Transcript Lilly Arg - Insulins

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
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 Cterminus 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 Bchain. 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 Bchain 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 rpHPLC 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 analog in conditions that mimic the commercial formulation of
insulin glargine: ~3.64 mg/ml of protein, 30 mg/mL (unless specified otherwise) of Zn 2+ (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
INSULIN ANALOG STRUCTURES
K
R G
S
I
A1 V
A chain
A20
S
E Q
G
C C
C
T S I C
Y
A5
R
N
S L Y Q L E
T
K
F
A10
S
S
V
A15
P R
B1 N Q
S
T
H
S
F Y
L C
F
B5
G S H
R G B25
L V E A L Y L V C G E
B10
B20
analog
B15
B chain
82: A0:K(R),B29:K(R),A21:G-HI
(LY2109967)
R
G
S
I
A1 V
A chain
A20
S
E Q
R
G
C C
C
R
T
S I C S L Y Q L E N Y
A5
R
T
K
F
A10
S
S
V
A15
P
B1 N Q
S
T
H
S
F Y
L C
F
B5
G S H
R G B25
L V E A L Y L V C G E
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)
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)
B31:R,B32:R,A21:G-HI (4) (n=4)
B31:R,B32:R-HI (43) (n=4)
175
150
80
125
100
75
50
analog 4
analog 67
analog 79
analog 39
analog 43
70
PBS Solubility (%)
Plasma Glucose (mg/dL)
200
60
50
40
30
20
10
25
0
0
0
20
40
2+
60
80
[Zn ] (g/mL)
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 Zn 2+ but the
similarly low solubility at 80 ug/mL Zn2+.
Figure 3: Time-Action Profiles of HI Analogs Acylated With Arg
at Three Amino Functionalities
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)
A0:R, B0:R, B29:K(R)-HI (39) (n=6)
200
175
150
125
100
75
50
Saline (n=5)
B31:R,B32:R,A21:G-HI (4) (n=6)
25
0
0
4
8
12
16
20
Time from Injection (hours)
Plasma Glucose (mg/dL)
Plasma Glucose (mg/dL)
Glucose Response to Soluble Insulin Formulations (somatostatin-treated dogs; 2 nmol/kg, sc)
200
175
150
125
100
75
50
A0:R,B0:R,B29:K(R), A21:D-HI (91) (n=6)
A0:R,B0:R,B29:K(R), A21:S-HI (72) (n=6)
25
24
0
0
4
8
12
16
20
Time from Injection (hours)
24
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-anhydrideintermediated 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 timeaction 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
Analogs Following SC Administration to Beagle Dogs
A0:R,A21:G,B0:R,B29:K(R)-HI (67) (30 ug/ml Zn; n=6)
A0:R,A21:G,B0:R,B29:K(R)-HI (67) (5 ug/ml Zn; n=6)
Immunoreactivity (pM)
200
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)
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
Arg at N terminus of the A chain and B29:Lys
Plasma Glucose (mg/dL)
Glucose Response to Soluble Insulin 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,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)
250
200
150
100
50
0
0
4
8
12
16
20
Time from Injection (hours)
24
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.
Figure 6: Time-Action Profiles of HI Analogs with Arg at N
terminus of the A chain and C terminus of the B chain
250
150
Plasma Insulin (pM)
Plasma Glucose (mg/dL)
175
125
100
75
50
200
150
100
50
25
0
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)
A0:R,A21:G,B31:R,B32:R-HI (98) (n=6)
A0:R,A21:G,B31:R HI (83) (n=4)
0
4
8
12
16
20
Time from Injection (hours)
24
0
0
4
8
12
16
20
24
Time from Injection (hours)
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
Analogs 4 and 82 in Somatostatin-Treated Dogs
500
4, (6 nmol/kg)
10.0
82, (6 nmol/kg)
7.5
4, (3 nmol/kg)
5.0
400
Insulin (pM)
12.5
4, (6 nmol/kg)
300
82, (6 nmol/kg)
200
4, (3 nmol/kg)
82, (3 nmol/kg)
82, (3 nmol/kg)
100
0.0
0.0
2.5
5.0
7.5
10.0
Time from Injection (hr)
8
6
4
2
0
12.5
82%
47%
15.0
0
0.0
Insulin AUC
(nmol hr/L; 0-10
hours)
2.5
Glucose Infused
(g/kg; 0-10
hours)
Glucose Infusion Rate
(mg/kg/min)
15.0
2.5
5.0
7.5
10.0
12.5
Time from Injection (hr)
5
4
3
15.0
109%
76%
2
1
0
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
Analogs 4 and 98 in Somatostatin-Treated Dogs
500
12.5
10.0
Insulin (pM)
400
300
4, (3 nmol/kg)
7.5
5.0
98, (3 nmol/kg)
100
4, (3 nmol/kg)
98, (3 nmol/kg)
2.5
0.0
Glucose Infused
(g/kg; 0-10 hr)
0.0
200
2.5
5.0
7.5
10.0
12.5
Time from Injection (hr)
8
6
81%
4
2
0
98
4
15.0
0
Insulin AUC
(nmol hr/L; 0-10 hr)
Glucose Infusion Rate
(mg/kg/min)
15.0
0.0
2.5
5.0
7.5
10.0
12.5
Time from Injection (hr)
15.0
5
4
169%
3
2
1
0
98
4
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 Data Correlation Analyses
Relative Metabolic Potency vs. Relative IR Affinity
Relative Mitogenic Potency vs. Relative IGF-1R Affinity
5
Relative Mitogenic Potency
Relative Metabolic Potency
1.2
1.0
0.8
0.6
0.4
r2 = 0.725
0.2
0.0
4
3
2
r2 = 0.969
1
0
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Relative IR Affinity
0
1
2
3
4
5
6
Relative IGF-1R Affinity
Isoelectric Point vs. PBS Solubility
7.4
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)
Isoelectric Point (pI)
7.2
7.0
6.8
6.6
6.4
6.2
r2 = 0.466
6.0
5.8
5.6
5.4
0
20
40
60
PBS Solubility (%)
80
100
CONCLUSIONS
• two insulin analogs (LY2109967, 82; and LY2116419, 98) were discovered which possessed 15- and 5fold,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 Therapeutics 23: 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 Experimental 40: 66-71.
4. Darrington, R.T and Anderson, B.D (1994) Pharm Res 11: 784-793.