 4Zn 2 (N B-tridecanoyl GA G des(B30) human insulin) 6 4Zn 2+, (N tB 2 9 tetradecanoyl AlA21 ln des(B30) human insulin) 6 4Zn 2 (N 6 2 9 -decanoyl AlaA21 ln des(B30) human insulin) 6 4Zn 2+, 2(N 629 -dodecanoyl AlaA21 ln des(B30) human insulin) 6 4Zn 2+, 2(N' 2 9 tridecanoyl Ala A2 Gi1 des(B30) human insulin) 6 4Zn

(NB

2 9 -terdcny A2 Gi 6 2e(+)hmn nui),4n~ (N ttr-decanoyl Ala Gi 6 des(B30) human insulin 6 4Zn 2 (N 29 -ddecanoyi Ala A21G 6 des(B3 human insulin) 6 4Zn 2 iB 2 9 -trdecanoyl Gin 6 3 2 des(B30) human insulin) 6 4Zn 2 0 N' 29 -terdcny Gin 6 dB30 hua2nsln 6 ,4n 2(N t 29 -decAnJ. Gin 63 G des(B30) human insulin )6Zn 2 (N -te-dodecanoyl Gin 63 G des(B30) human insulin) 6 4Zn (NI 8 29 recanoyl haA2'n B e(3hmninsulin) 6 ,4Zn,2 6 2 -trdecanoyl Gn dB human insulin) 6 )6,n 2

Z

*4 35(N 6 2 -decanoyl l eB0 human insulin) 6 4Zn (N 1 29 -dodecanoyl human insulin) 6 4 Zn'+, ("tridecanoyi GlyA 2 1 human insulin) 6 4Zn 2 (N B 2 9 -tetradecanoyl GlyA 2 I human insul in) 6 4 Zn 2 (N18 29 decanoyl Gl y 21 human insulin) 6 4Zn 2 (N'8 29 -dodecanoyl Gl yA 2 l human insulin) 6 4Zn 2 (N B 2 9 -tridecanoyl Gl yA 2 1 Gin'B 3 human insulin) 6 4Zn 2 (N'6 2 9 -tetradecanoyl G1lyA 2 1 Gin'B 3 human insulin) 6 4 Zn 2 (N 1 2 9 -decanoyl G lyA2i Gin'B 3 human insulin) 6 4Zn 2 (N I 2 9 -dodecanoyl Gl yA 2 1 Gin 9 3 human insulin) 6' 4Zn 2

(N"

2 9 tridecanoyl AlaA 1 human insulin) 6 4Zn

(N"

2 9 -terdcny AA 2 lhmnisi) 6 4Z 2 (N tetadecanoyl Ala human insulin) 6 4Zn

(NIB

29 -ddecanoyl Ala A 2 human insulin) 6 4Zn 2 9 -trdecanoyl AlaA~ Gi human insulin) 6 4Zn 2 9 -terdcny Aa 2 l Gin 3 hanisl), Z 2 (N tri-decanoyl Ala 1 Gin human insulin) 6 4Zn

(N"

2 9 -ddcny A A 2 Gin 3 hmn64n 2 1(N 2 -terdecanoylAl1 Gin human insulin) 6 4Zn (N82 -ttAecny GB 3 hmnisl),4n 2 2(N 2 -decanoylAl1 Gin human insulin) 6 4Zn (N 2 -dodecanoylAl1 Gin human insulin) 6 4 n 2

(N"

2 9 -rdcny Blu 3 0 hmnisl) 6 4 n 2 (N 2 -trdecanoyl Glu human insulin) 6 4Zn 2 (N 2 -tedecanoyl Gn human insulin) 6 4Zn S 25 (N 2 -ddecanoyl Glu 3 0 human insulin) 6 4Zn (N -trdecanoyl Glyn G 3 human insulin) 6 4Zn 2 29 -tetradecanoyl G lUB~ 30 human insulin 6 4Zn 2 2, (N 1 2 9 -decanoyl G lUAlu 30 human insulin) 6 4Zn 2(N 1 2 9 -dodecanoyi G lyUB u 30 human insulin) 6'n 2 Zn 2 3(NB 2 9 -tridecanoyi Gl yA2' GinB 3 Ghuman huaninsuli.n 624Z,~ 2 9 -tetradecanoyl G lyA 2 Gn 3 lUuB 30 human insulin) 6 4Zn

(NIB

2 9 -decanoyl Gl yA 2 l Gn 3 U1 9 30 human insulin) 6 4Zn 8N 2 9 -d cny GlyA 2 l GiUBu 30 human insulin) 6 4Zn 2 (N"-tridecanoyi GlaAI Glu 3 hmainln),4n, 3(N" 2 -tetradecanoyi G1A~ Ginu' 3 0 human insulin 4Zn

(NIB

29 -decanoyi AiaA 2 1 ln Glu 8 30 human insulin) 6 4Zzn2+ *(N'6 2 9 -decanoyl Ala A GiUB 30 human insulin)6' 4Zn 2 29 -tridecanoyl AlaA21 Gln" Glu B30 human insulin) 6 4Zn 2 (NB29-tetradecanoyl AlaA21 Gn" 3 Glu B3 0 human insulin) 6 4Zn 2 (N B 29 -decanoyl AlaA 2 1 Gln.

3 Glu B 30 human insulin)6, 4Zn 2

(NB

29 d-dodecanoyl AlaA 2 1 Gin" 3 Glu 3 0 human insulin) 6 4Zn 2 (N s 29 -tridecanoyl Gin" 3 Glu 30 human insulin) 6 4Zn 2

(NB

29 -tetradecanoyl Gin" 3 Glus 30 human insulin),, 4Zn 2 (N 29 -decanoyl Gin" 3 Glu s3 0 human insulin)6 4Zn 2 and

(N'B

29 -dodecanoyl Gin" 3 Glu 3 0 human insulin) 6 4Zn 2 BRIEF DESCRIPTION OF THE DRAWINGS The present invention is further illustrated with reference to the appended drawings wherein Fig. 1 shows the construction of the plasmid pEA5.3.2; Fig. 2 shows the construction of the plasmid pEA108; and Fig. 3 shows the construction of the plasmid pEA113.

DETAILED DESCRIPTION OF THE INVENTION i Terminology The three letter codes and one letter codes for the amino acid residues used herein are those stated in J. Biol. Chem. 243, p.

S3558 (1968).

S 20 In the DNA sequences, A is adenine, C is cytosine, G is guanine, and T is thymine.

The following acronyms are used: DMSO for dimethyl sulphoxide, DMF for dimethylformamide, Boc for tert-butoxycarbonyl, RP-HPLC for reversed phase high performance liquid chromatography, X-OSu is an Nhydroxysuccinimid ester, X is an acyl group, and TFA for trifluoroacetic acid.

ooo .go Preparation of lipophilic insulin derivatives The insulin derivatives according to the present invention can be prepared i.a. as described in the following: 1. Insulin derivatives featuring in position B30 an amino acid residue which can be coded for by the genetic code, e.g.

threonine (human insulin) or alanine (porcine insulin).

1.1 Starting from human insulin.

Human insulin is treated with a Boc-reagent di-tert-butyl dicarbonate) to form (Al,Bl)-diBoc human insulin, human o0 insulin in which the N-terminal end of both chains are protected by a Boc-group. After an optional purification, e.g.

by HPLC, an acyl group is introduced in the e-amino group of Lys" 2 by allowing the product to react with a Nhydroxysuccinimide ester of the formula X-OSu wherein X is the acyl group to be introduced. In the final step, TFA is used to remove the Boc-groups and the product, NaB 29 -X human insulin, is isolated.

1.2 Starting from a single chain insulin precursor.

A single chain insulin precursor, extended in position B1 with an extension (Ext) which is connected to Bl via an arginine residue and in which the bridge from B30 to Al is an arginine residue, i.e. a compound of the general formula Ext-Arg-B(l- 30)-Arg-A(l-21), can be used as starting material. Acylation of this starting material with a N-hydroxysuccinimide ester of the S 25 general formula X-OSu wherein X is an acyl group, introduces the acyl group X in the e-amino group of Lys B29 and in the Nterminal amino group of the precursor. On treating this acylated precursor of the formula (N' 5 29 -X),X-Ext-Arg-B(1-30)- *OOO4 Arg-A(l-21) with trypsin in a mixture of water and a suitable water-miscible organic solvent, e.g. DMF, DMSO or a lower alcohol, an intermediate of the formula (NR9-X),ArgB 3 1 insulin is obtained. Treating this intermediate with carboxypeptidase B yields the desired product, (N'8 29 insulin.

2. Insulin derivatives with no amino acid residue in position i.e. des(B30) insulins.

2.1 Starting from human insulin or porcine insulin.

On treatment with carboxypeptidase A in ammonium buffer, human 0o insulin and porcine insulin both yield des(B30) insulin. After an optional purification, the des(B30) insulin is treated with a Boc-reagent di-tert-butyl dicarbonate) to form (A1,Bl)diBoc des(B30) insulin, des(B30) insulin in which the Nterminal end of both chains are protected by a Boc-group. After an optional purification, e.g. by HPLC, an acyl group is introduced in the e-amino group of LysB 29 by allowing the product to react with a N-hydroxysuccinimide ester of the formula X-OSu wherein X is the acyl group to be introduced. In the final step, TFA is used to remove the Boc-groups and the product,

(NEB

29 des(B30) insulin, is isolated.

2.2 Starting from a single chain human insulin precursor.

A single chain human insulin precursor, which is extended in position Bl with an extension (Ext) which is connected to Bl via an arginine residue and which has a bridge from B30 to Al can be a useful starting material. Preferably, the bridge is a peptide of the formula Yn-Arg, where Y is a codable amino acid except lysine and arginine, and n is zero or an integer between 1 and 35. When n>l, the Y's may designate different amino acids. Preferred examples of the bridge from B30 to Al are: 30 AlaAlaArg, SerArg, SerAspAspAlaArg and Arg (European Patent No.

*ot 163529). Treatment of such a precursor of the general formula Ext-Arg-B(l-30)-Yn-Arg-A(1-21) with a lysyl endopeptidase, e.g.

Achromobacter lyticus protease, yields Ext-Arg-B(l-29) Thr-Y Arg-A(l-21) des(B30) insulin. Acylation of this intermediate with a N-hydroxysuccinimide ester of the general formula X-OSu wherein X is an acyl group, introduces the acyl group X in the e-amino group of Lys 6 29 and in the N-terminal amino group of the A-chain and the B-chain to give (NcB 29 X-Ext-Arg-B(l-29) X- Thr-Yn-Arg-A(l-21) des(B30) insulin. This intermediate on treatment with trypsin in mixture of water and a suitable organic solvent, e.g. DMF, DMSO or a lower alcohol, gives the desired derivative, (NcB 29 des(B30) human insulin.

Data on NEB 2 modified insulins.

Certain experimental data on NEB 29 modified insulins are given in Table 1.

The lipophilicity of an insulin derivative relative to human insulin, k're, was measured on a LiChrosorb RP18 (51.m, 250x4 mm) HPLC column by isocratic elution at 40°C using mixtures of A) 0.1 M sodium phosphate buffer, pH 7.3, containing acetonitrile, and B) 50% acetonitrile in water as eluents. The elution was monitored by following the UV absorption of the eluate at 214 nm. Void time, to, was found by injecting 0.1 mM sodium nitrate. Retention time for human insulin, thnn was adjusted to at least 2to by varying the ratio between the A and B solutions. k'reL= (tderivative-t) (th n-to) S..The degree of prolongation of the blood glucose lowering effect was studied in rabbits. Each insulin derivative was tested by subcutaneous injection of 12 nmol thereof in each of six f rabbits in the single day retardation test. Blood sampling for glucose analysis was performed before injection and at 1, 2, 4 Sand 6 hours after injection. The glucose values found are expressed as percent of initial values. The 'Index of Protraction, which was calculated from the blood glucose values, is the scaled Index of Protraction (prolongation), see p. 211 in Markussen et al., Protein Engineering 1 (1987) 205- 213. The formula has been scaled to render a value of 100 with bovine ultralente insulin and a value of 0 with Actrapid* insulin (Novo Nordisk A/S, 2880 Bagsvaerd, Denmark).

The insulin derivatives listed in Table 1 were administered in solutions containing 3 Zn2+ per insulin hexamer, except those specifically indicated to be Zn-free.

For the very protracted analogues the rabbit model is inadequate because the decrease in blood glucose from initial is too small to estimate the index of protraction. The prolongation of such analogues is better characterized by the disappearance rate in pigs. T 50 is the time when 50% of the A14 Tyr( 125 1) analogue has disappeared from the site of injection as measured with an external 7-counter (Ribel, U et al., The Pig as a Model for Subcutaneous Absorption in Man. In: M. serrano-Rios and P.J. Lefebre (Eds): Diabetes 1985; Proceedings of the 12th Congress of the International Diabetes Federation, Madrid, Spain, 1985 (Excerpta Medica, Amsterdam, (1986) 891-96).

O In Table 2 are given the T 50 values of a series of very protracted insulin analogues. The analogues were administered in solutions containing 3 Zn 2 per insulin hexamer.

*o Table 1 Insulin Derivative *)Relative Blood glucose, of initial Index of Lipophilici protraction ty 1h 2h 4h 6h

N'

62 9 -benzovl insulin 1.14 Ne8 29 -phenylacetyl insulin (Zn- 1.28 55.4 58.9 88.8 90.1 NIR29_C~oeyaey insulin 1.90 53.1 49.6 66.9 81128 NeB 2 9 cyclohexylpropionyl 3.29 55.5 47.6 61.5 73.0 39 insulin N,8 29 -cyclohexylvaleroy1 9.87 65.0 58.3 65.7 71.0 49 insulin

N,'B

29 -octanov1 insulin 3.97 57.1 54.8 69.0 78.9 33 NE8 29 -decanoy1, des(B30) 11.0 74.3 65.0 60.9 64.1 insulin

NEB

29 -decanovi insulin 12.3 73.3 59.4 64.9 68.0

NB

29 -undecanoy1, des(B30) 19.7 8.8.1 80.0 72.1 72.1 insulin

N'

62 9 -laurovl. des(B301 insulin 37.0 91.4 90.0 84.2 83.9 78 NB2 9 mvristovl insulin 113 98.5 92.0 83.9 84.5 97

N(B

2 9 -choloyl insulin 7.64 58.2 53.2 69.0 88.5 NtB 29 -7-deoxycholoy1 insulin 24.4 76.5 65.2 77.4 87.4 (Zn-free)__ N,8 2 9 -ithocholoy1 insulin (Zn- 51.6 98.3 92.3 100.5 93.4 115 free)

N'B

2 9 -4-benzoyl-phenylalanyl 2.51 53.9 58.7 74.4 89.0 14 insulin__ NE2 9 3.5diiodotrosvl insulin 1.07 53.9 48.3 60.8 27 1N( 2 9 Lthroxyl insulin 8.00 Table 2 Derivative of Relative Subcutaneous Human Insulin hydrophobicity disappearance pigs- 600 jiM, krel T 50 hours 3 Zn 2 +/hexamer, phenol 0.3%, glycerol 1.6%, N iB 29 decanoyl insulin 11.0 5.6 NtB 2 9 undecanoyl 19.7 6.9 insulin N IB 2 9 lauroyl 37 10.1 insulin N tB 2 9 tridecanoyl 65 12.9 insulin Nt8 29 myristoyl 113 13.8 insulin N tB 29 palmitoyl 346 12.4 des(B30) insulin N'8 2 9 succinimido- 10.5 13.6 myristic acid N tB 2 9 myristoyl 113 11.9 insulin Human NPH Solubility The solubility of all the N IB 29 modified insulins mentioned in Table 1, which contain 3 Zn 2 ions per insulin hexamer, exceeds 600 nmol/ml in a neutral (pH aqueous, pharmaceutical formulation which further comprises 0.3% phenol as preservative, and 1.6% glycerol to achieve' isotonicity. 600 nmol/ml is the concentration of human insulin found in the 100 IU/ml compositions usually employed in the clinic.

The e-B29 amino group can be a component of an amide bond, a sulphonamide bond, a carbamide, a thiocarbamide, or a carbamate. The lipophilic substituent carried by the e-B29 amino group can also be an alkyl group.

Pharmaceutical compositions containing a human insulin derivative according to the present invention may be administered parenterally to patients in need of such a treatment. Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means o0 of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump. A further option is a composition which may be a powder or a liquid for the administration of the human insulin derivative in the form of a nasal spray.

The injectable human insulin compositions of the invention can be prepared using the conventional techniques of the pharmaceutical industry which involves dissolving and mixing the ingredients as appropriate to give the desired end product.

Thus, according to one procedure, the human insulin derivative is dissolved in an amount of water which is somewhat less than the final volume of the composition to be prepared. An isotonic agent, a preservative and a buffer is added as required and the pH value of the solution is adjusted if necessary using an acid, e.g. hydrochloric acid, or a base, e.g. aqueous sodium 25 hydroxide as needed. Finally, the volume of the solution is adjusted with water to give the desired concentration of the ingredients.

Examples of isotonic agents are sodium chloride, mannitol and glycerol.

3o Examples of preservatives are phenol, m-cresol, methyl phydroxybenzoate and benzyl alcohol.

Examples of suitable buffers are sodium acetate and sodium phosphate.

A composition for nasal administration of an insulin derivative according to the present invention may, for example, be prepared as described in European Patent No. 272097 (to Novo Nordisk A/S).

The insulin compositions of this invention can be used in the treatment of diabetes. The optimal dose level for any patient will depend on a variety of factors including the efficacy of i0 the specific human insulin derivative employed, the age, body weight-, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the case of diabetes. It is recommended that the daily dosage of the human insulin derivative of this invention be determined for each individual patient by those skilled in the art in a similar way as for known insulin compositions.

Where expedient, the human insulin derivatives of this invention may be used in mixture with other types of insulin, e.g. human insulin or porcine insulin or insulin analogues with a more rapid onset of action. Examples of such insulin analogues are described e.g. in the European patent applications having the publication Nos. EP 214826 (Novo Nordisk EP 375437 (Novo Nordisk A/S) and EP 383472 (Eli SLilly Co.).

25 The present invention is further illustrated by the following examples which, however, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

ooee oo a*

EXAMPLES

Plasmids and DNA material All expression plasmids are of the cPOT type. Such plasmids are described in EP patent application No. 171 142 and are s characterized in containing the Schizosaccharomyces pombe triose phosphate isomerase gene (POT) for the purpose of plasmid selection and stabilization. A plasmid containing the POT-gene is available from a deposited E. coli strain (ATCC 39685). The plasmids furthermore contain the S. cerevisiae triose phosphate isomerase promoter and terminator (PTp, and TTP,). They are identical to pMT742 (Egel-Mitani, M. et al., Gene 73 (1988) 113-120) (see Fig. 1) except for the region defined by the ECoRI-XbaI restriction sites encompassing the coding region for signal/leader/product.

Synthetic DNA fragments were synthesized on an automatic

DNA

synthesizer (Applied Biosystems model 380A) using phosphoramidite chemistry and commercially available reagents (Beaucage, S.L. and Caruthers, Tetrahedron Letters 22 (1981) 1859-1869).

All other methods and materials used are common state of the O art knowledge (see, e.g. Sambrook, Fritsch, E.F. and Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989).

Analytical Molecular masses of the insulins prepared were obtained by MS (mass spectroscopy), either by PDMS (plasma desorption mass spectrometry) using a Bio-Ion 20 instrument (Bio-Ion Nordic AB, Uppsala, Sweden) or by ESMS (electrospray mass spectrometry) using an API III Biomolecular Mass Analyzer (Perkin-Elmer Sciex 30 Instruments, Thornhill, Canada). o.0* EXAMPLE 1 Synthesis of AlaA 2 1 AspB 3 human insulin precursor from Yeast strain yEAOO2 using the LaC2l2spx3 signal/leader.

The following oligonucleotides were synthesized: #98 CTTGGTTGAAG

CTTTGTACTTGGTTTGTGGTGAA

AGAGGTTTCTTCTACACTCCAJAGTCTGACGACGCT-

3 1 (SEQ ID NO:3) #128 5 '-CTGCGGGCTGCGTCTAAGCACAGTAGTTTTCCA-3JTGGTACkA

AGACAGATAGAAGTACAACATTGTTCAACGATACCCTTAGCGTC

GTCAGACTTTGG-31 (AlaA2) (SEQ ID NO:4) W126 5' -GTCGCCATGGCTAAGAGATTCGTTG-31

(A

(SEQ ID W16 5'-CTGCTCTAGAGCCTGCGGGCTGCGTCT-31 (SEQ ID NO: SB3) SB3) The following Polymerase Chain Reaction (PCR) was performed using the Gene Amp PCR reagent kit (Perkin Elmer, 761 Main Avewalk, CT 06859, USA) according to the manufacturer's instructions. In all cases, the PCR mixture was overlayed with 100 A.l of mineral oil (Sigma Chemical Co. St. Louis, MO, USA).

Al of oligonucleotide #98 (2.5 pmol) Al of oligonucleotide #128 (2.5 pmol) 10,41 of lOX PCR buffer 16 41 of dNTP mix 0.5 A~l of Taq enzyme 58.5 A~l of water one cycle was performed: 94*C for 45 sec., 49*C for 1 min, 72*C for 2 mmn.

Subsequently, 5A1 of oligonucleotides #16 and #126 was added 30 and 15 cycles were perf ormed: 94 *C f or 4 5 sec. 45 *C f or 1 min, 72*C for 1.5 min. The PCR mixture was loaded onto a 2.5 agarose gel and subjected to electrophoresis using standard techniques (Sambrook et al., Molecular cloning, Cold Spring Harbour Laboratory Press, 1989). The resulting DNA fragment was cut out of the agarose gel and isolated using the Gene Clean Kit (Bio 101 Inc., PO BOX 2284, La Jolla, CA 92038, USA) according to the manufacturer's instructions. The purified PCR DNA fragment was dissolved in 10 pl of water and restriction endonuclease buffer and cut with the restriction endonucleases NcoI and Xba I according to standard techniques, run on a agarose gel and purified using the Gene Clean Kit as described.

The plasmid pAK188 consists of a DNA sequence of 412 bp composed of a EcoRI/NcoI fragment encoding the synthetic yeast signal/leader gene LaC212spx3 (described in Example 3 of WO 89/02463) followed by a synthetic NcoI/XbaI fragment encoding the insulin precursor MI5, which has a SerAspAspAlaLys bridge connecting the B29 and the Al amino acid residues (see SEQ ID NOS. 14, 15 and 16), inserted into the EcoRI/XbaI fragment of the vector (phagemid) pBLUESCRIPT (Stratagene,

USA).

The plasmid pAK188 is shown in Fig. 1.

The plasmid pAK188 was also cut with the restriction endonucleases NcoI and XbaI and the vector fragment of 3139 bp isolated. The two DNA fragments were ligated together using T4 0 DNA ligase and standard conditions (Sambrook et al., Molecular Cloning, Cold Spring Harbour Laboratory Press, 1989). The 25 ligation mixture was transformed into a competent E. coli strain followed by selection for ampicillin resistance. Plasmids were isolated from the resulting E. coli colonies using standard DNA miniprep technique (Sambrook et al., Molecular Cloning, Cold Spring Harbour Laboratory Press, 1989), checked with appropriate restrictions endonucleases i.e.

EcoRI, Xba I, NcoI and HpaI. The selected plasmid was shown by DNA sequencing analyses (Sequenase, U.S. Biochemical Corp.) to contain the correct sequence for the AlaA 21 AspB 3 human insulin precursor and named pEA5.3.

The plasmid pKFN1627 is an E. coli S. cerevisiae shuttle vector, identical to plasmid pKFN1003 described in EP patent No. 375718, except for a short DNA sequence upstream from the unique XbaI site. In pKFN1003, this sequence is a 178 bp s fragment encoding a synthetic aprotinin gene fused in-frame to the yeast mating factor alpha 1 signal-leader sequence. In pKFN1627, the corresponding 184 bp sequence encodes the insulin precursor MI5 (GluB1, GluB 28 B(l-29, GluBl,GluB 28 SerAspAspAlaLys-A(l-21) fused in-frame to the mating factor o0 alpha 1 sequence (see SEQ ID NOS. 17, 18 and 19). The vector pKFN1627 is shown in Fig. 1.

pEA5.3 "Was cut with the restriction endonucleases EcoRI and XbaI and the resulting DNA fragment of 412 bp was isolated. The yeast expression vector pKFN1627 was cut with the restriction endonucleases NcoI and XbaI and with NcoI and EcoRI and the DNA fragment of 9273 bp was isolated from the first digestion and the DNA fragment of 1644 bp was isolated from the second. The 412 bp EcoRI/XbaI fragment was then ligated to the two other fragments, that is the 9273 bp NcoI I/XbaI fragment and the 1644 bp NcoI/EcoRI fragment using standard techniques.

The ligation mixture was transformed into E. coli as described above. Plasmid from the resulting E. coli was isolated using standard techniques, and checked with appropriate restriction endonucleases i.e. EcoRI, XbaI, NcoI, Hpa I. The selected 25 plasmid was shown by DNA sequence analysis (using the Sequenase kit as described by the manufacturer, U.S. Biochemical) to contain the correct sequence for the AlaA 2 1 AspB 3 human insulin precursor DNA and to be inserted after the DNA encoding the LaC212spx3 signal/leader DNA. The plasmid was named pEA5.3.2 and is shown in Fig. 1. The DNA sequence encoding the LaC212spx3 signal/leader/AlaA 2 1 AspB 3 human insulin precursor complex and the amino acid sequence thereof are SEQ ID NOS. 21 and 22. The plasmid pEA5.3.2 was transformed into S.

cerevisiae strain MT663 as described in European patent application having the publication No. 214826 and the resulting strain was named yEA002.

EXAMPLE 2 Synthesis of AlaA 21 ThrB 3 human insulin precursor from Yeast strain yEA005 using the LaC212spx3 signal/leader.

The following oligonucleotides were synthesized: #101

SGGTTGAAGCTTTGTACTTGGTTTGTGGTGAAAGAGGTTTCTTCTACA

CTCCAAAGTCTGACGACGCT-3 (Thr B 3 (SEQ ID NO:7) S128 5 -CTGCGGGCTGCGTCTAAGCACAGTAGTTTTCCAATTGGTACAAA

GAACAGATAGAAGTACAACATTGTTCAACGATACCCTTAGCGTCG

TCAGACTTTGG-3' (AlaA 2 1 (SEQ ID NO:4) 5'-GTCGCCATGGCTAAGAGATTCGTTA-3 1 (ThrB 3 (SEQ ID NO:8) #16 5'-CTGCTCTAGAGCCTGCGGGCTGCGTCT-3' (SEQ ID NO:6) The DNA encoding AlaA 2 1 ThrB 3 human insulin precursor was constructed in the same manner as described for the DNA encoding AlaA 2 1 AspB 3 human insulin precursor in Example 1. The DNA sequence encoding the LaC212spx3 signal/leader/AlaA 2 1 ThrB 3 human insulin precursor complex and the amino acid sequence thereof are SEQ ID NOS. 23, 24 and 25. The plasmid pEA8.1.1 was shown to contain the desired sequence, transformed into S.

cerevisiae strain MT663 as described in Example 1 and the 25 resulting strain was named yEA005.

EXAMPLE 3 S Synthesis of GlyA 2 1 AspB 3 human insulin precursor from Yeast strain yEA007 using the LaC212spx3 signal/leader.

o The following oligonucleotides were synthesized: 30 The following oligonucleotides were synthesized: #98 #127

GTTGAAGCTTTGTACTTGGTTTGTGGTGAAAGAGGTTTCTTCT

ACACTCCAAAGTCTGACGACGCT-3' (AspB 3 (SEQ ID NO:3)

AGAACAGATAGAAGTACAACATTGTTCAACGATACCCT

TAGCGTCGTCAGACTTTGG-3' (GlyA 2 1 (SEQ ID NO:9) 5'-GTCGCCATGGCTAAGAGATTCGTTG-3' (AspB 3 (SEQ ID #126 NO: #16 5'-CTGCTCTAGAGCCTGCGGGCTGCGTCT-3' (SEQ ID NO:6) The DNA encoding GlyA 2 l AspB3 human insulin precursor was constructed in the same manner as described for the DNA encoding AlaA 2 l AspB 3 human insulin precursor in Example 1. The DNA sequence encoding the LaC2l2spx3 signal/leader/GlyA 2 1 AspB 3 human insulin precursor complex and the amino acid sequence thereof are SEQ ID NOS. 26, 27 and 28. The plasmid pEA1.5.6 was shown to contain the desired sequence, transformed into S.

cerevisiae strain MT663 as described in Example 1 and the resulting strain was named yEA007.

EXAMPLE 4 Synthesis of GlyA 2 1 ThrB 3 human insulin precursor from Yeast strain yEA006 using the LaC2l2spx3 signal/leader.

The following oligonucleotides were synthesized: #101 25 GGTTGAAGCTTTGTACTTGGTTTGTGGTGAAAGAGGTTTCTTCTACA CTCCAAAGTCTGACGACGCT-3' (ThrB 3 (SEQ ID NO:7) #127

AGAACAGATAGAAGTACAACATTGTTCAACGATACCCT

TAGCGTCGTCAGACTTTGG-31 (GlyA 2 l) (SEQ ID NO:9) #15 5'-GTCGCCATGGCTAAGAGATTCGTTA-31 (ThrB 3 1 (SEO ID NO: 8) #16 (SEQ ID NO:6) 5'-CTGCTCTAGAGCCTGCGGGCTGCGTCT-3 The DNA encoding GlyA21 ThrB 3 human insulin precursor was constructed in the same manner as described for the DNA encoding AlaA 2 1 Asp B 3 human insulin precursor in Example 1. The DNA sequence encoding the LaC212spx3 signal/leader/GlyA 2 1 ThrB 3 human insulin precursor complex and the amino acid sequence thereof are SEQ ID NOS. 29, 30 and 31. The plasmid pEA4.4.11 was shown to contain the desired DNA sequence, transformed into S. cerevisiae strain MT663 as described in Example 1 and the resulting strain was named yEA006.

EXAMPLE Synthesis of Arg

B

1 B 3 1 Synthesis of ArgB ArgB 3 1 single chain human insulin precursor having an N-terminal extension (GluGluAlaGluAlaGluAlaArg) from Yeast strain yEA113 using the alpha factor leader.

A)

The following oligonucleotides were synthesized: #220 5'-ACGTACGTTCTAGAGCCTGCGGGCTGC-3' (SEQ ID #263 TTCTACACTCCAAAGACTAGAGGTATCGTTGAA-3' (SEQ ID NO:11) #307 5 -GCTAACGTCGCCATGGCTAAGAGAGAAGAAGCTGAAGCTGAAGCT AGATTCGTTAACCAACAC-3' (SEQ ID NO:12) The following Polymerase Chain Reaction (PCR) was performed using the Gene Amp PCR reagent kit (Perkin Elmer, 761 Main Avewalk, CT 06859, USA) according to the manufacturer's 25 instructions. In all cases, the PCR mixture was overlayed with 100 il of mineral oil (Sigma Chemical Co, St. Louis, MO, USA).

The plasmid pAK220 (which is identical to pAK188) consists of a DNA sequence of 412 bp encoding the synthetic yeast signal/leader LaC212spx3 (described in Example 3 of WO 30 89/02463) followed by the insulin precursor MI5 (see SEQ ID NOS. 14, 15 and 16) inserted into the vector (phagemid) pBLUESCRIPT (Stratagene,

USA).

ooee oo il of oligonucleotide #220 (100 pmol) il of oligonucleotide #263 (100 pmol) i1 of 10X PCR buffer 16 pg of dNTP mix 0.5 pl of Tag enzyme il of pAK220 plasmid (identical to pAK188) as template (0.2 .g of DNA) 63 il of water A total of 16 cycles were performed, each cycle comprising 1 minute at 95°C; 1 minute at 40°C; and 2 minutes at 72'C. The PCR mixture was then loaded onto a 2% agarose gel and subjected to electrophoresis using standard techniques. The resulting DNA fragment was cut out of the agarose gel and isolated using the Gene Clean kit (Bio 101 Inc., PO BOX 2284, La Jolla, CA 92038, USA) according to the manufacture's instructions. The purified PCR DNA fragment was dissolved in 10 Al of water and restriction endonuclease buffer and cut with the restriction endonucleases HindIII and XbaI according to standard techniques. The HindIII/XbaI DNA fragment was purified using The Gene Clean Kit as described.

The plasmid pAK406 consists of a DNA sequence of 520 bp comprising an EcoRI/HindIII fragment derived from pMT636 (described in WO 90/10075) encoding the yeast alpha factor leader and part of the insulin precursor ligated to the 25 HindIII/XbaI fragment from pAK188 encoding the rest of the insulin precursor MI5 (see SEQ ID NOS. 32, 33 and 34) inserted into the vector cPOT. The vector pAK406 is shown in Fig. 2.

The plasmid pAK233 consists of a DNA sequence of 412 bp encoding the synthetic yeast signal/leader LaC212spx3 30 (described in Example 3 of WO 89/02463) followed by the gene for the insulin precursor B(1-29)-GluLysArg-A(1-21) (A21-Gly) (see SEQ ID NOS. 35, 36 and 37) inserted into the vector cPOT.

The plasmid pAK233 is shown in Fig. 2.

The plasmid pAK233 was cut with the restriction endonucleases NcoI and XbaI and the vector fragment of 9273 bp isolated. The plasmid pAK406 was cut with the restriction endonucleases NcoI and HindIII and the vector fragment of 2012 bp isolated. These two DNA fragments were ligated together with the HindIII/XbaI PCR fragment using T4 DNA ligase and standard conditions. The ligation mixture was then transformed into a competent E. coli strain followed by selection for ampicillin resistance. Plasmids were isolated from the resulting E. coli to colonies using a standard DNA miniprep technique and checked with appropriate restriction endonucleases i.e. EcoRI, XbaI, NcoI, HindIII. The selected plasmid was shown by DNA sequencing analyses to contain the correct sequence for the ArgB 3 1 single chain human insulin precursor DNA and to be inserted after the DNA encoding the S. cerevisiae alpha factor DNA. The plasmid was named pEA108 and is shown in Fig. 2. The DNA sequence encoding the alpha factor leader/ArgB 3 1 single chain human insulin precursor complex and the amino acid sequence thereof are SEQ ID NOS. 38, 39 and 40. The plasmid pEA 108 was transformed into S. cerevisiae strain MT663 as described in Example 1 and the resulting strain was named yEA108.

B)

0 The following Polymerase Chain Reaction (PCR) was performed using the Gene Amp PCR reagent kit (Perkin Elmer, 761 Main 25 Avewalk, CT 06859, USA) according to the manufacturer's instructions. In all cases, the PCR mixture was overlayed with 100 sl of mineral oil (Sigma Chemical Co., St. Louis, MO, USA)

S

5 gl of oligonucleotide #220 (100 pmol) pM of oligonucleotide #307 (100 pmol) 30 10 Al of 10X PCR buffer 16 gl of dNTP mix A1 of Taq enzyme 0.2 g1 of pEA108 plasmid as template (0.1 ug DNA) 63 g1 of water A total of 16 cycles were performed, each cycle comprising 1 minute at 95'C; 1 minute at 40'C; and 2 minutes at 72'C. The PCR mixture was then loaded onto an 2% agarose gel and subjected to electrophoresis using standard techniques. The resulting DNA fragment was cut out of the agarose gel and isolated using the Gene Clean kit (Bio 101 Inc., PO BOX 2284, La Jolla, CA 92038, USA) according to the manufacture's instructions. The purified PCR DNA fragment was dissolved in Al of water and restriction endonuclease buffer and cut with the restriction endonucleases NcoI and XbaI according to standard techniques. The NcoI/XbaI DNA fragment was purified using The Gene Clean Kit as described.

The plasmid pAK401 consists of a DNA sequence of 523 bp composed of an EcoRI/NcoI fragment derived from pMT636 (described in WO 90/10075) (constructed by by introducing a NcoI site in the 3'-end of the alpha leader by site directed mutagenesis) encoding the alpha factor leader followed by a NcoI/XbaI fragment from pAK188 encoding the insulin precursor (see SEQ ID NOS. 41, 42 and 43) inserted into the vector (phagemid) pBLUESCRIPT (Stratagene, USA). The plasmid pAK401 is shown in Fig. 3.

The plasmid pAK401 was cut with the restriction endonucleases NcoI and XbaI and the vector fragment of 3254 bp isolated and ligated together with the NcoI/XbaI PCR fragment. The ligation mixture was then transformed into a competent E. coli strain and plasmids were isolated from the resulting E. coli colonies using a standard DNA miniprep technique and checked with appropriate restriction endonucleases i.e. EcoRI, XbaI, NcoI.

The selected plasmid, named p113A (shown in Fig. was cut 30 with EcoRI and XbaI and the fragment of 535 bp isolated.

The plasmid pAK233 was cut with the restriction endonucleases NcoI and XbaI, and with EcoRI/NcoI and the fragments of 9273 and 1644 bp isolated. These two DNA fragments were ligated together with the EcoRI/XbaI fragment from p113A using T4 DNA ligase and standard conditions. The ligation mixture was then transformed into a competent E. coli strain followed by selection for ampicillin resistance. Plasmids were isolated from the resulting E. coli colonies using a standard

DNA

s miniprep technique and checked with appropriate restriction endonucleases i.e. EcoRI, XbaI, NcoI, HindIII. The selected plasmid was shown by DNA sequencing analyses to contain the correct sequence for the ArgB 3 1 single chain human insulin precursor DNA with the N-terminal extension GluGluAlaGluAlaGluAlaArg and to be inserted after the DNA encoding the S. cerevisiae alpha factor DNA. The plasmid was named pEA113 and is shown in Fig. 3. The DNA sequence encoding the alpha factor leader/Arg

B

1 ArgB 31 single chain human insulin precursor having an N-terminal extension (GluGluAlaGluAlaGluAlaArg) and the amino acid sequence thereof are SEQ ID NOS. 44, 45 and 46. The plasmid pEA113 was transformed into S. cerevisiae strain MT663 as described in Example 1 and the resulting strain was named yEA113.

EXAMPLE 6 Synthesis of ArgB- 1 ArgB 3 1 single chain human insulin precursor having an N-terminal extension (GluGluAlaGluAlaGluAlaGluArg) from Yeast strain yEA136 using the alpha factor leader.

The following oligonucleotide was synthesized: 25 #389 CTGAAAGATTCGTTAACCAACAC-3 (SEQ ID NO:13) The following PCR was performed using the Gene Amp PCR reagent kit 5 sl of oligonucleotide #220 (100 pmol) 5 gl of oligonucleotide #389 (100 pmol) 10 gl of 10X PCR buffer 16 gl of dNTP mix /1 of Taq enzyme 2 gl of pEA113 plasmid as template (0.5 ug DNA) 63 Al of water A total of 12 cycles were performed, each cycle comprising 1 minute at 95°C; 1 minute at 37'C; and 2 minutes at 72'C.

The DNA encoding alpha factor leader/Arg B 1 ArgB 3 1 single chain human insulin precursor having an N-terminal extension (GluGluAlaGluAlaGluAlaGluArg) was constructed in the same manner as described for the DNA encoding alpha factor leader/Arg

B

1 ArgB31 single chain human insulin precursor having an N-terminal extension (GluGluAlaGluAlaGluAlaArg) in Example 5. The plasmid was named pEAl36. The DNA sequence encoding the alpha factor leader/Arg B 1 ArgB31 single chain human insulin precursor having an N-terminal extension (GluGluAlaGluAlaGluAlaGluArg) and the amino acid sequence thereof are SEQ ID NOS. 47, 48 and 49. The plasmid pEAl36 was transformed into S. cerevisiae strain MT663 as described in Example 1 and the resulting strain was named yEA136.

EXAMPLE 7 Synthesis of (A1,Bl)-diBoc human insulin.

5 g of zinc-free human insulin was dissolved in 41.3 ml of DMSO. To the solution was added 3.090 ml of acetic acid. The 25 reaction was conducted at room temperature and initiated by addition of 565 mg of di-tert-butyl pyrocarbonate dissolved in 5.650 ml of DMSO. The reaction was allowed to proceed for hour and then stopped by addition of 250 g1 of ethanolamine.

The product was precipitated by addition of 1500 ml of acetone.

30 The precipitate was isolated by centrifugation and dried in vacuum. A yield of 6.85 g material was obtained.

(Al,Bl)-diBoc insulin was purified by reversed phase HPLC as follows: The crude product was dissolved in 100 ml of ethanol in water, adjusted to pH 3.0 with HC1 and applied to a column (5 cm diameter, 30 cm high) packed with octadecyldimethylsilyl-substituted silica particles (mean particle size 15 m, pore size 100 A) and equilibrated with elution buffer. The elution was performed using mixtures of ethanol and 1 mM aqueous HC1, 0.3 M KC1 at a flow of 2 1/h. The insulin was eluted by increasing the ethanol content from to 45%. The appropriate fraction was diluted to 20% ethanol and precipitated at pH 4.8. The precipitated material was isolated by centrifugation and dried in vacuum. Thus 1.701 g of (A1,Bl)diBoc human insulin was obtained at a purity of 94.5%.

EXAMPLE 8 Synthesis of (NB 29 -benzoyl human insulin) 6 3Zn 2 400 mg of (Al,Bl)-diBoc human insulin was dissolved in 2 ml of DMSO. To the solution was added 748 ~l of a mixture of Nmethylmorpholine and DMSO The reaction was conducted at 15*C and initiated by addition of 14.6 mg of benzoic acid N-hydroxysuccinimide ester dissolved in 132 ul O1 DMF. The reaction was stopped after 2 hours by addition of 100 ml of acetone. The precipitated material was isolated by centrifugation and dried in vacuum. 343 mg of material was 25 collected.

The Boc protecting groups were eliminated by addition of 4 ml of TFA. The dissolved material was incubated for 30 minutes and oo then precipitated by addition of 50 ml of acetone. The precipitate was isolated by centrifugation and dried in vacuum.

N'B

29 -benzoyl human insulin was purified by reversed phase HPLC as described in Example 7. A yield of 230 mg was obtained.

Recrystallization from 15% aqueous ethanol containing 6 mM Zn 2 o*OO and 50 mM citrate at pH 5.5 gave crystals of the title compound which were isolated by centrifugation and dried in vacuum. The yield was 190 mg.

Molecular mass, found by MS: 5911, theory: 5911.

EXAMPLE 9 Synthesis of (Na 29 -lithocholoyl human insulin)6, 3Zn 2 400 mg of (Al,Bl)-diBoc human insulin was dissolved in 2 ml of DMSO. -To the solution was added 748 ul of a mixture of Nmethylmorpholine and DMSO The reaction was conducted at 15"C and initiated by addition of 31.94 mg of lithocholic acid N-hydroxysuccinimide ester dissolved in 300 pl of DMF. The reaction was stopped after 2 hours by addition of 100 ml of acetone. The precipitated material was isolated by centrifugation and dried in vacuum. 331 mg of material was obtained.

The Boc protecting groups were eliminated by addition of 4 ml of TFA. The dissolved material was incubated for 30 minutes and then precipitated by addition of 50 ml of acetone. The O 20 precipitate was isolated by centrifugation and dried in vacuum.

The yield was 376 mg.

B29-lithocholoyl insulin was purified by reversed phase HPLC as described in Example 7. A final yield of 67 mg was obtained at a purity of 94%. Recrystallization from 15% aqueous ethanol containing 6 mM Zn2 and 50 mM citrate at pH 5.5 gave crystals of the title compound which were isolated by centrifugation and dried in vacuum. The yield was 49 mg.

Molecular mass, found by MS: 6160, theory: 6166.

43 EXAMPLE Synthesis of (N'B 9 -decanoyl human insulin) 6 3Zn 2 400 mg of (A1,B1)-diBoc human insulin was dissolved in 2 ml of DMSO. To the solution was added 748 pl of a mixture of Nmethylmorpholine and DMSO The reaction was conducted at 15"C and initiated by addition of 18.0 mg of decanoic acid N-hydroxysuccinimide ester dissolved in 132 pl of DMF. The reaction was stopped after 60 minutes and the product o0 precipitated by addition of 100 ml of acetone. The precipitated material was isolated by centrifugation and dried in vacuum.

420 mg-~f intermediate product was collected.

The Boc protecting groups were eliminated by addition of 4 ml of TFA. The dissolved material was incubated for 30 minutes and the product was then precipitated by addition of 50 ml of acetone. The precipitate was isolated by centrifugation and dried in vacuum. The yield of crude product was 420 mg.

The crude product was purified by reversed phase HPLC as described in Example 7. A final yield of 254 mg of the title product was obtained. The purity was 96.1%. Recrystallization from 15% aqueous ethanol containing 6 mM Zn 2 and 50 mM citrate O at pH 5.5 gave crystals of the title compound which were isolated by centrifugation and dried in vacuum. The yield was 217 mg.

S 25 Molecular mass, found by MS: 5962, theory: 5962.

EXAMPLE 11 Synthesis of des(B30) human insulin.

Synthesis of des(B30) human insulin was carried out as 30 described by Markussen (Methods in diabetes research, Vol. I, a 44 Laboratory methods, part B, 404-410. Ed: J. Larner and S. Phol, John Wiley Sons, 1984). 5 g of human insulin was dissolved in 500 ml of water while the pH value of the solution was kept at 2.6 by addition of 0.5 M sulphuric acid. Subsequently, the insulin was salted out by addition of 100 g of ammonium sulphate and the precipitate was isolated by centrifugation.

The pellet was dissolved in 800 ml of 0.1 M ammonium hydrogen carbonate and the pH value of the solution was adjusted to 8.4 with 1 M ammonia.

50 mg of bovine carboxypeptidase A was suspended in 25 ml of water and isolated by centrifugation. The crystals were suspended in 25 ml of water and 1 M ammonia was added until a clear solution was obtained at a final pH of 10. The carboxypeptidase solution was added to the insulin solution and the reaction was allowed to proceed for 24 hours. A few drops of toluene were added to act as preservative during the reaction.

After 24 hours the des(B30) human insulin was crystallized by successive addition of 80 g of sodium chloride while the solution was stirred. The pH value was then adjusted to 8.3 and the crystallization was allowed to proceed for 20 hours with gentle stirring. The crystals were isolated on a 1.2 gm filter, washed with 250 ml of ice cold 2-propanol and finally dried in vacuum.

25 EXAMPLE 12 Synthesis of (A1,Bl)-diBoc des(B30) human insulin.

The title compound was synthesized by a method similar to that described in Example 7, using des(B30) porcine insulin as the 30 starting material. The crude product was precipitated by acetone and dried in vacuum. The (Al,Bl)-diBoc des(B30) human 0* insulin was purified by reversed phase HPLC as described in Example 7.

EXAMPLE 13 Synthesis of N'B 29 -decanoyl des(B30) human insulin.

400 mg of (A1,Bl)-diBoc des(B30) human insulin was used as starting material for the synthesis of N'' 29 -decanoyl human insulin, following the procedure described in Example The crude product was precipitated by acetone, dried in vacuum and ,deprotected using TFA. The resulting product was precipitated by acetone and dried in vacuum. N B 29 -decanoyl human insulin was then purified by reversed phase HPLC as described in Example Molecular mass, found by MS: 5856, theory: 5861.

EXAMPLE 14 Synthesis of N' 29 -dodecanoyl des(B30) human insulin.

a. Immobilization of A. lvticus protease 13 mg of A. lyticus protease, dissolved in 5 ml of aqueous 0.2 M NaHCO 3 buffer, pH 9.4, was mixed with 4 ml of settled MiniLeak* Medium gel, which had been washed with the same buffer (MiniLeak is a divinylsulfone activated Sepharose CL 6B, obtained from KemEnTec, Copenhagen). The gel was kept in suspension by gentle stirring for 24 hours at room temperature.

25 Then, the gel was isolated by filtration, washed with water, and suspended in 20 ml of 1 M ethanolamine buffer, pH 9.4, and kept in suspension for 24 hours at room temperature. Finally, the gel was washed with water followed by 0.1 M acetic acid and stored at 4*C. The enzyme activity in the filtrate was 13% of that in the initial solution, indicating a yield in the immobilization reaction of about 87%.

b. Immobilization of porcine trypsin Porcine trypsin was immobilized to MiniLeak" Low to a degree of s substitution of 1 mg per ml of gel, using the conditions described above for immobilization of A. lyticus.

c. Synthesis of Glu(GluAla) 3 Arg-B(1-29), ThrArg-A(l-21) insulin using immobilized A. lyticus protease To 200 Eg of Glu(GluAla) 3 Arg-B(1-29)-ThrArg-A(1-2 1 single-chain human insulin precursor, dissolved in 20 ml of 0.1 M NaHCO 3 buffer, pH 9.0, was added 4 ml of the gel carrying the immobilized A. lyticus protease. After the gel had been kept in suspension in the reaction mixture for 6 hours at room temperature the hydrolysis was complete, rendering Glu(GluAla) 3 Arg-B(l-29), ThrArg-A(1-21) human insulin (the reaction was followed by reversed phase HPLC). After the hydrolysis, the gel .was removed by filtration. To the filtrate was added 5 ml of ethanol and 15 gL of 1 M ZnCI 2 and the pH was adjusted to using HC1. The precipitation of the product was completed on standing overnight at 4°C with gentle stirring. The product was isolated by centrifugation. After one washing with 1 ml of ice cold 20% ethanol and drying in vacuo the yield was 190 mg.

d. Synthesis of NI',Np',NB29-tridodecanoyl Glu(GluAla) 3 Arg-B(1- 29), Thr-Arg-A(1-21) human insulin using dodecanoic acid Nhydroxysuccinimide ester 190 mg (30 gmol) of Glu(GluAla) 3 Arg-B(l-29) ThrArg-A(l-21) insulin was dissolved in 1 ml of DMSO and 1.05 ml of a 0.572 M solution of N,N-diisopropylethylamine in DMF. The solution was cooled to 15'C and 36 mg (120 pmol) of dodecanoic acid Nhydroxysuccinimide ester dissolved in 0.6 ml of DMSO was added.

*oo oooo* The reaction was completed within 24 hours. The lipophilic title compound was not isolated.

e. Synthesis of NIB2-dodecanoyl des(B30) insulin The product from the previous step, contained in approximately 2,65 ml of DMSO/DMF/N,N-diisopropylethylamine was diluted with 10.6 ml of a 50 mM glycine buffer comprising ethanol and the pH adjusted to 10 with NaOH. After standing for 1 hour at room temperature 1 ml of MiniLeak gel, carrying 1 mg of immobilized trypsin per ml of gel, was added. The reaction mixture was stirred gently for 48 hours at room temperature. In order to isolate the desired product, the reaction mixture was applied to a reversed phase HPLC column (5 cm in diameter, cm high), packed with octadecyldimethylsilyl-substituted silica particles (mean particle size 15 gm, pore size 100 For the elution was used 20 mM Tris/HCl buffers, adjusted to pH 7.7 and comprising an increasing concentration of ethanol, from 40% to 44% at a rate of 2000 ml/h. The major peak eluting at about 43-44% of ethanol contained the title compound. The fractions containing the major peak were pooled, water was added to reduce the ethanol concentration to 20% and the pH was adjusted to 5.5. The solution was left overnight at whereby the product precipitated. The precipitate was isolated by centrifugation at -8'C and dried in vacuo. The yield of the title compound was 90 mg.

oo Molecular mass, found by MS: 5892, theory: 5890.

EXAMPLE Synthesis of N 29 (N-myristoyl-a-glutamyl) human insulin.

500 mg of (A1,Bl)-diBoc human insulin was dissolved in 2.5 ml of DMSO and 428 ml of ethyl diisopropylamine, diluted with ml of DMSO/DMF 1/1 was added. The temperature was 6 48 adjusted to 15'C and 85 mg of N-myristoyl-Glu(OBut)

N-

hydroxysuccinimide ester, dissolved in 2.5 ml of DMSO/DMF 1/1 was added. After 30 min the reaction mixture was poured into 60 ml of water, the pH adjusted to 5 and the precipitate s isolated by centrifugation. The precipitate was dried in vacuo.

The dried reaction mixture was dissolved in 25 ml of TFA, and the solution was left for 30 min at room temperature. The TFA was removed by evaporation in vacuo. The gelatinous residue was dissolved in 60 ml of water and the pH was adjusted to 11.2 using concentrated ammonia. The title compound was crystallized from this solution by adjustment of the pH to 8.5 using 6 N HC1. The product was isolated by centrifugation, washed once by ml of water, and dried in vacuo. Yield 356 mg. Purity by HPLC 94%.

The product of this example is thus human insulin wherein the e-amino group of Lys B29 has a substituent of the following structure:

CH

3 (CH2) 1 2

CONHCH(CH

2

CH

2

COOH)CO-.

Molecular mass, found by MS: 6146, theory: 6148.

EXAMPLE 16 Synthesis of NB 29 -undecanoyl des(B30) human insulin.

The title compound was synthesized analogously to NW 829 dodecanoyl des(B30) human insulin as described in Example 14, by using undecanoic acid N-hydroxysuccinimide ester instead of 25 dodecanoic acid N-hydroxysuccinimide ester.

Molecular mass of the product found by MS: 5876, theory: 5876.

49 EXAMPLE 17 Synthesis of NEB 29 -tridecanoyl des(B30) human insulin.

The title compound was synthesized analogously to NIB 29 dodecanoyl des(B30) human insulin as described in Example 14, by using tridecanoic acid N-hydroxysuccinimide ester instead of dodecanoic acid N-hydroxysuccinimide ester.

Molecular mass of the product found by MS: 5899, theory: 5904.

EXAMPLE 18 Synthesis of NtB 29 -myristoyl des(B30) human insulin.

The title compound was synthesized analogously to N(B 29 dodecanoyl des(B30) human insulin as described in Example 14, by using myristic acid N-hydroxysuccinimide ester instead of dodecanoic acid N-hydroxysuccinimide ester.

Molecular mass of the product found by MS: 5923, theory: 5918.

EXAMPLE 19 Synthesis of NB 29 -palmitoyl des(B30) human insulin.

20 The title compound was synthesized analogously to N B29 dodecanoyl des(B30) human insulin as described in Example 14, by using palmitic acid N-hydroxysuccinimide ester instead of Sdodecanoic acid N-hydroxysuccinimide ester.

i Molecular mass of the product found by MS: 5944, theory: 5946.

EXAMPLE Synthesis of N' 829 -suberoyl-D-thyroxine human insulin.

a. Preparation of N-(succinimidylsuberovl)-D-thyroxine.

Disuccinimidyl suberate (1.0 g, Pierce) was dissolved in DMF ml), and D-thyroxine (2.0 g, Aldrich) was added with stirring at 20'C. The thyroxine slowly dissolved, and after hours the solvent was removed by evaporation in vacuo. The oily residue was crystallized from 2-propanol to yield 0.6 g of N- (succinimidylsuberoyl)-D-thyroxine, m.p. 128-133*C.

b. Reaction of (Al,Bl)-diBoc human insulin with N- (succinimidylsuberoyl)-D-thvroxine.

(Al,Bl)-diBoc human insulin (200 mg) was dissolved in dry DMF ml) by addition of triethylamine (20 gl) at room temperature. Then, N-(succinimidylsuberoyl)-D-thyroxine (80 mg) was added. The reaction was monitored by reversed phase HPLC and when the reaction was about 90% complete, the solvent was removed in vacuo. To the evaporation residue, anhydrous trifluoroacetic acid (5 ml) was added, and the solution was kept. for 1 hour at room temperature. After removal of the trifluoroacetic acid in vacuo, the residue was dissolved in a mixture of 1M acetic acid (5 ml) and acetonitrile (1.5 ml), O purified by preparative reversed phase HPLC and desalted on a column. The yield of N' 29 -suberoyl-D-thyroxine human insulin was 50 mg.

The product of this example is thus human insulin wherein the e-amino group of Lys 829 has a substituent of the following structure: Thyrox-CO(CH 2 6 CO-, wherein Thyrox is thyroxine which is bound to the octanedioic acid moiety via an amide bond to its a-amino group.

Molecular mass of the product found by MS: 6724, theory: 6723.

*ooo EXAMPLE 21 Synthesis of N' 829 -(2-succinylamido)myristic acid human insulin.

a. Preparation of a-aminomyristic acid methyl ester.HC1.

To methanol (5 ml, Merck) at -10'C, thionyl chloride (0.2 ml, Aldrich) was added dropwise while stirring vigorously. Then, aaminomyristic acid (0.7 g, prepared from the a-bromo acid by reaction with ammonia) was added. The reaction mixture was stirred at room temperature overnight, and then evaporated to dryness. The crude product (0.7 g) was used directly in step b.

b. Preparation of N-succinoyl-a-aminomyristic acid methyl ester.

a-Aminomyristic acid methyl ester,HC1 (0.7 g) was dissolved in chloroform (25 ml, Merck). Triethylamine (0.35 ml, Fluka) was added, followed by succinic anhydride (0.3 g, Fluka). The reaction mixture was stirred at room temperature for 2 hours, concentrated to dryness, and the residue recrystallized from ethyl acetate/petroleum ether Yield: 0.8 g.

c. Preparation of N-(succinimidvlsuccinoyl)-a-aminomvristic acid methyl ester.

N-succinoyl-a-aminomyristic acid methyl ester (0.8 g) was dissolved in dry DMF (10 ml, Merck, dried over 4A molecular sieve). Dry pyridine (80 -il, Merck), and di(N-succinimidyl)carbonate (1.8 g, Fluka) were added, and the reaction 25 mixture was stirred overnight at room temperature. The evaporation residue was purified by flash chromatography on silica gel 60 (Merck), and recrystallized from 2propanol/petroleum ether Yield of N- (succinimidylsuccinoyl)-a-aminomyristic acid methyl ester: 0.13 g, m.p. 64-66°C.

*o d. Reaction of (Al.Bl)-diBoc human insulin with N- (succinimidylsuccinoyl)-a-aminomyristic acid methyl ester.

The reaction was carried out as in Example 20 but using N- (succinimidylsuccinoyl)-a-aminomyristic acid methyl ester (16 mg) instead of N-(succinimidylsuberoyl)-D-thyroxine. After removal of the trifluoroacetic acid in vacuo, the evaporation residue was treated with 0.1M sodium hydroxide at 0*C to saponify the methyl ester. When the saponification was judged to be complete by reversed phase HPLC, the pH value in the o0 solution was adjusted to 3, and the solution was lyophilized.

After purification by preparative reversed phase HPLC and desalting on a PD-10 column, the yield of N'" 29 succinylamido)myristic acid human insulin was 39 mg.

The product of this example is thus human insulin wherein the 1is -amino group of Lys 829 has a substituent of the following structure:

CH

3 (CH) 11 CH (COOH)NHCOCH 2

CH

2

CO-.

Molecular mass of the product found by MS: 6130, theory: 6133.

EXAMPLE 22 Synthesis of NEB 29 -octyloxycarbonyl human insulin.

The synthesis was carried out as in Example 20 but using noctyloxycarbonyl N-hydroxysuccinimide (9 mg, prepared from noctyl chloroformate (Aldrich) and N-hydroxysuccinimide), instead of N-(succinimidylsuberoyl)-D-thyroxine. The yield of 25 N' -octyloxycarbonyl human insulin was 86 mg.

The product of this example is thus human insulin wherein the e-amino group of Lys" 29 has a substituent of the following structure: CH (CH 2 7 0CO-.

Molecular mass of the product found by MS: 5960, theory: 5964.

o*oo EXAMPLE 23 Synthesis of N,89- (2-succinylamido)palmitic acid human insulin.

a. Preparation of N-(succinimidylsuccinoyl)-a-amino palmitic acid methyl ester.

This compound -was prepared as described in Example 21 using a-amino palmitic acid instead of a-amino myristic acid.

b. Reaction of (A1,B1)-diBoc human insulin with N- (succinimidylsuccinoyl)-a-aminolpalmitictic acid methyl ester.

The reaction was carried out as in Example 21 but using N- (succirrimidylsuccinoyl)-a-aminopalmitic acid methyl ester instead of N-(succinimidylsuccinoyl)-a-aminopalmitic acid methyl ester to give N(B? 9 -(2-succinylamido)palmitic acid human insulin.

The product of this example is thus human insulin wherein the E-amino group of LySB29 has a substituent of the following structure:

CH

3

(CH

2 13

CH(COOH)NHCOCH

2 CH 2

CO-.

EXAMPLE 24 Synthesis of NBI9-(2-succinylamidoethyloxy)palmitic acid human S 20 insulin.

a. Preparation of N-(succinimidylsuccinoyl)-2-aminoethyloxy palmitic acid methyl ester.

This compound was prepared as described in Example 21 but using 2-aminoethyloxy palmitic acid (synthesized by the general procedure described by R. TenBrink, J. Org. Chem. 52 (1987) 418-422 instead of a-amino myristic acid.

Reaction of (A1.Bl')-diBoc human insulin with N- (succiflimidylsuccinoyl) -2-aminoethyloxypalmitictic acid methyl ester.

The reaction was carried out as in Example 21 but using N- (succinimidy2.succinoyl) -2-aminoethyloxypalmitic acid methyl ester instead of N- (succinimidylsuccinoyl) -ca-aminomyristic acid methyl ester to give NIB 2 9 (2succinylamidoethyloxy) palmitic acid human insulin.

The product of this example is thus human insulin wherein. the l0 e-amino group of LySB2 9 has a substituent of the following structure:

CH

3

(CH

2 13 CH (COOH) NHCH- 2

CH

2

OCOCH-

2

CH

2

CO-.

EXA1MPLE Synthesis of N 18 2 9 1ithocholoyl-cr-glutamyl des(B30) human insulin.

The synthesis was carried out as in Example 13 using N-lithocholoyl-L-glutamic acid a-N-hydroxysuccinimide ester, -ytert-butyl ester instead of decanoic acid N-hydroxysuccinimide ester.

The product of this example is thus des(B30) human insulin wherein the E-amino group of Lys B 29 has a substituent of the following structure: lithocholoyl-NHCH(CH 2

CH

2 COOH) Ca-.

Molecular mass of the product found by MS: 6194, theory: 6193.

EXAMPLE 26 Synthesis of NcB--3 3 ,5,5 '-tetraiodothyroacetyl human insulin.

The synthesis was carried out as in Example 10 using tetraiodothyroacetic acid N-hydroxysuccinimide ester, instead of decanoic acid N-hydroxysuccinimide ester.

Molecular mass of the product found by MS: 6536, theory: 6538.

EXAMPLE 27 Synthesis of NCB29-L-thyroxyl human insulin.

The synthesis was carried out as in Example 10 using Boc-Lthyroxine N-hydroxysuccinimide ester, instead of decanoic acid N-hydroxysuccinimide ester.

Molecular mass of the product found by MS: 6572, theory: 6567.

EXAMPLE 28 A pharmaceutical composition comprising 600 nmol/ml of N c

B

29 0 decanoyl des(B30) human insulin, 1/3Zn2+ in solution.

NB

29 -decanoyl des(B30) human insulin (1.2 .mol) was dissolved in 20 water (0.8 ml) and the pH value was adjusted to 7.5 by addition of 0.2 M sodium hydroxide. 0.01 M zinc acetate (60 gl) and a solution containing 0.75% of phenol and 4% of glycerol (0.8 ml) was added. The pH value of the solution was adjusted to using 0.2 M sodium hydroxide and the volume of the solution was 25 adjusted to 2 ml with water.

The resulting solution was sterilized by filtration and transferred aseptically to a cartridge or a vial.

EXAMPLE 29 A pharmaceutical composition comprising 600 nmol/ml of N' 829 decanoyl human insulin, ,Zn 2 in solution.

1.2 gmol of the title compound was dissolved in water (0.8 ml) and the pH value was adjusted to 7.5 by addition of 0.2 M sodium hydroxide. A solution containing 0.75% of phenol and 1.75% of sodium chloride (0.8 ml) was added. The pH value of the solution was adjusted to 7.5 using 0.2 M sodium hydroxide S o and the volume of the solution was adjusted to 2 ml with water.

The resulting solution was sterilized by filtration and transferred aseptically to a cartridge or a vial.

EXAMPLE A pharmaceutical composition comprising 600 nmol/ml of NB 29 lithocholoyl human insulin in solution.

1.2 gmol of the title compound was suspended in water (0.8 ml) and dissolved by adjusting the pH value of the solution to using 0.2 M sodium hydroxide. To the solution was then added 0.8 ml of a stock solution containing 0.75 cresol and 4% glycerol in water. Finally, the pH value was again adjusted to 8.5 and the volume of the solution was adjusted to 2 ml with water.

The resulting solution was sterilized by filtration and 25 transferred aseptically to a cartridge or a vial.

0* SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Novo Nordisk A/S STREET: Novo Alle CITY: DK-2880 Bagsvaerd COUNTRY: Denmark TELEPHONE: +45 44448888 TELEFAX: +45 44490555 TELEX: 37173 (ii) TITLE OF INVENTION: ACYLATED INSULIN (iii) NUMBER OF SEQUENCES: 49 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Novo Nordisk A/S Corporate Patents STREET: Novo Alle CITY: DK-2880 Bagsvaerd COUNTRY: Denmark COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentin Release Version #1.25 (vi) CURRENT APPLICATION

DATA:

APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBERS: DK 1044/93 and US 08/190,829 FILING DATES: 09-SEP-1993 and 02-FEB-1994 (viii) ATTORNEY/AGENT

INFORMATION:

NAME: Jorgensen, Dan et al.

REFERENCE/DOCKET NUMBER: 3985.204-WO,DJ (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: +45 44448888 TELEFAX: +45 44493256 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 21 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Gly Ile Val Glu Gin Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gin Leu 1 5 10 Glu Asn Tyr Cys Xaa INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 30 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Xaa Val Xaa Gin His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Xaa 25 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 110 base pairs TYPE: nucleic acid 0 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TGGCTAAGAG ATTCGTTGAC CAACACTTGT GCGGTTCTCA CTTGGTTGAA GCTTTGTACT TGGTTTGTGG TGAAAGAGGT TTCTTCTACA CTCCAAAGTC TGACGACGCT 110 INFORMATION FOR SEQ ID NO:4: SEQUENCE

CHARACTERISTICS:

LENGTH: 100 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CTGCGGGCTG CGTCTAAGCA CAGTAGTTTT CCAATTGGTA CAAAGAACAG ATAGAAGTAC AACATTGTTC AACGATACCC TTAGCGTCGT CAGACTTTGG 100 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID GTCGCCATGG CTAAGAGATT CGTTG INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: SCTGCTCTAGA GCCTGCGGGC TGCGTCT 27 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 110 base pairs TYPE: nucleic acid S: STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TGGCTAAGAG ATTCGTTACT CAACACTTGT GCGGTTCTCA CTTGGTTGAA GCTTTGTACT TGGTTTGTGG TGAAAGAGGT TTCTTCTACA CTCCAAAGTC TGACGACGCT 110 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GTCGCCATGG CTAAGAGATT CGTTA INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: S(A) LENGTH: 100 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CTGCGGGCTG CGTCTAACCA CAGTAGTTTT CCAATTGGTA CAAAGAACAG ATAGAAGTAC AACATTGTTC AACGATACCC TTAGCGTCGT CAGACTTTGG 100 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID ACGTACGTTC TAGAGCCTGC GGGCTGC 27 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 78 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA oo*** o*o (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CACTTGGTTG AAGCTTTGTA CTTGGTTTGT GGTGAAAGAG GTTTCTTCTA CACTCCAAAG ACTAGAGGTA TCGTTGAA 78 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 63 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA 9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GCTAACGTCG CCATGGCTAA GAGAGAAGAA GCTGAAGCTG AAGCTAGATT CGTTAACCAA CAC 63 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 65 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GCTAACGTCG CCATGGCTAA GAGAGAAGAA GCTGAAGCGA AGCTGAAAGA TTCGTTAACC AACAC INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 415 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 80..391 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 62 ATCGAATTCC ATTCAAGAAT AGTTCAAACA AGAAGATTAC AAACTATCAA TTTCATACAC AATATAAACG ACCAAAAGA ATG MAG GCT GTT TIC TTG GTT TTG TCC TTG ATC Met Lys Ala Val Phe Leu Val Leu Ser Leu Ile 1 5 GGA TTC TGC TGG Gly Phe Cys Trp ATI CCG GMA GAG Ile Pro Glu Giu GTC GCC ATG GCT Val Ala Met Ala TTG GTT &AM GCT Leu Val Giu Ala ACT CCA MAG TCT Thr Pro Lys Ser TCT ATC TGT TCT Ser Ilie Cys Ser CCGCAGGCTC TAGA

INFORMATION

GCC

Al a

TCT

Se r

MAG

Lys

TG

Le u

GAC

Asp

TG

Le u

CMA

Gin

CTG

Leu

AGA

Arg

TAC

Tyr 65

GAC

Asp

TAC

Tyr

CCA

Pro

ATC

Ile

TIC

Phe 50

TG

Leu

GCT

Ala

CMA

Gin

GTC

Val1 AT C Ilie

GTT

Val1

GTT

Val

MAG

Lys TT G Leu

ACT

Thr 20

GCT

Ala

MAC

Asn

TGT

Cys

GGT

Gi y

GMA

Giu 100

GGC

Gly

GMA

Glu

CMA

Gin

GGT

Gly

ATC

lIe, 85

AAC

As n

GAT

Asp

MAC

As n

CAC

His

GMA

Giu 70

GTT

Val

TAC

Tyr

GMA

Gi u

ACC

Thr

TTG

Leu

AGA

Arg

GMA

Glu

TGT

Cys TCT Gil GAG Ser Val Giu TIG GCT MAC Leu Ala Asn GGT TCT CAC Gly Ser His TTC TIC TAC Phe Phe Tyr TGT TGT ACT Cys Cys Thr

TAGACGCAGC

160 208 256 352 415 FOR SEQ ID i)SEQUENCE CHARACTERISTICS: LENGTH: 104 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Ala Val Phe Leu Val Leu Ser Leu Ilie Gly Phe Cys lrp Ala 1 5 10 Gin Pro Val Thr Gly Asp Giu Ser Ser Val Giu Ile Pro Giu Giu Ser 25 Leu -lie Ilie Ala Giu Asn Thr Thr Leu Ala Asn Val Aia Met Ala Lys 35 40 Arg Phe Val Asn Gin His Leu Cys Gly Ser His Leu Val Giu Ala Leu 50 55 Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Ser Asp 70 75 Asp Ala Lys Gly Ile Val Glu Gin Cys Cys Thr Ser Ile Cys Ser Leu 90 Tyr Gin Leu Glu Asn Tyr Cys Asn 100 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 415 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii4 MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TAGCTTAAGG TAAGTTCTTA TCAAGTTTGT TCTTCTAATG

TTTGATAGTT

TTATATTTGC TGGTTTTCTT ACTTCCGACA AAAGAACCAA

AACAGGAACT

GACCCGGGTT GGTCAGTGAC CGCTACTTAG TAGACAACTC

TAAGGCCTTC

GTAGCGACTT TTGTGGTGAA ACCGATTGCA GCGGTACCGA TTCTCTAAGC GAACACGCCA AGAGTGAACC AACTTCGAAA CATGAACCAA

ACACCACTTT

GATGTGAGGT TTCAGACTGC TGCGATTCCC ATAGCAACTT

GTTACAACAT

AAGAAACATG GTTAACCTTT TGATGACATT GATCTGCGTC

GGGCGTCCGA

AAAGTATGTG

AGCCTAAGAC

TCAGAGACTA

AATTGGTTGT

CTCCAAAGAA

GAAGATAGAC

GATCT

120 180 240 300 360 415 999 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 523 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 80..499 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: ATCGAATTCC ATTCAAGAAT AGTTCAAACA AGAAGATTAC AAACTATCAA

TTTCATACAC

64 AATATAAACG ATTAAAAGA ATG AGA UTT CCT TCA ATT TTT ACT GCA GTT TTA Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu 1 5 TTC GCA Phe Ala GAT GMA Asp Giu TTA GMA Leu Glu MAT MAC Asn Asn AAA GMA Lys Giu TGC GGT Cys Gly GGT TTC Gly Phe CMA TGT Gin Cys 125

GCA

Ala

ACG

Thr

GGG

Gly

GGG

Gi y

GMA

Giu

TCT

Ser

TTC

Phe 110

TGT

Cys

TCC

Ser

GCA

Ala

GAT

Asp

TTA

Leu

GGG

Gi y

CAC

His

TAC

Tyr

ACT

Thr

TCC

Ser

CMA

Gin

TTC

Phe

TTG

Le u

GTA

Val1

TTG

Le u

ACT

Thr

TCT

Ser

GCA

Ala

AT

Ilie

GAT

Asp

TTT

Phe 65

TCT

Ser

GTT

Val1

GMA

Giu

ATC

Ile

TTA

Leu

CCG

Pro

GTT

Val 50

ATA

Ile

TTG

Leu

GMA

Glu

MAG

Lys

GCT

Ala

GCT

Ala 35

GCT

Ala

MAT

As n

GAT

Asp

GCT

Ala

TCT

Ser 115

GCT

Ala 20

GMA

Giu

GTT

Val1

ACT

Th r

MAG

Lys

TTG

Leu 100

GAC

Asp

CCA

Pro

GCT

Ala

TTG

Le u

ACT

Th r

AGA

Arg 85

TAC

Tyr

GAC

Asp

GTC

Val

GTC

Val

CCA

Pro

ATT

Ile 70

GMA

Gi u

TTG

Leu

GCT

Ala MAC ACT ACA ACA GMA As n

ATC

Ile

T

Phe

GCC

Ala

GTT

Val

GTT

Val1

MAG

Lys

TTG

Leu 135 Thr

GGT

Gi y

TCC

Ser

AGC

Ser

MAC

Asn

TGT

Cys

GGT

Gi y 120 Thr

TAC

Tyr

MAC

As n

ATT

Ilie

CMA

Gin

GGT

Gi y 105

ATC

Ile Thr

TCA

Ser

AGC

Ser

GCT

Aia

CAC

His

GMA

Giu

GTT

Vali Gi u

GAT

Asp

ACA

Thr

GCT

Ala

UTG

Leu

AGA

Arg

GMA

Giu 160 208 256 304 352 400 448 496 TGT TCT TTG TAC CMA Cys Ser Leu Tyr Gin GMA MC TAC TGT Giu Asn Tyr Cys 130

MAC

Asn 140 TAGACGCAGC CCGCAGGCTC

TAGA

523

B

B

INFORMATION FOR SEQ ID NO:18: ()SEQUENCE

CHARACTERISTICS:

LENGTH: 140 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Met Arg Phe Pro Ser Ilie Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Giu Asp Glu Thr Al a Gi n Gly Asp Phe Ile Pro Ala Asp Val Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Val Leu Pro Phe Ile Phe 55 Ala Ser Asn Ser Thr Asn Lys Asn Gly Leu Leu Giu Glu Gly Val Asn Thr Thr Ile 70 Glu Ser Ile Ala Ala 75 Leu Ser Leu Asp Lys Ar g Tyr Val Asn Gin Cys Gly Ser Hi s Leu Val Giu Ala Gi u Lys 7Ser 115 Ilie Cys Ser 130 Leu 100 Asp Leu Val Cys Gi y 105 Ile Arg Gly Phe Phe Tyr Thr 110 Cys Thr Ser Asp Ala Lys Gly 120 Glu Val Giu Gin Leu Tyr Gin Leu 135 Asn Tyr Cys Asn 140 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 523 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 9 9* 9 9- 9 9* 9 9 9 9 9 9 9* 9999 9 9999 TAG CTTAAGG

TTATATTTGC

TAG GAGGCGT

CCGACTTCGA

TAAAAGGTTG

ACGATTTCTT

AGTdAACCAA

CAGACTGCTG

TAACCTTTTG

TAAGTTCTTA

TAATTTTCTT

AATCGACGAG

CAGTAGCCAA

TC GTGTTTAT

CTTCCCCATA

CTTCGAAACA

CGATTCCCAT

ATGACATTGA

TCAAGTTTGT

ACTCTAAAGG

GTCAGTTGTG

TGAGTCTAAA

TGCCCAATAA

GAAACCTATT

TGAACCAAAC

AGCAACTTGT

TCTGCGTCGG

TCTTCTAATG

AAGTTAAAAA

ATGTTGTCTT

TCTTCCCCTA

CAAATATTTA

CTCTCTTCAA

ACCACTTTCT

TACAACATGA

GCGTCCGAGA

TTTGATAGTT

TGACGTCAAA

CTACTTTGCC

AAGCTACAAC

TGATGATAAC

TTGGTTGTGA

CCAAAGAAGA

AGATAGACAA

TCT

MAAGTATGTG

ATAAGCGTCG

GTGTTTAAGG

GACAAAACGG

GGTCGTAACG

ACACGCCAAG

TGTGACTTT

GAAACATGGT

120 180 240 300 360 420 480 523 999999 9 *99999 9 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 415 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 80..391 (xi) SEQUENCE DESCRIPTION: SEQ ID ATCGAATTCC ATTCAAGAAT AGTTCAAACA AGAAGATTAC AAACTATCAA

TTTCATACAC

AATATAAAGG ACCAAAAGA ATG AAG GCT GTT Met Lys Ala 1 GCC CAA CCA GTC Ala Gin Pro Val SVa 1 TTC TTG GTT TTG TCC TTG ATC Phe Leu Val Leu Ser Leu lle

GGA

Gly TTC TGC Phe Cys

TGG

Trp ACT GGC GAT GAA TCA TCT GTT GAG Thr Gly Asp Glu Ser Ser Val Glu 20 ATT CCG GAA lle Pro Glu GAG TCT CTG ATC Glu Ser Leu lle

ATC

lle GCT GAA AAC ACC Ala Glu Asn Thr TTG GCT AAC Leu Ala Asn GTC GCC Val Ala ATG GCT AAG AGA Met Ala Lys Arg GTT GAC CAA CAC Val Asp Gin His

TTG

Leu TGC GGT TCT CAC Cys Gly Ser His

TTG

Leu GTT GAA GCT TTG Val Glu Ala Leu TTG GTT TGT GGT Leu Val Cys Gly GAA AGA GGT TTC TTC TAC Glu Arg Gly Phe Phe Tyr 70 256 304 352 401 ACT CCA AAG TCT Thr Pro Lys Ser

GAC

Asp 80 GAC GCT AAG GGT Asp Ala Lys Gly GTT GAA CAA TGT Val Glu Gin Cys TGT ACT Cys Thr

S

S

S

S.

S

S

**SS

TCT ATC TGT Ser lle Cys

TCT

Ser TTG TAC CAA TTG Leu Tyr Gin Leu AAC TAC TGT GCT Asn Tyr Cys Ala

TAGACGCAGC

CCGCAGGCTC TAGA INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 104 amino acids TYPE: amino acid TOPOLOGY: linear 415 (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:21: Met

I

Gin Le u Lys Ala Val Pro Val Thr Ilie Ilie Ala Phe 5 Leu Val Leu Gly Asp Glu Ser Ser Leu 10 Ser Val 25 Leu Ala Ilie Gly Phe Cys Glu Ilie Pro Glu Met Trp Al a GI u Ser Al a Lys Giu Asn Thr Arg Phe Vai Thr 40 Cys Asn Val Ala Val1 Tyr As p Asp Gin His Cys Giy Glu 70 Leu 55 Arg Gly Ser His Leu Thr Gl u Al a Leu Leu Val Ai a Lys Gly Phe Phe Tyr 75 Thr Pro Lys Ser Asp Gly Ilie Vai Giu Gin Giu Asn Tyr Cys Ala 100 Cys Ser Ilie Cys Ser Leu Tyr Gin Leu INFORMATION FOR SEQ ID NO:22: i)SEQUENCE CHARACTERISTICS: LENGTH: 415 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: TAGCTTAAGG TAAGTTCTTA TCAAGTTTGT TCTTCTAATG

TTTGATAGTT

TTATATTTGC TGGTTTTCTT ACTTCCGACA AAAGAACCAA

AACAGGAACT

GACCCGGGTT GGTCAGTGAC CGCTACTTAG TAGACAACTC

TAAGGCCTTC

GTAGCGACTT TTGTGGTGAA ACCGATTGCA GCGGTACCGA'

TTCTCTAAGC

GAACACGCCA AGAGTGAACC AACTTCGAAA CATGAACCMA

ACACCACTTT

GATGTGAGGT TTCAGACTGC TGCGATTCCC ATAGCAACTT

GTTACAACAT

AAGAAACATG GTTAACCTTT TGATGACACG AATCTG CGTC GGGCGTCCGA o p.

AAAGTATGTG

AGCCTAAGAC

TCAGAGACTA

AACTGGTTGT

CTCCAAAGAA

GAAGATAGAC

GATCT

120 180 240 300 360 415 INFORMATION FOR SEQ ID NO:23: ()SEQUENCE CHARACTERISTICS: LENGTH: 415 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:

NAME/KEY:

LOCATION:

CDS

80..391 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: ATCGAATTCC ATTCAAGAAT AGTTCAAACA AGAAGATTAC AAACTATCAA TTTCATACAC AATATAAACG ACCAAAAGA ATG AAG GCT GTT Met Lys Ala Val 1 TTC TTG GTT Phe Leu Val 5 TTG TCC TTG ATC Leu Ser Leu Ile GGA TTC TGC Gly Phe Cys ATT CCG GAA Ile Pro Glu

TGG

Trp GCC CAA CCA GTC Ala Gin Pro Val

ACT

Thr 20 GGC GAT GAA TCA Gly Asp Glu Ser TCT GTT GAG Ser Val Glu TTG GCT AAC Leu Ala Asn 160 208 GAG TCT CTG ATC Glu Ser Leu lle GCT GAA AAC ACC Ala Glu Asn Thr

ACT

Thr GTC GCC Val Ala ATG GCT AAG AGA Met Ala Lys Arg

TTC

Phe GTT ACT CAA CAC Val Thr Gin His TGC GGT TCT CAC Cys Gly Ser His

TTG

Leu GTT GAA GCT TTG Val Glu Ala Leu

TAC

Tyr 65 TTG GTT TGT Leu Val Cys GGT GAA Gly Glu AGA GGT TTC TTC Arg Gly Phe Phe 256 304 352 ACT CCA AAG TCT Thr Pro Lys Ser

GAC

Asp GAC GCT AAG GGT Asp Ala Lys Gly

ATC

lie 85 GTT GAA CAA TGT Val Glu Gin Cys TGT ACT Cys Thr TCT ATC TGT Ser Ile Cys TTG TAC CAA TTG Leu Tyr Gin Leu AAC TAC TGT GCT Asn Tyr Cys Ala

TAGACGCAGC

401 e g c S S0 S

*SSS

S

CCGCAGGCTC TAGA INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 104 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 415

S.

*c e e Met 1 Lys Ala Val Phe 5 Leu Val Leu Ser Leu Ile Gly Phe Cys 10 Trp Ala Gin Pro Val Leu Ile lle Arg Phe Val Thr Ala Gly Asp Glu Ser Ser Val 25 Leu Ala Glu lie Pro Glu Asn Thr Asn Val Ala Val Glu Glu Ser Met Ala Lys Glu Ala Leu Thr Gin His Tyr Leu Leu 55 Arg Gly Ser His Leu Thr Val Cys Gly Asp Glu 70 Val Gly Phe Phe Tyr 75 Thr Pro Lys Ser Asp Leu Ala Lys Gly Glu Gin Cys Ser lie Cys Ser Tyr Gin Leu Glu 100 Tyr Cys Ala INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 415 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID TAGCTTAAGG TAAGTTCTTA TCAAGTTTGT TCTTCTAATG

TTTGATAGTT

TTATATTTGC TGGTTTTCTT ACTTCCGACA AAAGAACCAA AACAGGAACT GACCCGGGTT GGTCAGTGAC CGCTACTTAG TAGACAACTC

TAAGGCCTTC

GTAGCGACTT TTGTGGTGAA ACCGATTGCA GCGGTACCGA

TTCTCTAAGC

GAACACGCCA AGAGTGAACC AACTTCGAAA CATGAACCAA ACACCACTTT GATGTGAGGT TTCAGACTGC TGCGATTCCC ATAGCAACTT

GTTACAACAT

AAGAAACATG GTTAACCTTT TGATGACACG AATCTGCGTC GGGCGTCCGA 9*

AAAGTATGTG

AGCCTAAGAC

TCAGAGACTA

AATGAGTTGT

CTCCAAAGAA

GAAGATAGAC

GATCT

120 180 240 300 360 415 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 415 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 80..391 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: ATCGAATTCC ATTCAAGAAT AGTTCAAACA AGAAGATTAC AAACTATCAA TTTCATACAC AATATAAACG ACCAAAAGA ATG AAG GCT GTT TTC TTG GTT TTG TCC TTG ATC 112 Met Lys Ala Val Phe Leu Val Leu Ser Leu lle 1 5 SGGA TTC TGC TGG GCC CAA CCA GTC ACT GGC GAT GAA TCA TCT GTT GAG 160 Gly Phe Cys Trp Ala Gin Pro Val Thr Gly Asp Glu Ser Ser Val Glu 20 ATT CCG GAA GAG TCT CTG ATC ATC GCT GAA AAC ACC ACT TTG GCT AAC 208 lie Pro Glu Glu Ser Leu Ile Ile Ala Glu Asn Thr Thr Leu Ala Asn 35 GTC GCC ATG GCT AAG AGA TTC GTT GAC CAA CAC TTG TGC GGT TCT CAC 256 Val Ala Met Ala Lys Arg Phe Val Asp Gin His Leu Cys Gly Ser His 50 TTG GTT GAA GCT TTG TAC TTG GTT TGT GGT GAA AGA GGT TTC TTC TAC 304 Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr 65 70 ACT CCA AAG TCT GAC GAC GCT AAG GGT ATC GTT GAA CAA TGT TGT ACT 352 Thr Pro Lys Ser Asp Asp Ala Lys Gly Ile Val Glu Gin Cys Cys Thr 85 TCT ATC TGT TCT TTG TAC CAA TTG GAA AAC TAC TGT GGT TAGACGCAGC 401 Ser lie Cys Ser Leu Tyr Gin Leu Glu Asn Tyr Cys Gly 100 CCGCAGGCTC TAGA 415 415 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 104 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Met Lys Ala Val Phe Leu Val Leu Ser Leu Ile Gly Phe Cys Trp Ala 1 5 10 *o 0:o~ Gin Pro Val Leu Ile Ile Arg Phe Val Tyr Leu Val Asp Ala Lys Tyr Gin Leu Thr Gly Asp Ala Glu Asn Glu Ser Ser 25 Thr Thr Leu Val Glu Ile Pro Ala Asn Val 40 Ala Val Glu Glu Ser Met Ala Lys Glu Ala Leu Asp Gin His Cys Gly Glu 70 Cys Gly Ser His Leu Thr Arg Gly Phe Phe Tyr 75 Thr Pro Lys Ser Asp Leu Gly Ile Glu Asn 100 Val Glu Gin Cys Ser Ile Cys Ser Tyr Cys Gly INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 415 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: TAGCTTAAGG TAAGTTCTTA TCAAGTTTGT TCTTCTAATG

TTTGATAGTT

TTATATTTGC TGGTTTTCTT ACTTCCGACA AAAGAACCAA

AACAGGAACT

GACCCGGGTT GGTCAGTGAC CGCTACTTAG TAGACAACTC TAAGGCCTTC GTAGCGACTT TTGTGGTGAA ACCGATTGCA GCGGTACCGA

TTCTCTAAGC

GAACACGCCA AGAGTGAACC AACTTCGAAA CATGAACCAA

ACACCACTTT

GATGTGAGGT TTCAGACTGC TGCGATTCCC ATAGCAACTT

GTTACAACAT

AAGAAACATG GTTAACCTTT TGATGACACC AATCTGCGTC GGGCGTCCGA

AAAGTATGTG

AGCCTAAGAC

TCAGAGACTA

AACTGGTTGT

CTCCAAAGAA

GAAGATAGAC

GATCT

120 180 240 300 360 415 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 415 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 80..391 SEQUENCE DESCRIPTION: SEQ ID NO:29: ATCGAATTCC ATTCAAGAAT AGTTCAAACA AGAAGATTAC AAACTATCAA

TTTCATACAC

AATATAAACG ACCAAAAGA ATG MAG GCT GTT TIC TIG GTT HTG TCC TTG ATC Met Lys Ala Val Phe Leu Val Leu Ser Leu Ile 1 5 112 GGA TIC TGC TGG Gly Phe Cys Trp ATT CCG GMA GAG Ile Pro Glu Giu GTC GOC ATG GCT Val Ala Met Ala TTG GTT GMA GCT Leu Val Glu Ala ACT CCA MAG ICT Thr Pro Lys Ser TCT ATC IGT TCT Ser le. Cys Ser CCGCAGGCTC

TAGA

GCC

Ala

TCT

Ser

MAG

Lys

TTG

Leu

GAC

Asp

TTG

Leu

CMA

Gin

CTG

Le u

AGA

Arg

TAC

Tyr 65

GAC

Asp

TAC

Tyr CCA GTC Pro Val ATC ATC Ilie Ilie 35 TTC GTT Phe Val 50 TTG GTT Leu Val GCT MAG Ala Lys CMA TTG Gin Leu

ACT

Thr 20

GCT

Ala

ACT

Thr

TGT

Cys

GGT

Gly

GMA

Giu 100

GGC

Gly

GMA

Giu

CAA

Gin

GGT

Gly

ATC

Ilie 85

MAC

Asn

GAT

Asp

MAC

Asn

CAC

His

GMA

Giu 70

GTT

Val

TAC

Tyr

GMA

Giu

ACC

Thr

HTG

Leu

AGA

Arg

GMA

Glu

TGT

Cys

TCA

Ser

ACT

Thr

TGC

Cys

GGT

Gly

CMA

Gin

GGT

Gi y TCT GHT GAG Ser Val Giu TTG GCT MAC Leu Ala Asn GGT TCT CAC Gly Ser His TIC TIC TAC Phe Phe Tyr TGT TGT ACT Cys Cys Thr

TAGACGCAGC

160 208 256 304 352 401 415 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 104 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Ala Val Phe Leu Val Leu Ser Leu Ilie Gly Phe Cys Trp Ala 1 5 10 Gin Pro Val Thr Gly Asp Glu Ser Ser Val Giu Ilie Pro Glu Giu Ser 20 25 Leu lie lie Ala Glu Asn Thr Thr 40 Leu Ala Asn Val Ala Met Ala Lys Arg Phe Tyr Leu Val Thr Gin His Val Cys Gly Glu 70 Leu Cys 55 Gly Ser His Leu Thr Val Glu Ala Leu Arg Gly Phe Phe Tyr 75 Thr Pro Lys Ser Asp Leu Asp Ala Lys Gly lie Val Glu Gin Tyr Gin Leu Glu Asn Tyr Cys Gly 100 Cys Ser Ile Cys Ser INFORMATION FOR SEQ ID NO:31: SEQUENCE

CHARACTERISTICS:

LENGTH: 415 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TAGCTTAAGG TAAGTTCTTA TCAAGTTTGT TCTTCTAATG

TTTGATAGTT

TTATATTTGC TGGTTTTCTT ACTTCCGACA AAAGAACCAA

AACAGGAACT

GACCCGGGTT GGTCAGTGAC CGCTACTTAG TAGACAACTC

TAAGGCCTTC

GTAGCGACTT TTGTGGTGAA ACCGATTGCA GCGGTACCGA

TTCTCTAAGC

GAACACGCCA AGAGTGAACC AACTTCGAAA CATGAACCAA

ACACCACTTT

GATGTGAGGT TTCAGACTGC TGCGATTCCC ATAGCAACTT

GTTACAACAT

AAGAAACATG GTTAACCTTT TGATGACACC AATCTGCGTC

GGGCGTCCGA

AAAGTATGTG

AGCCTAAGAC

TCAGAGACTA

AATGAGTTGT

CTCCAAAGAA

GAAGATAGAC

GATCT

120 180 240 300 360 415 a a INFORMATION FOR SEQ ID NO:32: SEQUENCE

CHARACTERISTICS:

LENGTH: 523 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:

CDS

a a LOCATION: 80..499 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: ATCGAATTCC ATTCAAGAAT AGTTCAAACA AGAAGATTAC AAACTATCAA

TTTCATACAC

AATATAAACG ATTAAAAGA ATG AGA TTT CCT TCA ATT TTT ACT GCA GTT TTA Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu 1 5 112 TTC GCA GCA TCC TCC GCA Phe Ala Ala Ser Ser Ala I TTA GCT GCT CCA GTC AAC AC1

GAT

Asp

TTA

Leu

AAT

Asn

AAA

Lys

TGC

Cys

GGT

Gly

CAA

Gin GAA ACG SGlu Thr

GAA-GGG

Glu Gly AAC GGG Asn Gly GAA GAA Glu Glu GGT TCT Gly Ser TTC TTC Phe Phe 110 TGT TGT Cys Cys 125

GCA

Ala

GAT

Asp

TTA

Leu

GGG

Gly

CAC

His

TAC

Tyr

ACT

Thr

CAA

Gin

TTC

Phe

TTG

Leu

GTA

Val

TTG

Leu

ACT

Thr

TCT

Ser

ATT

Ile

GAT

Asp

TTT

Phe 65

TCT

Ser

GTT

Val

CCA

Pro

ATC

Ile SLeu Ala Ala Pro Val Asn Thr 20 CCG GCT GAA GCT GTC ATC GG1 Pro Ala Glu Ala Val Ile G13 35 GTT GCT GTT TTG CCA TTT TCC Val Ala Val Leu Pro Phe Ser 50 ATA AAT ACT ACT ATT GCC AGC Ile Asn Thr Thr lie Ala Ser 70 TTG GAT AAG AGA TTC GTT AAC Leu Asp Lys Arg Phe Val Asn 85 GAA GCT TTG TAC TTG GTT TGT Glu Ala Leu Tyr Leu Val Cys 100 AAG TCT GAC GAC GCT AAG GGT Lys Ser Asp Asp Ala Lys Gly 115 120 r

T

y

ACA

Thr

TAC

Tyr

AAC

Asn

ATT

Ile

CAA

Gin

GGT

Gly 105

ATC

lle

ACA

Thr

TCA

Ser

AGC

Ser

GCT

Ala

CAC

His

GAA

Glu GTT Val

GAA

Glu

GAT

Asp

ACA

Thr

GCT

Ala

TTG

Leu

AGA

Arg 3AA ilu 160 208 256 304 352 400 448 496 TGT TCT TTG TAC Cys Ser Leu Tyr 130 CAA TTG Gin Leu 135 GAA AAC TAC TGT Glu Asn Tyr Cys

AAC

Asn 140 (2) TAGACGCAGC CCGCAGGCTC

TAGA

INFORMATION FOR SEQ ID NO:33: SEQUENCE

CHARACTERISTICS:

LENGTH: 140 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 523 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser Al a Leu Ala Ala Pro Ala Gin Val Asn Thr Thr Thr Tyr Ser Glu Asp Glu Thr Ile Pro Ala Asp Val Ala Glu Ala Val Ile 61 y 40 Asp Leu Giu As n Gly Asp Phe Gly Leu Leu Val Leu Pro Phe Al a Ser Asn Ser Thr Ph e Ilie Asn Thr Thr Ilie 70 Ser Ile Ala Ala 75 Giu Glu Gly Val Ser Leu Asp Lys Arg Tyr Phe Val Asn Gin Leu Cys Giy Ser His Leu Val Giu Ala Pro Lys Ser 115 Ilie Cys Ser 130 Le u 100 Asp Leu Leu Val Cys Gi y 105 Ile Arg Gly Phe Phe Tyr Thr 110 Cys Thr Ser Asp Aia Lys Gly 120 Tyr Gin Leu Giu Val Glu Gin Cys 125 Asn Tyr Cys INFORMATION FOR SEQ ID NO:34:

S

S

S.

S

SEQUENCE CHARACTERISTICS: LENGTH: 523 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: TAGCTTAAGG TAAGTTCTTA TCAAGTTTGT TCTTCTAATG

TTTGATAGTT

TTATATTTGC TAATTTTCTT ACTCTAAAGG AAGTTAAAAA

TGACGTCAAA

TAGGAGGCGT AATCGACGAG GTCAGTTGTG ATGTTGTCTT

CTACTTTGCC

CCGACTTCGA CAGTAGCCAA TGAGTCTAAA TCTTCCCCTA

AAGCTACAAC

TAAAAGGTTG TCGTGTTAT TGCCCAATAA CAAATATTTA

TGATGATAAC

ACGATTTCTT CTTCCCCATA GAAACCTATT CTCTAAGCAA

TTGGTTGTGA

AGTGAACCAA CTTCGAAACA TGAACCAAAC ACCACTTTCT

CCAAAGAAGA

CAGACTGCTG CGATTCCCAT AGCAACTTGT TACAACATGA

AGATAGACAA

AAAGTATGTG

ATAAGCGTCG

GTGTTTAAGG

GACAAAACGG

GGTCGTAACG

ACACGCCAAG

TGTGAGGTT

GAAACAiGGT 120 180 240 300 360 420 480 TAACCTTTTG ATGACATTGA TCTGCGTCGG GCGTCCGAGA

TCT

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 409 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 80..385 (xi) SEQUENCE DESCRIPTION: SEQ ID ATCGAATTCC ATTCAAGAAT AGTTCAAACA AGAAGATTAC AAACTATCAA

TTTCATACAC

AATATAAACG ACCAAAAGA ATG AAG GCT GTT TTC TTG GTT TTG TCC TTG ATC Met Lys Ala Val Phe Leu Val Leu Ser Leu Ile 1 5 523 112 GGA TTC TGC TGG GCC CAA CCA GTC ACT GGC Gly Phe Cys Trp Ala Gin Pro Val Thr Gly 20 ATT CCG GAA GAG TCT CTG ATC ATC GCT GAA Ile Pro Glu Glu Ser Leu Ile Ile Ala Glu 35 GTC GCC ATG GCT AAG AGA TTC GTT AAC CAA Val Ala Met Ala Lys Arg Phe Val Asn Gin 50 TTG GTT GAA GCT TTG TAC TTG GTT TGT GGT Leu Val Glu Ala Leu Tyr Leu Val Cys Gly 60 65 ACT CCT AAG GAA AAG AGA GGT ATC GTT GAA Thr Pro Lys Glu Lys Arg Gly Ile Val Glu 85 TGT TCT TTG TAC CAA TTG GAA AAC TAC TGT Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys 100

TAGA

INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids

GAT

Asp

AAC

Asn

CAC

His

GAA

Glu 70

CAA

Gin

GGT

Gly GAA TCA TCT GTT GAG Glu Ser Ser Val Glu ACC ACT TTG GCT AAC Thr Thr Leu Ala Asn TTG TGC GGT TCT CAC Leu Cys Gly Ser His AGA GGT TTC TTC TAC Arg Gly Phe Phe Tyr TGT TGT ACT TCT ATC Cys Cys Thr Ser Ile TAGACGCAGC

CCGCAGGCTC

160 208 256 304 352 405 409 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE (xi) SEQUENCE Met Lys Ala Val Phe 1 5 Gin Pro Val Thr Giv TYPE: protein DESCRIPTION: SEQ ID NO:36: Leu Val Leu Ser Leu Ile Gly Phe 10 Ala Leu Ile Ile Arg Phe Val Tyr Leu Val Aso tG1u Sce' Giu Asn Thr Thr 40 Ser Val 25 Leu Alia Giu Ile Pro CYs Trp Ala Giu Glu Ser Met Aia Lys Giu Aia Leu Asn Val Ala Val1 Asn Gin His Leu Cys 55 Gly Ser His Leu Th r Cys Gly Glu Arg Gly Phe Phe Tyr Pro Lys Giu Lys Gl n Arg Gly Ile Val Giu Gin Cys Cys Thr Cys Gly Ile Cys Ser Leu Tyr Leu Giu Asn Tyr 100 INFORMATION FOR SEQ ID NO:37: SEQUENCE

CHARACTERISTICS:

LENGTH: 409 base pairs TYPE: nucieic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TAGCTTAAGG TAAGTTCTTA TCAAGTTTGT TCTTCTAATG

TTTGATAGTT

TTATATTTGC TGGTTTTCTT ACTTCCGACA AAAGAACCAA

AACAGGAACT

GACCCGGGTT GGTCAGTGAC CGCTACTTAG TAGACAACTC

TAAGGCCTTC

GTAGCGACTT TTGTGGTGAA ACCGATTGCA GCGGTACCGA

TTCTCTAAGC

GAACACGCCA AGAGTGAACC AACTTCGAAA CATGAACCAA

ACACCACTTT

GATGTGAGGA TTCCTTTTCT CTCCATAGCA ACTTGTTACA

ACATGAAGAT

CATGGTTAAC CTTTTGATGA CACCAATCTG CGTCGGGCGT

CCGAGATCT

AAAGTATGTG

AGCCTAAGAC

TCAGAGACTA

AATTGGTTGT

CTCCAAAGAA

AGACAAGAAA

120 180 240 300 360 409 INFORMATION FOR SEQ ID NO:38: SEQUENCE

CHARACTERISTICS:

LENGTH: 511 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 77. .487 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: GAATTCCATT CAAGAATAGT TCAAACAAGA AGATTACAAA CTATCAATTT

CATACACAAT

ATAAACGATT AAAAGA ATG AGA TTT CCT TCA ATT TTT ACT GCA GTT T-TA Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu 109 a.

a a. a a.

a a a.

TTC

Phe

GAT

Asp

TTA

Leu

MAT

As n

AAA

Lys

TGC

Cys

GGT

G y_

ACT

Thr

GCA

Ala

GMA

Glu

GMA

Glu

MAC

As n

GMA

Glu

GGT

Gl y

TTC

Phe

TCT

Ser

GCA

Ala

ACG

Thr

GGG

Gly

GGG

Gly

GMA

Glu

TCC

Ser

TTC

Phe 110

ATC

Ile TCC TCC Ser Ser GCA CMA Ala Gln GAT TTC Asp Phe TTA TTG Leu Leu GGG GTA Gly Val 80 CAC HTG His Leu 95 TAC ACT Tyr Thr TGT TCT Cys Ser

GCA

Ala

ATT

Ile

GAT

Asp

TTT

Phe 65

TCC

Ser

GTT

Val

CCA

Pro

TTG

HTA

Leu

CCG

Pro

GTT

Val 50

ATA

Ile

ATG

Met

GMA

Gl u

MAG

Lys

GCT

Ala

GCT

Ala 35

GCT

Ala

MAT

Asn

GCT

Ala

GCT

Ala

ACT

Thr 115

GCT

Ala 20

GMA

Glu

GTT

Val1

ACT

Thr

MAG

Lys

HTG

Leu 100

AGA

Ar g

CCA

Pro G CT Al a

TTG

Leu

ACT

Thr

AGA

Arg 85

TAC

Tyr

GGT

Gl y

GMA

Gl u GTC MAC ACT ACA Val Asn Thr Thr GTC ATC GGT TAC Val Ile Gly Tyr CCA TTT TCC MAC Pro Phe Ser Asn AUT GCC AGC AUT Ile Ala Ser Ile 70 TTC GTT MAC CMA Phe Val Asn Gln TTG GTT TGT GGT Leu Val Cys Gly 105 ATC GTT GMA CMA Ile Val Glu Gln

TCA

Ser

AGC

Ser

GCT

Ala

CAC

His

GMA

Glu

TGT

Cys

GAT

Asp

ACA

Thr

GCT

Ala

HTG

Leu

AGA

Arg

TGT

Cys 205 253 301 349 397 445 487 ACA GMA Thr Gl u TAC CAA TTG 120 MAC TAC TGC Asn Tyr-Cys 135 Leu Tyr Gln Leu 130 125 a TAGACGCAGC CCGCAGGCTC TAGA 511 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTE

RISTICS:

LENGTH: 137 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE (xi) SEQUENCE Arg Phe Pro Ser 5 TYPE: protein DESCRIPTION: SEQ ID NO:39: Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 10 Met 1 Ala Leu Al a Ala Pro Ile Pro Ala Giu Ala Asp Val Ala Val Leu Phe Ile Asn Thr Thr IVal Asn Val Ile Pro Phe Ile Ala 70 Phe Val Thr Thr Thr Gi u Asp GI u Thr Al a Gi n 25 Gly Tyr Ser Asp Leu Glu Gly Asp Phe 40 Ser Asn Ser Thr Asn Asn Gly Leu Leu Ser Ilie Ala Ala Lys Giu Glu Gly Val Ser Met Ala Lys 75 Asn Gin His Leu Arg Cys Gly Ser Val Glu Ala Leu Tyr 100 Leu Val Cys 90 Gly Glu 105 Hi s Leu Tyr Thr Arg Gly Phe Pro LYS Thr Arg Gly 115 Leu Tyr Gin Leu Glu 130 Ilie Val Glu Gin Cys Cys Thr Ser 120 125 Asn Tyr Cys Asn 135 Ilie Cys Ser

S.

S S

OSO*

*SSS

S. S S 5 INFORMATION FOR SEQ ID i)SEQUENCE

CHARACTERISTICS:

LENGTH: 511 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear Iii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID N10:40: CTTAAGGTMA GTTCTTATCA AGTTTGTTCT TCTAATGTTT GATAGTTAAA GTATGTG

TTA

TATTTGCTMA TTTTCTTACT CTAAAGGAAG TTAAAAATGA CGTCAAAATA

AGCGTCGTAG

S.

S. S 6O

S

.S.SSS

S

0

CSS*S*

0 120 GAGGCGTAAT CGACGAGGTC AGTTGTGATG TTGTCTTCTA CTTTGCCGTG TTTAAGGCCG 180 ACTTCGACAG TAGCCAATGA GTCTAAATCT TCCCCTAAAG CTACAACGAC AAAACGGTAA 240 AAGGTTGTCG TGTTTATTGC CCAATAACAA ATATTTATGA TGATAACGGT CGTAACGACG 300 ATTTCTTCTT CCCCATAGGT ACCGATTCTC TAAGCAATTG GTTGTGAACA CGCCAAGGGT 360 GAACCAACTT CGAAACATGA ACCAAACACC ACTTTCTCCA AAGAAGATGT GAGGTTTCTG 420 ATCTCCATAG CAACTTGTTA CAACATGAAG ATAGACAAGA AACATGGTTA ACCTTTTGAT 480 GACGTTGATC TGCGTCGGGC GTCCGAGATC T 511 INFORMATION FOR SEQ ID NO:41: SEQUENCE

CHARACTERISTICS:

LENGTH: 523 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:

CDS

LOCATION: 80..499 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: ATCGAATTCC ATTCAAGAAT AGTTCAAACA AGAAGATTAC AAACTATCAA TTTCATACAC AATATAAACG ATTAAAAGA ATG AGA TTT CCT TCA ATT TTT ACT GCA GTT TTA 112 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu 1 5 TTC GCA GCA TCC TCC GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA 160 Phe Ala Ala Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu 20 GAT GAA ACG GCA CAA ATT CCG GCT GAA GCT GTC ATC GGT TAC TCA GAT 208 Asp Glu Thr Ala Gin Ile Pro Ala Glu Ala Val lie Gly Tyr Ser Asp 35 TTA GAA GGG GAT TTC GAT GTT GCT GTT TTG CCA TTT TCC MAC AGC ACA 256 Leu Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr 50 AAT-AAC GGG TTA TTG TTT ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT 304 Asn Asn Gly Leu Leu Phe le Asn Thr Thr lie Ala Ser Ile Ala Ala 65 70 AAA GAA GAA GGG GTA TCC ATG GCT AAG AGA TTC GTT AAC CAA CAC UG 352 Lys Glu Glu Gly Val Ser Met Ala Lys Arg Phe Val Asn Gin His Leu 85

TGC

Cys

GGT

Gi y

CMA

Gin

MAC

Asn 140 (2) GGT TCC CAC TTG GTT GMA GCT TIG Gly Ser His Leu Val Giu Ala Leu 100 TTC TTC TAC ACT CCT MG TCT GAC Phe Phe Tyr Thr Pro Lys Ser Asp 110 115 TGC TGT ACC TCC ATC TGC TCC TTG Cys Cys Thr Ser Ile Cys Ser Leu 125 130 TAGACGCAGC CCGCAGGCTC

TAGA

TAC TTG GTT TGC GGT GMA AGA Tyr Leu Val Cys Gly Giu Arg 105 GAT GCT MAG GGT ATT GTC GAG Asp Ala Lys Gly Ile Val Giu 120 TAC CAA TTG GMA MC TAC TGC Tyr Gin Leu Glu Asn Tyr.Cys 135 400 448 496 INFO-RMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 140 amino acids TYPE: amino acid TOPOLOGY: linear Met Al a Ile Asp Phe 65 Ser Val Pro (i i) (xi) Arg Phe Leu Al a Pro Ala Val Ala 50 Ile Asn Met Ala Giu Ala Lys Ser 115

SEQUENCE

Pro Ser Ala Pro Giu Ala Val Leu Thr Thr Lys Arg 85 Leu Tyr 100 Asp Asp MOLECULE TYPE: protein DESCRIPTION: SEQ ID NO:42: Ilie Phe Thr Ala Val Leu Phe Ala Ala Ser Val Val Pro Ile 70 Phe Leu Al a Asn Ile Phe 55 Ala Val Val Lys Leu 135 Thr Gly 40 Ser Ser As n Cys Gly 120 Thr 25 Tyr Asn Ile Gin Gi y 105 Ile 10 Th r Ser Ser Ala His 90 Glu Val Glu Asp Th r Ala 75 Leu Arg Glu Asp Le u As n Lys Cys Gi y Gi n Asn 140 Giu Glu Asn Giu Giy Phe Cys Thr Gi y Gly Giu Ser Phe 110 Cys Ala Asp Leu Gly His Tyr Thr Gln Phe Leu Val Leu Thr Ser Ile Cys 130 Ser Leu Tyr Gin Glu Asn Tyr Cys INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 523 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: TAGCTTAAGG

TAAGTTCTTA

TTATATTTGC

TAGGAGGCGT

CCGACTTeGA

TAAAAGGTTG

ACGATTTCTT

GGTGAACCAA

CAGACTGCTA

TAACCTTTTG

TAATTTTCTT

AATCGAC GAG

CAGTAGCCAA

TCGTGTTTAT

CTTCCCCATA

CTTCGAAACA

CGATTCCCAT

ATGACGTTGA

TCAAGTTGT

ACTCTAAAGG

GTCAGTTGTG

TGAGTCTAAA

TGCCCAATAA

GGTACCGATT

TGAAC CAAAC

AACAGCTCGT

TCTGCGTCGG

TCTTCTAATG

AAGTTAAAAA

ATGTTGTCTT

TCTTCCCCTA

CAAATATTTA

CTCTAAGCAA

GCCACTTTCT

TACGACATGG

GCGTCCGAGA

TTTGATAGTT

TGACGTCAAA

CTACTTTGCC

AAGCTACAAC

TGATGATAAC

TTGGTTGTGA

CCAAAGAAGA

AGGTAGACGA

TCT

AAAGTATGTG

ATAAGCGTCG

GTGTTAAGG

GACAAAAC GG

GGTCGTAACG

ACACGCCAAG

TGTGAGGATT

GGAACATGGT

120 180 240 300 360 420 480 523 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 535 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 77..511 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: GAATTCCATT CAAGAATAGT TCAAACAAGA AGATTACAAA CTATCAATTT

CATACACAAT

ATAAACGATT AAAAGA ATG AGA TTT CCT TCA ATT MT ACT GCA GTT TTA Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu 1 5 TTC GCA GCA TCC TCC GCA TTA GCT GCT CCA GTC MAC ACT ACA ACA GMA Phe Al a Al a Ser Ser Al a Leu Al a Al a Pro Val Asn Thr Thr Thr G! u 20 109 157 GAT GMA ACG GCA CMA AUT CCG GCT GAA Asp Giu Thr Ala Gin Ile Pro Ala Glu

HTA

Leu

MAT

Asn

AMA

Lys

GCT

Ala

TTG

Leu

AGA

Ar g

TTG

Leu 140

GMA

Giu

MAC

Asn

GMA

Glu

AGA

Arg

TAC

Tyr

GGT

Gly 125

GMA

Giu

GGG

Gly

GGG

Gly

GMA

Giu

TTC

Rh e

HTG

Le u 110

ATC

Ile

MAC

Asn

GAT

Asp

HTA

Leu

GGG

Gi y

GTT

Vai 95

GTT

Val

GTT

Val

TAC

Tyr

TTC

Phe

TTG

Leu

GTA

Vai

MAC

As n

TGT

Cys

GMA

Glu

GAT

Asp

HT

Phe 65

TCC

Ser

CAA

Gin

GGT

Giy

CMA

Gin

GTT

Vai 50

ATA

Ile

ATG

Met

CAC

His

GMA

Giu

TGT

Cys 130 35

GCT

Aia

MAT

Asn

GCT

Ala

TTG

Leu

AGA

Arg 115

TGT

Cys

GTT

Val1

ACT

Thr

MAG

Lys

TGC

Cys 100

GGT

Gi y

ACT

Th r 83

GCT

Ala

TTG

Leu

ACT

Thr

AGA

Arg 85

GGT

Gi y

TTC

Phe

TCT

Ser GTC ATC GGT Val Ilie Gly CCA UTT TCC Pro Phe Ser ATT GCC AGC Ile Ala Ser 70 GMA GMA GCT Giu Giu Ala TCC CAC TTG Ser His Leu TTC TAC ACT Phe Tyr Thr 120 ATC TGT TCT Ilie Cys Ser 135 TAC TCA GAT Tyr Ser Asp

MAC

As n

AUT

Ile

GMA

Giu

GTT

Val 105

CCA

Pro

TTG

Le u

AGC

Ser

GCT

Ala

GCT

Ai a

GMA

Giu

MAG

Lys

TAC

Tyr

ACA

Th r

GCT

Ala

GMA

Glu

GCT

Ala

ACT

Th r

CMA

Gin 253 301 349 397 445 493 535 TGC MAC Cys Asn 145 TAGACGCAGC CCGCAGGCTC

TAGA

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 145 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Arg Phe Pro Ser Ilie Phe Thr Ala Vai Leu Phe Aia Ala Ser Ser 1 5 10 Ala Leu Aia Ala Pro Val Asn Thr Thr Thr Giu Asp Glu Thr Ala Gin 25 Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35 40 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 55 Phe Ilie Asn Ser Met Ala Thr Thr Ilie Ala 70 Lys Arg Giu Glu Ser Ilie Ala Al a Lys 75 Giu Ala Glu Giu Gly Val Arg Phe Val Asn Ala Glu Gly Ala 90 Gi u Gin His Leu Gly Glu Arg 115 Gin Cys Cys 130 Ser His Leu Val1 105 Pro Ala Leu Tyr Leu Val Cys 110 Ilie Val Giu Asn Tyr Cys Phe Phe Tyr Thr 120 Ser Lys Thr Arg Giy 125 Giu Thr Ser Ilie Cys 135 Leu Tyr Gin As n 145 INFORMATION FOR SEQ ID NO:46: i)SEQUENCE

CHARACTERISTICS:

LENGTH: 535 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: CTTAAGGTAA

GTTCTTATCA

TATTTGCTM

.TTTTCTTACT

GAGGCGTAAT

CGACGAGGTC

ACTTCGACAG

TAGCCAATGA

AAGGTTGTCG

TGTTTATTGC

ATTTCTTCTT

CCCCATAGGT

ATTGGTTGTG

AACACGCCAA

TCCAAAGAAG

ATGTGAGGTT

AAGAAACATG GTTAACCTTM

AGTTTGTTCT

CTAAAGGAAG

AGTTGTGATG

GTCTAAATCT

CCAATAACAA

ACCGATTCTC

GGGTGAACCA

TC TGAT CTC C

TGATGACGT

TCTAATGTTT

TTAAAAATGA

TTGTCTTCTA

TCCCCTAAAG

ATATTTATGA

TCTTCTTCGA

ACTTCGAAAC

ATAGCAACTT

GATCTGCGTC

GATAGTTAAA

CGTCAAAATA

CTTTGCCGTG

CTACAACGAC

TGATAACGGT

CTTCGACTTC

ATGAACCAAA

GTTACAACAT

GGGCGTCCGA

GTATGTGTTA

AGC GTCGTAG

TTTAAGGCCG

AAAACGGTAA

CGTAACGACG

GATCTAAGCA

CACCACTTC

GAAGATAGAC

GATCT

120 180 240 300 360 420 480 535 INFORMATION FOR SEQ ID NO:47: i)SEQUENCE

CHARACTERISTICS:

LENGTH: 538 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 77..514 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: GAATTCCATT CAAGAATAGT TCAAACAAGA AGATTACAAA CTATCAATTT

CATACACAAT

ATAMACGATT AAAAGA ATG AGA UTT CCT TCA AUTTTM ACT GCA GTT HTA Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu 109

TTC

Phe

GAT

Asp

HTA

Leu

MAT

As n

A

Lys

GCT

Ala

GCT

Ala

ACT

Th r

CAA

Gin 140 GCA GCA Ala Ala GMA ACG Glu Thr GMA GGG Giu Gly MAC GGG Asn Gly GMA GMA Glu Glu GMA AGA Glu Arg TTG TAC Leu Tyr 110 AGA GGT Arg Gly 125 HTG GMA Leu Glu

TCC

Ser

GCA

Ala

GAT

Asp

TTA

Leu

GGG

Gi y

TTC

Phe

TTG

Leu

ATC

Ile

MAC

As n TCC GCA HTA Ser Ala Leu CMA ATT CCG Gin Ile Pro TTC GAT GTT Phe Asp Val 50 HTG UTT ATA Leu Phe Ile 65 GTA TCC ATG Val Ser Met GTT MAC CMA Val Asn Gin GTT TGT GGT Val Cys Gly GTT GMA CMA Val Glu Gin 130 TAC TGC MAC Tyr Cys Asn 145

GCT

Ala

GCT

Ala 35

GCT

Ala

MAT

As n

GCT

Ala

CAC

His

GMA

Gi u 115

TGT

Cys

*GCT

Ala 20

GMA

Glu

GTT

Val

ACT

Thr

MAG

Lys

TTG

Leu 100

AGA

Ar g

TGT

Cys

CCA

Pro

GCT

Ala

TTG

Leu

ACT

Thr

AGA

Arg 85

TGC

Cys

GGT

Gi y

ACT

Thr GTC MAC ACT Val Asn Thr GTC ATC GGT Val Ile Gly CCA TTT TCC Pro Phe Ser ATT GCC AGC Ilie Ala Ser 70 GMA GMA GCT Glu Giu Ala GGT TCC CAC Gly Ser His TTC TTC TAC Phe Phe Tyr 120 TCT ATC TGT Ser Ilie Cys

ACA

Thr

TAC

Tyr

MAC

Asn

AUT

Ile

GMA

Giu

HTG

Leu 105

ACT

Thr

ACA

Thr

TCA

Ser

AGC

Ser

GCT

Ala

GCT

Ala

GTT

Val1

CCA

Pro

GMA

Giu

GAT

Asp

ACA

Thr

GCT

Ala

GMA

Gi u

GMA

Gi u

MAG

Lys 157 205 253 301 349 397 445 493 538 a a.a a a a a *.aa*a a aaa* a TCT TTG TAC Ser Leu Tyr 135 TAGACGCAGC CCGCAGGCTC TAGA INFORMATION FOR SEQ ID NO:48: ()SEQUENCE CHARACTERISTICS: LENGTH: 146 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE (xi) SEQUENCE TYPE: protein DESCRIPTION: SEQ ID NO:48: Met 1 Al a Arg Phe Pro Ser Ile Phe Thr Ala Leu Phe Ala Ala Ser Ser.

Leu Al a Al a Val Asn Thr Thr 25 Tyr Glu Asp Glu Ilie Pro Ala Asp Vai Ala Phe Ilie Asn Glu Ala Val Ile Gi y Ser Ser Asp Leu Thr Al a GI n Gly Asp Phe Gly Leu Leu Val Leu Pro Thr Thr Ilie 70 Lys Arg Glu Asn Ser Thr Asn Lys Ser Ilie Ala Ala 75 Giu Giu Glu Gly Vai Ser Met Ala Asn Gin His Cys Giy Giu 115 Giu Gin Cys 130 Cys Asn 145 Glu Ala Giu Ala GIlu Arg Phe Val Leu 100 Ar g Giy Ser His Leu 105 Thr Giu Ala Leu Gly Phe Phe Tyr 120 Cys Pro Lys Thr Tyr Leu Val 110 Gly Ilie Val Giu Asn Tyr Cys Thr Ser Ser Leu Tyr Gin 140 e S S *5

S.-

S

*SS*

S

S.

S

S.

S

INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 538 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: CTTAAGGTAA GTTCTTATCA AGTTTGTTCT TCTAATGTTT GATAGTTAAA GTATGTGTTA TATTTGCTAA TTTTCTTACT CTAAAGGAAG TTAAAAATGA CGTCAAAATA AGCGTCGTAG GAGGCGTAAT CGACGAGGTC AGTTGTGATG TTGTCTTCTA CTTTGCCGTG TTTAAGdCCG 120 180

ACTTCGACAG

AAGGTTGTCG

ATTTCTTCTT

GCAATTGGTT

TTCTCCAAAG

GACAAGAAAC

TAGCCAATGA

TGTTTATTGC

CCCCATAGGT

GTGAACACGC

AAGATGTGAG

ATGGTTAACC

GTCTAAATCT

CCAATAACAA

ACCGATTCTC

CAAGGGTGAA

GTTTCTGATC

MTTGATGAC

TCCCCTAAAG

ATATTTATGA

TCTTCTTCGA

CCAACTTCGA

TCCATAGCAA

GrTGATCTGC

CTACAACGAC

TGATAACGGT

CTTCGACTTC

AACATGAACC

CTTGTTACAA

GTCGGGCGTC

AAAACGGTAA

CGTAACGACG

GACTTTCTAA

AAACACCACT

CATGAAGATA

CGAGATCT

240 300 360 420 480 538 .:000: 00 0 be 00 90 e 0 *0 .0.0 0 0000 0 *00000 0 0

Claims (10)

1. An insulin derivative which is NEB 29 -tetradecanoyl des(B30) human insulin.

2. An insulin derivative which is any Zn 2 complex of NEB 29 -tetradecanoyl human insulin.

3. An insulin derivative which is a Zn 2 complex of NEB 29 -tetradecanoyl des(B30) human insulin containing 2, 3, or 4 Zn 2 ions per insulin hexamer.

4. An insulin derivative which is NEB 29 -(lithocholoyl-glutamyl) human insulin.

5. An insulin derivative which is any Zn 2 complex of NEB 29 -(lithocholoyl- glutamyl) des(B30) human insulin.

6. An insulin derivative which is a Zn2+ complex of NEB 29 -(lithocholoyl- glutamyl) des(B30) human insulin containing 2, 3 or 4 Zn 2 ions per insulin 20 hexamer.

7. A pharmaceutical composition for the treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of an insulin derivative according to any one of claims 1 to 6, together with a S: 25 pharmaceutically acceptable carrier.

8. A pharmaceutical composition for the treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of an insulin derivative according to any one of claims 1 to 6, in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier. Y:\SPECI\51960-OO.doc 89

9. A method of treating diabetes in a patient, comprising administering to the patient a therapeutically effective amount of an insulin derivative according to any one of claims 1 to 6, or of a composition according to claim 7.

10. A method of treating diabetes in a patient, comprising administering to the patient a therapeutically effective amount of an insulin derivative according to any one of claims 1 to 6, in mixture with an insulin or an insulin analogue which has a rapid onset of action, or of a composition according to claim 8. DATED: 8 March 2001 PHILLIPS ORMONDE FITZPATRICK Attorneys for: NOVO NORDISK A/S ooo Y:\SPEC51960-00.doc