INSULIN ANALOGS WITH EXTENDED TIME-ACTION AND HIGH SELECTIVITY FOR INSULIN
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INSULIN ANALOGS WITH EXTENDED TIME-ACTION AND HIGH SELECTIVITY FOR INSULIN vs IGF-1 RECEPTOR

Wayne Kohn, Radmila Micanovic, Sharon Myers, Andrew Vick, Steven Kahl, Lianshan Zhang, Beth Strifler, Shun Li, Jing Shang, John Beals, John Mayer and Richard DiMarchi, from Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, 46285


ABSTRACT SELECTIVITY FOR INSULIN

Attempts to identify a basal insulin product with a peakless profile and 24 hr duration have resulted in little success prior to discovery of insulin glargine, which represents a pharmacokinetic improvement, but with potential limitations such as increased mitogenicity. We conducted a structure-function analysis to identify a superior pI-shifted basal insulin with a receptor affinity profile more comparable to native human insulin. In particular, we have compared the functional effects of basic residues added selectively and combinatorially to the N-terminus of the A- and B-chains and to the C-terminus of the B-chain. IGF-1 receptor affinity was significantly enhanced by addition of basic residues at the C-terminus of the B-chain. Arginine additions at the N-terminus of the A-chain appreciably decreased IGF-1 receptor affinity when incorporated concomitantly with arginines at the C-terminus of the B-chain. Substitutions at position A21 also affected receptor selectivity. Analogs were functionally tested for glucose uptake in 3T3-L1 adipocytes and stimulation of proliferation of human mammary epithelial cells. A strong positive correlation between receptor affinities and respective metabolic and mitogenic potencies was observed. An in vitro assay that estimated solubility of the insulin analogs under physiological conditions was predictive of the time-action in an SRIF dog model. Relative to glargine, two analogs LY2116419 and LY2109967 possessed five and fifteen fold greater insulin receptor selectivity, respectively, with correspondingly five and ten fold lower in vitro mitogenic potency, respectively. In the dog model, both analogs displayed a peakless PK/PD profile, which was similar to that of glargine in duration.

INTRODUCTION

An insulin formulation with a peakless activity profile and 24 hr duration has been long recognized as an important objective for optimizing glucose control in diabetes. The advent of rapid-acting insulins to cover mealtime glucose excursions has intensified the requirement for such a basal insulin , as slow-release formulations of wild-type insulins have been found inadequate. A recently introduced basal insulin, insulin glargine (1) contains one amino acid


substitution relative to human insulin and two additional Arg residues which extend the duration of action via an increased isoelectric point (pI-shift approach) from 5.6 to 7.0. The peptide is formulated at pH 4 and precipitates upon subcutaneous injection. The precipitated peptide acts as a depot that is redissolved and absorbed over an extended period. While glargine possesses an attractive pharmacokinetic profile, it also demonstrates significantly increased mitogenic potential relative to human insulin and appreciable intra- and inter-patient variability. We have performed an extensive structure-function analysis to identify additional basal insulin analogs, which utilize the pI-shift approach to achieve a protracted time action yet maintain a receptor affinity profile that compares more favorably with native insulin. Through this investigation, we intended to identify the ideal number of basic amino acids and their optimal placement in the molecule in order to obtain the desirable pharmacological and physical properties. Effects of basic residues added at the N-terminus of the A-chain, and/or the N-terminus of the B-chain and/or the C-terminus of the B-chain were measured in terms of insulin and IGF-1 receptor binding, stimulation of glucose uptake in differentiated mouse 3T3-L1 adipocytes and stimulation of cell proliferation in human mammary epithelial cells. To identify analogs that may have a protracted time-action profile in vivo, solubility of the insulin analogs was measured in pH 7 PBS buffer. Four objectives of our basal insulin design include: (1) extended peakless PK/PD profile; (2) IR vs. IGF-1R selectivity close to that of human insulin; (3) adequate biopotency; and (4) chemical stability in pH 4 formulation.

ABBREVIATIONS

IR, insulin receptor; IGF-1R, insulin-like growth factor 1 receptor; HI, human insulin; rp-HPLC, reversed-phase high performance liquid chromatography; pI, isoelectric point; PBS, phosphate-buffered

saline

NOMENCLATURE

Human insulin is a 51 aa protein composed of a 21 aa A chain and a 30 aa B chain held together by two

disulfide bonds. The positions within the chains are designated A1 to A21 and B1 to B30, respectively.

Addition of an extra amino acid(s) at either N terminus are given the notation of position 0, -1, etc.

For example, addition of an Arg-Arg dipeptide sequence to the N terminus of the A chain would give

the analog A0:R, A(-1):R-HI. Similarly, additional amino acids at the C termini are denoted A22, A23, etc.


and B31, B32, etc. Addition of amino acid or other acylating agent to Lys e-amine is indicated

in brackets after the position of the amino acid. For example, acylation of the B29:Lys with Arg yields

the analog B29:K(R)-HI.

METHODS

Insulin analogs were prepared by acylation of one or more of the three amino groups (the two N-terminal amines and

the side chain amine of Lysine at position B29) of an insulin “template” with protected amino acids or dipeptides

activated as N-hydroxysuccinimide (NHS) esters with diisopropylcarbodiimide. Agents used were Boc-Arg(Pbf)-NHS;

Boc-Arg(Pbf)-Arg(Pbf)-NHS, and Boc-Lys(Boc-Arg(Pbf))-NHS. Insulin templates HI; A21:G-HI; A21:Xaa-HI;

A21:G,B31:R-HI, and A21:G,B31:R;B32:R-HI were obtained from expression in E. coli via recombinant DNA technology.

Singlechain precursors were solublized from inclusion bodies and refolded under redox conditions. Trypsin treatment cleaved

the leader sequence, which ends with an Arg, and excised the C peptide. Carboxypeptidase B was used to trim Arg residues

from the B chain C terminus as appropriate. Acylation reactions were performed in water/ACN or water/DMF mixtures at

room temperature. The product distribution was modulated via adjustment of the pH, the equivalents of reagent added, and

the length of reaction time. Purification was performed in one of two ways: (1) purification of the reaction mixture by rp-

HPLC followed by protecting-group removal and final rp-HPLC purification, or (2) the reaction mixture was diluted with

water and lyophilized, followed by protecting group removal, purification by cation exchange chromatography and final

purification/desalting by rp-HPLC. Product identity was confirmed by a combination of LC-MS (verification of purity

and molecular weight), N-terminal protein sequencing, and LC-MS analysis of Staph. Aureus V8 protease digest, which

yields characteristic fragments via specific cleavage on the carboxyl side of Glu residues (2).

Receptor binding assays were performed on P1 membrane preparations from stably transfected 293EBNA cells

overexpressing the HI or hIGF-1 receptor. Binding affinities were determined from a competitive binding assay using

the respective native ligand, radiolabeled with 125I. The assay was performed in 96 well plates using scintillation

proximity assay mode. Metabolic potency was determined by measurement of uptake of 14C-deoxyglucose by

differentiated mouse 3T3-L1 adipocytes over 1 hr at 37 oC. Mitogenic potency was determined by the incorporation of

14C-thymidine in human mammary epithelial cells over a 48 hr incubation time. In all assays, the activity relative to HI

control was determined within each experiment and then averaged over the number of experiments. Therefore comparison

of the average EC50 or IC50 for an analog with the average value for HI will not generate the same relative activity value.


PBS solubility assay was performed by formulating each agent to Lys analog in conditions that mimic the commercial formulation of

insulin glargine: ~3.64 mg/ml of protein, 30 mg/mL (unless specified otherwise) of Zn2+ (as ZnCl2) , 2.7 mg/mL m-cresol,

and 20 mg/ml 85 % glycerol, adjusted to pH 4 with HCl. A small aliquot was diluted 10 fold with PBS and allowed to sit

15 min, then spun down for 5 min at 14,000 rpm and r.t. The amount of protein remaining in solution was quantitated

from the peak area on rp-HPLC. Solubility was expressed as a percentage of that observed for HI diluted 10 fold in 0.1

HCl. Isoelectric points were determined with isoelectric focusing gel electrophoresis on Novex IEF gels of pH 3-10,

offering a pI performance range of 3.5-8.5.

In vivo experiments to evaluate the time-action profiles of insulin analogs were conducted in overnight-fasted, cannulated

male and female beagles. On the day of the experiment, indwelling vascular access ports were accessed and an arterial

blood sample was drawn for determination of fasting insulin and glucose concentrations (time = -30 minutes). A continuous

venous infusion (0.65 mg/kg/min) of cyclic somatostatin was initiated and continued for 24.5 hr to inhibit endogenous

insulin secretion. Thirty minutes after the start of the infusion (time = 0), an arterial sample was drawn and a sc bolus of

saline or an insulin preparation (2 nmol/kg) was injected into the dorsal aspect of the neck. Peptides were formulated

as described under the solubility assay. Arterial blood samples were taken periodically thereafter for the determination

of plasma glucose and insulin concentrations. Plasma glucose concentrations were determined the day of the study

using a glucose oxidase method in a Beckman Glucose Analyzer II. Plasma samples were stored at –80oC until time

for insulin analysis. Insulin levels were determined using commercially available radioimmunoassay kits sensitive to

human insulin and analogs.

The biopotency of insulin analogs was determined from a 10-hour euglycemic clamp study, in a set of five normal

dogs. A single subcutaneous dose (3 or 6 nmol/kg; the molar equivalent of 0.5 U/kg) was administered. Animals

were infused intravenously with cyclic somatostatin during the experiment to inhibit endogenous insulin secretion.

Experiments were conducted as previously described (3) using a randomized cross-over design, with a week between

studies in individual dogs.


Figure 1: SCHEMATIC of BASAL agent to Lys

INSULIN ANALOG STRUCTURES

K

R

G

S

S

I

A1

V

A chain

A20

S

S

E

Q

G

C

C

C

T

S

Y

A5

R

I

N

C

S

E

L

Q

Y

L

T

K

F

A10

S

S

S

S

V

A15

P

R

B1

N

S

S

T

Q

Y

H

S

S

F

L

F

C

G

B5

B5

G

R

B25

S

E

H

G

L

C

V

L

V

A

E

L

Y

B10

B20

analog 82: A0:K(R),B29:K(R),A21:G-HI

(LY2109967)

B15

B chain

R

G

S

S

I

A1

V

A chain

A20

S

S

E

Q

R

G

C

C

C

R

T

S

Y

A5

R

I

N

C

S

E

L

Q

Y

L

T

K

F

A10

S

S

S

S

V

A15

P

B1

N

S

S

T

Q

Y

H

S

S

F

L

F

C

G

B5

B5

G

R

B25

S

E

H

G

L

C

V

L

V

A

E

L

Y

B10

B20

B15

B chain

analog 98: A0:R,B31:R,B32:R,A21:G-HI

(LY2116419)


Figure 2: Effect of A21:G Substitution and LysPro Inversion on

Time-Action Profile and Solubility of B31:R,B32:R-HI

Glucose Concentrations in Somatostatin-treated, Normal,

Fasted Dogs after Treatment with Insulin Analogs

(2 nmol/kg, sc)

200

Effect of [Zn2+] on PBS Solubility

Humulin R (0.75 nmol/kg; historical; n=5)

B28:K,B29:P,B31:R,B32:R,A21:G-HI (6) (n=2)

175

B31:R,B32:R,A21:G-HI (4) (n=4)

B31:R,B32:R-HI (43) (n=4)

150

125

Plasma Glucose (mg/dL)

100

75

50

25

0

0

2

4

6

8

10

12

14

16

18

20

22

24

Time from Injection (hours)

These results indicate a dramatic increase of time-action upon A21:G substitution. In contrast, the B28:K,B29:P

inversion decreases the time action. Two animals treated with 6 were removed from the study after 1 hr due to extremely

low glucose levels. The rank order of the PBS solubility values for analogs 4, 6, and 43 (Table 1) correlate with their time

action profiles above. The dramatic effects of [Zn2+] on PBS solubility is shown on the right.

The effect of A21:G substitution on solubility is illustrated graphically for analogs 4 versus 43 and 67 versus 39,

respectively. Removal of B0:R from 67 results in 79, which displays increased solubility at 30 ug/mL Zn2+ but the

similarly low solubility at 80 ug/mL Zn2+.


Glucose Response to Soluble Insulin on

Formulations

(somatostatin-treated dogs; 2 nmol/kg, sc)

Saline (n=5)

B31:R,B32:R,A21:G-HI (4)(n=6)

A0:R,A21:G,B0:R,B29:K(R)-HI (67) (5 ug/ml Zn; n=6)

A0:R,A21:G,B0:R,B29:K(R)-HI (67) (30 ug/ml Zn; n=6)

200

200

A0:R, B0:R, B29:K(R)-HI (39) (n=6)

175

175

150

150

125

Plasma Glucose (mg/dL)

125

Plasma Glucose (mg/dL)

100

100

75

75

50

50

A0:R,B0:R,B29:K(R), A21:D-HI (91) (n=6)

25

A0:R,B0:R,B29:K(R), A21:S-HI (72) (n=6)

25

0

0

0

4

8

12

16

20

24

24

Time from Injection (hours)

Time from Injection (hours)

0

4

8

12

16

20

Figure 3: Time-Action Profiles of HI Analogs Acylated With Arg

at Three Amino Functionalities

Compound 39 displayed a favorable time-action profile similar to that of 4, but with somewhat lower potency. However,

this analog retains the wild-type Asn at position A21, which is unstable under acidic conditions due to aspart-anhydride-

intermediated degradation (4). Substitution of Gly at A21, resulting in 67, surprisingly led to a shorter time action,

unlike what occurred with the same substitution in 43 (Fig 2). Decreasing [Zn2+ ] in the formulation shortened the time-

action further. In this case the results do not correlate with the expected behavior based on the PBS solubilities (Table 1).

Substitution of Asp or Ser in 39 resulting in 91, and 72, respectively again resulted in less sustained glucodynamic effect,

although better tha that for 67. The time-action seems somewhat more prolonged for 91 than 72. The time-action profiles

roughly correlate with the PBS solubilities (Table 1) (i.e. higher PBS solubility for 72 and 91 than 39 result in a less

prolonged glucodynamic effect.


Figure 4: Pharmacokinetic Profiles of Arg-Derivatized HI on

Analogs Following SC Administration to Beagle Dogs

A0:R,A21:G,B0:R,B29:K(R)-HI (67) (30 ug/ml Zn; n=6)

200

A0:R,A21:G,B0:R,B29:K(R)-HI (67) (5 ug/ml Zn; n=6)

A0:R,A21:S,B0:R,B29:K(R)-HI (72) (n=6)

A0:R, B0:R, B29:K(R)-HI (39) (n=6)

150

B31:R,B32:R,A21:G-HI (4) (n=12)

Immunoreactivity (pM)

100

50

0

0

4

8

12

16

20

24

Time (hr)

These PK profiles correlate well with the plasma glucose effects observed for these analogs in Fig. 3. In particular, the

PK profile for 39 is quite prolonged showing greater levels past 16 hr than 4. The poor PD performance of 67 at two

different Zn2+ concentrations (Fig. 3) correlates well with the PK profile above. The AUC for this analog is very low

(749 pM*hr vs 1442 and 1603 for 39 and 4, respectively)


Figure 5: Time-Action Profiles of HI Analogs Containing on

Arg at N terminus of the A chain and B29:Lys

Saline (n=5)

B31:R,B32:R,A21:G-HI (4) (n=6)

A0:R,A21:G,B29:K(R)-HI (79) (30 ug/ml Zn; n=6)

A0:R,A21:G,B29:K(R)-HI (79) (80 ug/ml Zn; n=6)

A0:K(R),A21:G,B29:K(R)-HI (82) (n=6)

Glucose Response to Soluble Insulin

Formulations

(somatostatin-treated dogs; 2 nmol/kg, sc)

250

200

150

Plasma Glucose (mg/dL)

100

50

0

0

4

8

12

16

20

24

Time from Injection (hours)

Removal of the Arg at B0 from 67, resulted in 79, which displayed a time-action almost as prolonged as 4. Increasing

Zn2+ concentration in the formulation to 80 ug/mL increased the time-action to almost the same as 4. Importantly, a peak

of activity observed in the 30ug/mL Zn2+ formulation from 0-2 hr was also blunted in the high Zn formulation.Analog 82,

which contains an N terminal Lys acylated with Arg (addition of three basic groups relative to HI), displayed a time-action

profile in the standard formulation very similar to 4.


Table 1: on In Vitro Results Summary for Insulin Analogs Containing Two or Three Arg Additions to N

termini of A or B chain and C terminus of B chain

Saline (n=7)

A21:G,B31:R,B32:R-HI (4) (n=6)

A(-1):R,A0:R,A21:G-HI (86) (n=6)

A(-1):R,A0:R,A21:G,B31:R-HI (106) (n=6)

175

250

A0:R,A21:G,B31:R,B32:R-HI (98) (n=6)

A0:R,A21:G,B31:R HI (83) (n=4)

150

200

125

150

100

Plasma Glucose (mg/dL)

Plasma Insulin (pM)

75

100

50

50

25

0

0

0

4

8

12

16

20

24

0

4

8

12

16

20

24

Time from Injection (hours)

Time from Injection (hours)

Figure 6: Time-Action Profiles of HI Analogs with Arg at N

terminus of the A chain and C terminus of the B chain

The combinatorial placement of two or three Arg residues at the A chain N terminus and B chain C terminus resulted

in dramatically differing PK/PD profiles. Unlike 79 (Fig. 5), the two diArg analogs 83 and 86 displayed short time-action

punctuated by a distinct peak in the PK profile of 86 between 0-4 hr. Addition of a third Arg, resulting in either 98 or 106

resulted in two disparate analogs. For 106, the bioavailability is very low (about 1/2 that of 4) and the resulting

plasma glucose effect is small and diminsihes quickly. Analog 98, displays a remarkably flat PK profile and a prolonged

PD response, suggesting this analog could perform exceptionally well as a basal insulin. Insulin AUC of 98 was found to

be about 150% that of 4 in the above experiment.


Figure 7: 10 hr Euglycemic Clamp Results of on

Analogs 4 and 82 in Somatostatin-Treated Dogs

500

15.0

4, (6 nmol/kg)

12.5

400

4, (6 nmol/kg)

10.0

82, (6 nmol/kg)

300

Insulin (pM)

(mg/kg/min)

Glucose Infusion Rate

82, (6 nmol/kg)

4, (3 nmol/kg)

7.5

200

4, (3 nmol/kg)

5.0

82, (3 nmol/kg)

82, (3 nmol/kg)

100

2.5

0.0

0

0.0

2.5

5.0

7.5

10.0

12.5

15.0

0.0

2.5

5.0

7.5

10.0

12.5

15.0

Time from Injection (hr)

Time from Injection (hr)

8

5

82%

109%

4

hr/L; 0-10

6

76%

47%

Glucose Infused

hours)

(g/kg; 0-10

3

Insulin AUC

hours)

4

·

2

(nmol

2

1

0

0

4, 3

4, 3

82, 3

82, 6

82, 3

82, 6

4, 6

4, 6

Analog 82 had a more rapid onset of action than 4 (square wave time-action profile), and this was more

pronounced at the higher dose. The glucose infused over 10 hr due to 82 was 47% and 82% that due to 4 at 3

and 6 nmol/kg doses, respectively. Taking the total insulin detected over the 10 hr in to account the biopotency

of 82 relative to that of 4 is approximately 59% and 75% at the low and high doses, respectively.


Figure 8: 10 hr Euglycemic Clamp Results of on

Analogs 4 and 98 in Somatostatin-Treated Dogs

15.0

500

12.5

400

10.0

300

Glucose Infusion Rate

(mg/kg/min)

Insulin (pM)

4, (3 nmol/kg)

7.5

98, (3 nmol/kg)

200

5.0

98, (3 nmol/kg)

100

4, (3 nmol/kg)

2.5

0

0.0

0.0

2.5

5.0

7.5

10.0

12.5

15.0

0.0

2.5

5.0

7.5

10.0

12.5

15.0

Time from Injection (hr)

Time from Injection (hr)

8

5

4

6

81%

hr/L; 0-10 hr)

169%

3

(g/kg; 0-10 hr)

Glucose Infused

4

Insulin AUC

2

·

2

1

(nmol

0

0

4

4

98

98

Analog 98 and 4 have similar glucose infusion rate profiles over 10 hr. The total glucose infused over 10 hr due

to 98 was 81% that due to 4 . Taking the total insulin detected over the 10 hr in to account the biopotency

of 98 relative to that of 4 is approximately 48 %.


In Vitro on Data Correlation Analyses

r2 = 0.969

r2 = 0.725

There is a much stronger correlation between relative

IGF-1R affinity and mitogenic potency than there is

between relative IR affinity and metabolic potency.

There is very poor correlation between isoelectric point

and PBS solubility in the standard formulation conditions

containing 30 ug/mL Zn2+ (correlations were performed

with a least-squares linear regression on Sigmaplot)

r2 = 0.466


CONCLUSIONS on

  • two insulin analogs (LY2109967, 82; and LY2116419, 98)were discovered which possessed 15- and 5-

  • fold,respectively, greater IR / IGF-1R selectivity than insulin glargine and the requisite PK/PD profile to

  • support effective once daily dosing.

  • an in vitro solubility assay was used to screen peptides for the possibility of increased time action and

  • found to have some predictive power but many false positives were also identified with the assay.

  • substitutions at position A21 had profound effects on the potency and time-action of the analogs.

  • pI did not correlate well with PBS solubility or, more importantly, in vivo time action.

REFERENCES

1. Campbell, R.K. et al. (2001) Clinical Therapeutics23: 1938-1957.

2. Nakagawa, S.H. and Tager, H.S. (1991) J. Biol. Chem.266: 11502-11509.

3. Myers, S.R. et al. (1991) Metabolism, Clinical and Experimental40: 66-71.

4. Darrington, R.T and Anderson, B.D (1994) Pharm Res11: 784-793.


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