U.S. patent application number 13/310118 was filed with the patent office on 2012-05-17 for long-acting formulations of insulins.
This patent application is currently assigned to SANOFI. Invention is credited to Reinhard Becker, Peter Boderke, Annke Frick, Christiane Furst, Petra Loos, Werner Muller, Isabell Schottle, Katrin Tertsch, Ulrich Werner.
Application Number | 20120122774 13/310118 |
Document ID | / |
Family ID | 44148980 |
Filed Date | 2012-05-17 |
United States Patent
Application |
20120122774 |
Kind Code |
A1 |
Becker; Reinhard ; et
al. |
May 17, 2012 |
LONG-ACTING FORMULATIONS OF INSULINS
Abstract
The application relates to an aqueous pharmaceutical formulation
comprising 200-1000 U/mL [equimolar to 200-1000 IU human insulin]
of insulin glargine.
Inventors: |
Becker; Reinhard; (Frankfurt
am Main, DE) ; Frick; Annke; (Frankfurt am Main,
DE) ; Boderke; Peter; (Schwalbach, DE) ;
Furst; Christiane; (Frankfurt Am Main, DE) ; Muller;
Werner; (Frankfurt Am Main, DE) ; Tertsch;
Katrin; (Frankfurt Am Main, DE) ; Werner; Ulrich;
(Frankfurt Am Main, DE) ; Loos; Petra; (Frankfurt
Am Main, DE) ; Schottle; Isabell; (Frankfurt Am Main,
DE) |
Assignee: |
SANOFI
Paris
FR
|
Family ID: |
44148980 |
Appl. No.: |
13/310118 |
Filed: |
December 2, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13110568 |
May 18, 2011 |
|
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13310118 |
|
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61411608 |
Nov 9, 2010 |
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61429936 |
Jan 5, 2011 |
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Current U.S.
Class: |
514/6.2 |
Current CPC
Class: |
A61P 5/48 20180101; A61P
3/08 20180101; A61K 38/28 20130101; A61K 47/02 20130101; A61K 47/10
20130101; A61K 38/26 20130101; A61P 5/50 20180101; A61K 38/2278
20130101; A61P 3/10 20180101; A61K 47/26 20130101; A61K 9/0019
20130101; A61K 9/08 20130101 |
Class at
Publication: |
514/6.2 |
International
Class: |
A61K 38/28 20060101
A61K038/28; A61P 3/10 20060101 A61P003/10 |
Foreign Application Data
Date |
Code |
Application Number |
May 19, 2010 |
EP |
10305532.3 |
Jul 13, 2010 |
EP |
10305780.8 |
Feb 10, 2011 |
EP |
11305140.3 |
Claims
1. An aqueous pharmaceutical formulation comprising 300 U/mL of
insulin glargine [equimolar to 300 IU human insulin].
2. The aqueous pharmaceutical formulation of claim 1 comprising an
analogue of exendin-4.
3. The aqueous pharmaceutical formulation of claim 2, wherein the
analogue of exedin-4 is selected from a group comprising
lixisentatide, exenatide and liraglutide.
4. The aqueous pharmaceutical formulation of claim 3 comprising 0.1
.mu.g to 10 .mu.g lixisenatide per U insulin glargine.
5. The aqueous pharmaceutical formulation of claim 4 comprising 0.2
to 1 .mu.g lixisenatide per U insulin glargine.
6. The aqueous pharmaceutical formulation of claim 5 comprising
0.25 .mu.g to 0.7 .mu.g lixisenatide per U insulin glargine.
7. The aqueous pharmaceutical formulation of claim 1 comprising at
least one excipient selected from the group comprising zinc,
m-cresol, glycerol, polysorbate 20 and sodium.
8. The aqueous pharmaceutical formulation of claim 7 comprising 90
.mu.g/mL zinc, 2.7 mg/mL m-cresol and 20 mg/ml glycerol 85%.
9. The aqueous pharmaceutical formulation of claim 7 comprising 90
.mu.g/mL zinc, 2.7 mg/mL m-cresol, 20 .mu.g/mL polysorbate 20 and
20 mg/mL glycerol 85%.
10. The aqueous pharmaceutical formulation of claim 1, wherein the
pH is between 3.4 and 4.6.
11. The aqueous pharmaceutical formulation of claim 10, wherein the
pH is 4.
12. The aqueous pharmaceutical formulation of claim 10, wherein the
pH is 4.5.
13. A method of treating Type I and Type II Diabetes Mellitus in a
patient comprising administering to said patient an aqueous
pharmaceutical formulation comprising 300 U/mL of insulin glargine
[equimolar to 300 IU human insulin].
14. The method of claim 13 wherein said aqueous pharmaceutical
formulation further comprises an analogue of exendin-4.
15. The method of claim 14 wherein the analogue of exendin-4 is
selected from a group comprising lixisentatide, exenatide and
liraglutide.
16. The method of claim 15 comprising 0.1 .mu.g to 10 .mu.g
lixisenatide per U insulin glargine.
17. The method of claim 16 comprising 0.2 to 1 .mu.g lixisenatide
per U insulin glargine.
18. The method of claim 17 comprising 0.25 .mu.g to 0.7 .mu.g
lixisenatide per U insulin glargine.
19. The method of claim 13 further comprising at least one
excipient selected from the group comprising zinc, m-cresol,
glycerol, polysorbate 20 and sodium.
20. The method of claim 19 comprising 90 .mu.g/mL zinc, 2.7 mg/mL
m-cresol and 20 mg/mL glycerol 85%.
21. The method of claim 19 comprising 90 .mu.g/mL zinc, 2.7 mg/mL
m-cresol, 20 .mu.g/mL polysorbate 20 and 20 mg/mL glycerol 85%.
22. The method of claim 13 wherein the pH is between 3.4 and
4.6.
23. The method of claim 22 wherein the pH is 4.
24. The method of claim 22 wherein the pH is 4.5.
25. An aqueous pharmaceutical formulation comprising 300 U/mL of
insulin glargine [equimolar to 300 IU human insulin], 90 .mu.g
zinc, 2.7 mg m-cresol, 20 mg glycerol 85%, HCL and NaOH ad and
having a pH of 4.0.
Description
[0001] This application is a continuation application of U.S. Ser.
No. 13/110,568, filed May 18, 2011, which claims benefit to U.S.
Provisional Appln. Nos. 61/411,608, filed Nov. 9, 2010 and
61/429,936, filed Jan. 5, 2011.
[0002] The application relates to an aqueous pharmaceutical
formulation comprising 200-1000 U/mL [equimolar to 200-1000 IU
human insulin] of insulin glargine, and its use.
[0003] Insulin glargine is 31.sup.B-32.sup.B-Di-Arg human insulin,
an analogue of human insulin, with further substitution of
asparagine in position A21 by glycine.
[0004] Lantus.RTM. is an insulin product containing insulin
glargine providing 24 hour basal insulin supply after single dose
subcutaneous injection.
[0005] The glucodynamic effect of Lantus.RTM. is distinguished from
other currently marketed insulin products by virtue of a delayed
and predictable absorption of insulin glargine from the
subcutaneous injection site resulting in a smooth, 24 hour
time-concentration and action profile without a definite peak.
Lantus.RTM. was developed to meet the medical need for a
long-acting insulin product that can be administered as a single
daily injection to yield normal or near-normal blood glucose
control with a basal insulin profile that is as smooth as possible
over a 24-hour period. Such a preparation provides good control of
blood glucose all day, while minimizing the tendency to produce
hypoglycemia seen with other insulin preparations with a more
definite "peak" effect.
[0006] A considerable number of patients, in particular those with
increased insulin resistance due to obesity, use large doses to
control blood glucose. For example, a dose of 100 U requires
injection of 1 mL Lantus.RTM. U100, which may confer some
discomfort; each mL Lantus.RTM. U100 contains 100 U (3.6378 mg)
insulin glargine. To reduce the volume of injection, a formulation
containing 300 U insulin glargine per mL has been developed.
Although the invention is not limited to an insulin glargine U 300
formulation, the clinical studies described herein were performed
with an insulin glargine U 300 formulation; each mL insulin
glargine U300 contains 300 U (10.9134 mg) insulin glargine. This
formulation would allow patients to inject the same number of units
of insulin glargine at one third the volume of injection.
[0007] Both insulin glargine formulations, U100 and U300, were
expected to provide the same insulin exposure and the same
effectiveness, i.e. time profiles.
Figures
[0008] The figures below effectively show the surprising and
unexpected differences in exposure (PK) and activity (PD) between
Lantus U100 und Lantus U300 formulations (insulin glargine U100 und
insulin glargine U300 formulations) after the same s.c. dose given
to healthy subjects, at the same time as blood glucose (PD) was
constant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1: Glucose Infusion Rate (GIR) Lantus U100.
[0010] FIG. 2: Glucose Infusion Rate (GIR) Lantus U300.
[0011] FIGS. 3A and 3B: Serum Insulin Concentrations; Lantus U100
(3A) and U300 (3B).
[0012] FIGS. 4A and 4B: Blood Glucose U100; (4A) replicates=1, (4B)
replicates=2.
[0013] FIGS. 5A and 5B: Blood Glucose U300; (5A) replicates=1, (5B)
replicates=2.
[0014] FIGS. 6A, 6B and 6C: Results of a randomized, 4-sequence,
cross-over, double-blind, dose response study of 0.4, 0.6 and 0.9
U/kg HOE-901-U300 (insulin glargine U300) compared to 0.4 U/kg
Lantus.RTM. U100 (insulin glargine U100) in patients with diabetes
mellitus type 1 using the euglycemic clamp technique. FIG. 6A:
insulin glargine concentration (mU/L), FIG. 6B: blood glucose (BG,
mg/dL), FIG. 6C: glucose infusion rate (GIR, mgkg.sup.-1min.sup.-1)
The curves display LOWESS smoothed averages of all data points of
all subjects (population averages); LOWESS is a data analysis
technique for producing a "smooth" set of values from a time series
which has been contaminated with noise, or from a scatter plot with
a "noisy" relationship between the 2 variables.
[0015] FIG. 7: Glucose infusion rate (GIR, mgkg.sup.-1min.sup.-1)
The curves display LOWESS smoothed averages of all data points of
all subjects (population averages); LOWESS is a data analysis
technique for producing a "smooth" set of values from a time series
which has been contaminated with noise, or from a scatter plot with
a "noisy" relationship between the 2 variables.
[0016] FIG. 7: Legend: Profiles 1 to 3 (from top to bottom).
[0017] Results of a randomized, double-blind, parallel group dose
response study of 0.4, 0.6 and 1.2 U/kg Lantus.RTM. U100 (insulin
glargine U100) in patients with diabetes mellitus type 1 using the
euglycemic clamp technique.
[0018] FIG. 7: Legend: Profiles 4 to 7 (from top to bottom).
[0019] Results of a randomized, 4-sequence, cross-over,
double-blind, dose response study of 0.4, 0.6 and 0.9 U/kg
HOE-901-U300 (insulin glargine U300) compared to 0.4 U/kg
Lantus.RTM. U100 (insulin glargine U100) in patients with diabetes
mellitus type 1 using the euglycemic clamp technique.
[0020] FIGS. 8A, 8B, 8C, 8D and 8E: Optical microscope pictures of
precipitates of insulin glargine formulations with increasing
concentrations:
[0021] FIG. 8A: 100 U/mL, FIG. 8B: 300 U/mL, FIG. 8C: 500 U/mL,
FIG. 8D: 700 U/mL and FIG. 8E: 1000 U/mL,
with the magnitude of 100.times. and including the maximum
diameters.
[0022] All precipitations are performed with 60U of insulin
glargine.
[0023] FIG. 9: Time-action profile of insulin glargine U-100 vs.
U-300 in normoglycemic dogs.
DETAILED DESCRIPTION
[0024] Exposure and activity of insulin glargine U300, the test (T)
medication, was tested in non-diabetic healthy subjects in
euglycemic clamps for equivalence in exposure and activity to
Lantus U100, the approved reference (R) product. To account for the
long duration of action of insulin glargine after subcutaneous
administration 30 hours were selected. Exposure was assessed from
insulin glargine concentration time profiles after subcutaneous
administration while activity was simultaneously assessed as
glucose utilization per unit insulin.
[0025] A replicate design allowed limiting the number of subjects
for assessing bioequivalence and variability as recommended by the
FDA guideline "Guidance for Industry, Statistical Approaches to
Establishing Bioequivalence".
[0026] The respective clinical study was expected to establish
equivalence in exposure and activity.
[0027] A dose of 0.4 U/kg was selected for this study; it
corresponds to the average basal insulin dose in patients. In
non-diabetic healthy subjects this dose produces a sizeable
elevation in plasma insulin concentration and a lasting glucose
lowering effect that can be quantified in euglycemic clamp
settings.
[0028] The replicate design favored by guidelines requires two
replicate single dose injections of either IP (R: Lantus.RTM. U100,
T: insulin glargine U300) in predefined four way cross-over
sequences (RTTR or TRRT) as allotted by the randomization plan.
This was executed in Periods (P) 1-4 at four different days. As a
result, each subject received two replicate single subcutaneous
doses of 0.4 U/kg Lantus.RTM. U100 (R) and insulin glargine U300
(T), alternating between two opposite sites of the periumbilical
area.
[0029] A washout period of 4 to 18 days separated each dosing day.
The length of the wash-out period varied individually allowing both
the participant and the Investigator to adjust to their needs. By
experience, 4 days comprise a minimum period for recovery, enabling
1 clamp per week for a participant, while 18 days represent a break
of 3 weeks between clamp days, allowing subjects more freedom to
fulfill non-study related obligations.
[0030] Prior to the euglycemic clamp visits, at SCR (screening
visit), subjects have been screened for eligibility, and in EOS
(end-of-study) visit subjects have come in for a final examination
to ensure normal health status. Screening and P1 have not be
separated by more than 21 days, while the EOS visits occurred no
earlier than the same week day as Day 1 of P4 the following week,
i.e. after an additional 4 days, and no later than a fortnight
after Day 2 of P4, i.e. after an additional 14 days.
[0031] This has been a single dose study with in total 4 replicate
administrations. The effect of the IPs was to last about 24 hours,
which is why the subjects have been confined to the institute for 2
days. Subjects have been exposed to treatment 4 times.
[0032] The primary objective of the study was to assess the average
bioequivalence (ABE) of Lantus.RTM. U100 (commercial formulation)
and insulin glargine U300 in bioavailability (exposure) and
bioefficacy (activity) using the euglycemic clamp technique.
[0033] The secondary objective of the study was to assess safety
and tolerability of insulin glargine U300.
[0034] As mentioned above, both insulin glargine formulations, U100
and U300, were expected to provide the same insulin exposure and
the same effectiveness. However, surprisingly insulin exposure and
effectiveness were shown to be not the same. Insulin glargine U 100
and insulin glargine U 300 are not equivalent in bio-availability
(exposure) and bio-efficacy (activity). Exposure and activity after
administration of insulin glargine U300 were less by about 40% as
compared to exposure and activity after administration of the same
amount (0.4 U/kg) from insulin glargine U100.
[0035] Insulin glargine U300 did, however, show an even flatter PK
(exposure) and PD (activity) profile than insulin glargine U100, as
would be desired for a basal insulin. These surprising and
unexpected differences in exposure and activity between insulin
glargine U100 and insulin glargine U300 formulations after the same
s.c. dose to healthy subjects are effectively shown in the figures
below. Of note, at the same time blood glucose was constant.
[0036] The blood glucose lowering effect of insulin glargine was
additionally evaluated in healthy, normoglycemic Beagle dogs. With
increasing insulin glargine concentration the mean time of action
increased from 6.8 h (U100) to 7.69 h (U300), respectively. By
increasing the glargine concentration from 100 to 300 U/mL the
blood glucose decreasing time-action profile was changed towards a
flatter and prolonged activity in the dog. The current data in dogs
is consistent with data in humans showing that higher drug
concentrations of insulin glargine are positively correlated with
profile and longer duration of action.
[0037] Additionally, the precipitates of insulin glargine
formulations having concentrations of 100 U/mL, 300 U/mL, 500 U/mL
700 U/mL and 1000 U/mL have been investigated by microscopy. These
investigations revealed differences in the precipitations
characteristics, leading to remarkable bigger particles with
increasing concentrations.
[0038] Furthermore, the influence of the higher concentrations of
insulin glargine formulations with regard to dissolution properties
are investigated by using an in-vitro test system. To do so,
precipitation studies were performed using a phosphate buffer with
a pH of 7.4, simulating the in-vivo conditions.
[0039] The supernatant of the precipitated insulin was investigated
using HPLC technique to determine the insulin glargine content.
[0040] The present invention is not limited to an insulin glargine
U 300 formulation and is effective with other higher concentrated
formulations of insulin glargine as outlined in detail in the
specification, the clinical studies described herein were performed
with an insulin glargine U 300 formulation.
[0041] Specifically, the insulin glargine formulations of the
present invention exhibit a flatter PK (exposure) and flatter PD
(activity) profile than insulin glargine U100 and surprisingly act
as improved basal insulins compared to U100 glargine insulin and
therefore impart extended duration of exposure and reduce the
incidence of hypoglycemia in the treatment of Type I and Type II
diabetes, for example.
[0042] 1 mL of insulin glargine U 300 formulation contains 10.913
mg 21.sup.A-Gly-30.sup.B.alpha.-L-Arg-30.sup.Bb-L-Arg human insulin
[equimolar to 300 IU human insulin], 90 .mu.g zinc, 2.7 mg
m-cresol, 20 mg glycerol 85%, HCl and NaOH ad pH 4.0; specific
gravity 1.006 g/mL
[0043] However, variations with regard to the kind of excipients
and their concentrations are possible.
[0044] The pharmaceutical formulation of the present invention
contains 200-1000 U/mL of insulin glargine [equimolar to 200-1000
IU human insulin], preferably 250-500 U/mL of insulin glargine
[equimolar to 250-500 IU human insulin], more preferred 270-330
U/mL of insulin glargine [equimolar to 270-330 IU human insulin],
and even more preferred 300 U/mL of insulin glargine [equimolar to
300 IU human insulin].
[0045] In one embodiment, the present invention is directed to an
aqueous pharmaceutical formulation comprising insulin glargine in
the range of 200-1000 U/mL [equimolar to 200-1000 IU human
insulin], preferably 200 U/ml to 650 U/mL, still preferably 700
U/mL to 1000 U/ml, more preferably 270-330 U/mL and most preferably
in a concentration of 300 U/mL.
[0046] In one embodiment, the present invention is directed to an
aqueous formulation comprising 200-1000 U/mL [equimolar to 200 to
1000/U human insulin] of insulin glargine, with the proviso that
the concentration of insulin in said formulation is not 684 U/ml of
insulin glargine.
[0047] In another embodiment, the pharmaceutical formulation of the
present invention contains 200 U/mL of insulin glargine (equimolar
to 200 IU human insulin] or 300 U/mL of insulin glargine [equimolar
to 300 IU human insulin] or 400 U/mL of insulin glargine [equimolar
to 400 IU human insulin] or 500 U/mL of insulin glargine [equimolar
to 500 IU human insulin] or 600 U/mL of insulin glargine [equimolar
to 600 IU human insulin] or 700 U/mL of insulin glargine [equimolar
to 700 IU human insulin] or 800 U/mL of insulin glargine [equimolar
to 800 IU human insulin] or 900 U/mL of insulin glargine [equimolar
to 900 IU human insulin] or 1000 U/mL of insulin glargine
[equimolar to 1000 IU human insulin].
[0048] Surfactants can be added to pharmaceutical formulation, for
example, inter alia, non-ionic surfactants. In particular,
pharmaceutically customary surfactants are preferred, such as, for
example:
partial and fatty acid esters and ethers of polyhydric alcohols
such as of glycerol, sorbitol and the like (Span.RTM., Tween.RTM.,
in particular Tween.RTM. 20 and Tween.RTM. 80, Myrj.RTM.,
Brij.RTM.), Cremophor.RTM. or poloxamers. The surfactants are
present in the pharmaceutical composition in a concentration of
5-200 .mu.g/ml, preferably of 5-120 .mu.g/ml and particularly
preferably of 20-75 .mu.g/ml.
[0049] The formulation of the present invention can additionally
contain preservatives (e.g. phenol, m-cresol, p-cresol, parabens),
isotonic agents (e.g. mannitol, sorbitol, lactose, dextrose,
trehalose, sodium chloride, glycerol), buffer substances, salts,
acids and alkalis and also further excipients. These substances can
in each case be present individually or alternatively as
mixtures.
[0050] Glycerol, dextrose, lactose, sorbitol and mannitol can be
present in the pharmaceutical preparation in a concentration of
100-250 mM, NaCl in a concentration of up to 150 mM. Buffer
substances, such as, for example, phosphate, acetate, citrate,
arginine, glycylglycine or TRIS (i.e.
2-amino-2-hydroxymethyl-1,3-propanediol) buffer and corresponding
salts, are present in a concentration of 5-250 mM, preferably
10-100 mM. Further excipients can be, inter alia, salts or
arginine.
[0051] The zinc concentration of the formulation of the present
invention is in the range of the concentration which is reached by
the presence of 0-1000 .mu.g/mL, preferably 20-400 .mu.g/mL zinc,
most preferably 90 .mu.g/mL. However, the zinc may be present in
form of zinc chloride, but the salt is not limited to be zinc
chloride.
[0052] In the pharmaceutical formulation glycerol and/or mannitol
can be present in a concentration of 100-250 mmol/L, and/or NaCl is
preferably present in a concentration of up to 150 mmol/L.
[0053] In the pharmaceutical formulation a buffer substance can be
present in a concentration of 5-250 mmol/L.
[0054] A further subject of the invention is a pharmaceutical
insulin formulation which contains further additives such as, for
example, salts which delay the release of insulin. Mixtures of such
delayed-release insulins with formulations described above are
included therein.
[0055] For producing the formulations of the present invention the
ingredients are dissolved in water and the pH is adjusted by using
HCl and/or NaOH; and likewise by methods known in the art.
Likewise, a further subject of the invention is directed to the use
of such formulations for the treatment of diabetes mellitus.
[0056] A further subject of the invention is directed to the use or
the addition of surfactants as stabilizers during the process for
the production of insulin, insulin analogs or insulin derivatives
or their preparations.
[0057] The invention further relates to a formulation as described
above which additionally comprises also a glucagon-like peptide-1
(GLP1) or an analogue or derivative thereof, or exendin-3 or -4 or
an analogue or derivative thereof, preferably exendin-4.
[0058] The invention further relates to a formulation as described
above in which an
analogue of exendin-4 is selected from a group comprising
H-desPro.sup.36-exendin-4-Lys.sub.6-NH.sub.2,
H-des(Pro.sup.36,37)-exendin-4-Lys.sub.4-NH.sub.2 and
H-des(Pro.sup.36,37)-exendin-4-Lys.sub.5-NH.sub.2, or a
pharmacologically tolerable salt thereof.
[0059] The invention further relates to a formulation as described
above in which an
analogue of exendin-4 is selected from a group comprising
desPro.sup.36 [Asp.sup.28]exendin-4 (1-39), desPro.sup.36
[IsoAsp.sup.28]exendin-4 (1-39), desPro.sup.36 [Met(O).sup.14,
Asp.sup.28]exendin-4 (1-39), desPro.sup.36 [Met(O).sup.14,
IsoAsp.sup.28]exendin-4 (1-39), desPro.sup.36[Trp(O.sub.2).sup.25,
Asp.sup.28]exendin-2 (1-39), desPro.sup.36[Trp(O.sub.2).sup.25,
IsoAsp.sup.28]exendin-2 (1-39), desPro.sup.36
[Met(O).sup.14Trp(O.sub.2).sup.25, Asp.sup.28]exendin-4 (1-39) and
desPro.sup.36 [Met(O).sup.14Trp(O.sub.2).sup.25,
IsoAsp.sup.28]exendin-4 (1-39), or a pharmacologically tolerable
salt thereof.
[0060] The invention further relates to a formulation as described
in the preceding paragraph, in which the peptide-Lys.sub.6-NH.sub.2
is attached to the C termini of the analogues of exendin-4.
[0061] The invention further relates to a formulation as described
above in which an
analogue of exendin-4 is selected from a group comprising
H-(Lys).sub.6-des Pro.sup.36 [Asp.sup.28]exendin-4
(1-39)-Lys.sub.6-NH.sub.2 des Asp.sup.28Pro.sup.36, Pro.sup.37,
Pro.sub.38 exendin-4 (1-39)-NH.sub.2, H-(Lys).sub.6-des Pro.sup.36,
Pro.sup.37, Pro.sup.38 [Asp.sup.28]exendin-4 (1-39)-NH.sub.2,
H-Asn-(Glu).sub.5 des Pro.sup.36, Pro.sup.37, Pro.sup.38
[Asp.sup.28]exendin-4(1-39)-NH.sub.2, des Pro.sup.36, Pro.sup.37,
Pro.sup.38 [Asp.sup.28]exendin-4(1-39)-(Lys).sub.6-NH.sub.2,
H-(Lys).sub.6-des Pro.sup.36, Pro.sup.37, Pro.sup.38
[Asp.sup.28]exendin-4(1-39)-(Lys).sub.6-NH.sub.2,
H-Asn-(Glu).sub.5-des Pro.sup.36, Pro.sup.37, Pro.sup.38
[Asp.sup.28]exendin-4(1-39)-(Lys).sub.6-NH.sub.2, H-(Lys).sub.6-des
Pro.sup.36 [Trp(O.sub.2).sup.25,
Asp.sup.28]exendin-4(1-39)-Lys.sub.6-NH.sub.2, H-des Asp.sup.28
Pro.sup.36, Pro.sup.37, Pro.sup.38
[Trp(O.sub.2).sup.25]exendin-4(1-39)-NH.sub.2, H-(Lys).sub.6-des
Pro.sup.36, Pro.sup.37, Pro.sup.38 [Trp(O.sub.2).sup.25,
Asp.sup.28]exendin-4(1-39)-NH.sub.2, H-Asn-(Glu).sub.5-des
Pro.sup.36, Pro.sup.37, Pro.sup.38[Trp(O.sub.2).sup.25,
Asp.sup.28]exendin-4(1-39)-NH.sub.2, des Pro.sup.36, Pro.sup.37,
Pro.sup.38 [Trp(O.sub.2).sup.25,
Asp.sup.28]exendin-4(1-39)-(Lys).sub.6-NH.sub.2, H-(Lys).sub.6-des
Pro.sup.36, Pro.sup.37, Pro.sup.38 [Trp(O.sub.2).sup.25,
Asp.sup.28]exendin-4(1-39)-(Lys).sub.6-NH.sub.2,
H-Asn-(Glu).sub.5-des Pro.sup.36, Pro.sup.37, Pro.sup.38
[Trp(O.sub.2).sup.25,
Asp.sup.28]exendin-4(1-39)-(Lys).sub.6-NH.sub.2, H-(Lys).sub.6-des
Pro.sup.36 [Met(O).sup.14,
Asp.sup.28]exendin-4(1-39)-Lys.sub.6-NH.sub.2, des Met(O).sup.14
Asp.sup.28 Pro.sup.36, Pro.sup.37, Pro.sup.38
exendin-4(1-39)-NH.sub.2, H-(Lys).sub.6-des Pro.sup.36, Pro.sup.37,
Pro.sup.38 [Met(O).sup.14, Asp.sup.28]exendin-4(1-39)-NH.sub.2,
H-Asn-(Glu).sub.5-des Pro.sup.36, Pro.sup.37, Pro.sup.38
[Met(O).sup.14, Asp.sup.28]exendin-4(1-39)-NH.sub.2, des
Pro.sup.36, Pro.sup.37, Pro.sup.38 [Met(O).sup.14,
Asp.sup.28]exendin-4(1-39)-(Lys).sub.6-NH.sub.2, H-(Lys).sub.6-des
Pro.sup.36, Pro.sup.37, Pro.sup.38 [Met(O).sup.14,
Asp.sup.28]exendin-4(1-39)-Lys.sub.6-NH.sub.2,
H-Asn-(Glu).sub.6-des Pro.sup.36, Pro.sup.37, Pro.sup.38
[Met(O).sup.14, Asp.sup.28]exendin-4(1-39)-(Lys).sub.6-NH.sub.2,
H-(Lys).sub.6-des Pro.sup.36 [Met(O).sup.14, Trp(O.sub.2).sup.25,
Asp.sup.28]exendin-4(1-39)-Lys.sub.6-NH.sub.2, des Asp.sup.28
Pro.sup.36, Pro.sup.37, Pro.sup.38 [Met(O).sup.14,
Trp(O.sub.2).sup.25]exendin-4(1-39)-NH.sub.2, H-(Lys).sub.6-des
Pro.sup.36, Pro.sup.37, Pro.sup.38 [Met(O).sup.14,
Trp(O.sub.2).sup.25, Asp.sup.28]exendin-4(1-39)-NH.sub.2,
H-Asn-(Glu).sub.5-des Pro.sup.36, Pro.sup.37, Pro.sup.38
[Met(O).sup.14, Asp.sup.28]exendin-4(1-39)-NH.sub.2, des
Pro.sup.36, Pro.sup.37, Pro.sup.38 [Met(O).sup.14,
Trp(O.sub.2).sup.25,
Asp.sup.28]exendin-4(1-39)-(Lys).sub.6-NH.sub.2, H-(Lys).sub.6-des
Pro.sup.36, Pro.sup.37, Pro.sup.38 [Met(O).sup.14,
Trp(O.sub.2).sup.25,
Asp.sup.28]exendin-4(1-39)-(Lys).sub.6-NH.sub.2,
H-Asn-(Glu).sub.5-des Pro.sup.36, Pro.sup.37, Pro.sup.38
[Met(O).sup.14, Trp(O.sub.2).sup.25,
Asp.sup.28]exendin-4(1-39)-(Lys).sub.6-NH.sub.2, or a
pharmacologically tolerable salt thereof.
[0062] The invention further relates to a formulation as described
above which additionally comprises Arg.sup.34, Lys.sup.26
(N.sup..epsilon.(.gamma.-glutamyl(N.sup..alpha.-hexadecanoyl)))
GLP-1 (7-37) [liraglutide] or a pharmacologically tolerable salt
thereof.
[0063] Additionally, the formulation of the present invention can
also comprise an analogue of exendin-4, such, for example,
lixisentatide, exenatide and liraglutide. These exendin-4 analogues
are present in the formulation in the range of 0.1 .mu.g to 10
.mu.g per U insulin glargine, preferably 0.2 to 1 .mu.g per U
insulin glargine, and more preferably 0.25 .mu.g to 0.7 .mu.g per U
insulin glargine. Lixisenatide is preferred.
[0064] Additionally, the aqueous pharmaceutical formulation can
comprise one or more excipients selected from a group comprising
zinc, m-cresol, glycerol, polysorbate 20 and sodium. Specifically,
the aqueous pharmaceutical formulation can comprise 90 .mu.g/mL
zinc, 2.7 mg/mL m-cresol and 20 mg/ml glycerol 85%. Optionally, the
aqueous pharmaceutical formulation can comprise 20 .mu.g/mL
polysorbate 20.
[0065] The pH of the aqueous pharmaceutical formulation is between
3.4 and 4.6, preferably 4 or 4.5.
[0066] The present invention is directed to a method of treating
Type I and Type II Diabetes Mellitus comprising administering to
said patient the aqueous pharmaceutical composition of the present
invention to a diabetic patient. Preferred among the various
disclosed concentration ranges is a concentration of 300 U/mL and
the preferred insulin analogue is insulin glargine. Further the
aqueous pharmaceutical formulation also can comprise zinc,
m-cresol, glycerol, polysorbate 20 and sodium and mixtures thereof
in the ranges disclosed herein in relation to the aqueous
pharmaceutical formulation of the present invention. In a preferred
embodiment the aqueous pharmaceutical formulation also comprises
0.1 .mu.g to 10 .mu.g lixisenatide per U insulin glargine.
[0067] The insulin is administered preferably once daily but can be
administered twice daily as needed. Dosage requirements are a
function of the needs of the individual patient determined by the
achievement of normal or acceptable blood glucose levels.
[0068] The present invention is also directed to a method of
extending the duration of exposure of insulin glargine in the
treatment of Type I and Type II Diabetes Mellitus in a patient
comprising administering to said patient the aqueous pharmaceutical
formulation of the present invention. Preferred among the various
disclosed concentration ranges is a concentration of 300 U/mL.
Further the aqueous pharmaceutical formulation also can comprise
zinc, m-cresol, glycerol, polysorbate 20 and sodium and mixtures
thereof in the ranges disclosed herein in relation to the aqueous
pharmaceutical formulation of the present invention.
[0069] In a preferred embodiment the aqueous pharmaceutical
formulation also comprises 0.1 .mu.g to 10 .mu.g lixisenatide per U
insulin glargine.
[0070] The present invention is also directed to a method of
reducing the incidence of hypoglycemia in the treatment of Type I
and Type II Diabetes Mellitus in a patient with insulin glargine
comprising administering to said patient the aqueous pharmaceutical
formulation of the present invention. Preferred among the various
disclosed concentration ranges is a concentration of 300 U/mL.
Further the aqueous pharmaceutical formulation also can comprise
zinc, m-cresol, glycerol, polysorbate 20 and sodium and mixtures
thereof in the ranges disclosed herein in relation to the aqueous
pharmaceutical formulation of the present invention. In a preferred
embodiment the aqueous pharmaceutical formulation also comprises
0.1 .mu.g to 10 .mu.g lixisenatide per U insulin glargine.
[0071] The present invention is also directed to a method of
providing a peakless long acting basal insulin in the treatment of
Type I and Type II Diabetes Mellitus in a patient with comprising
administering to said patient the aqueous pharmaceutical
formulation of the present invention. Preferred among the various
disclosed concentration ranges is a concentration of 300 U/mL.
Further the aqueous pharmaceutical formulation also can comprise
zinc, m-cresol, glycerol, polysorbate 20 and sodium and mixtures
thereof in the ranges disclosed herein in relation to the aqueous
pharmaceutical formulation of the present invention. In a preferred
embodiment the aqueous pharmaceutical formulation also comprises
0.1 .mu.g to 10 .mu.g lixisenatide per U insulin glargine.
[0072] Use of an aqueous formulation according to any of the
foregoing items in the treatment of Type 1 Diabetes Mellitus and
Type 2 Diabetes Mellitus.
[0073] The application is described below with the aid of some
examples, which are in no way intended to act restrictively.
Example 1
Description of the Protocol
[0074] This study was a single center, randomized, controlled,
single-blind, four-period, 2-treatment, 2-sequence crossover study
in healthy subjects with six visits:
Visit 1: Screening (SCR)
[0075] Visit 2 to 5, Period (P) 1-4: Treatment, euglycemic clamp
period
Visit 6: End-of-study (EOS)
[0076] Subjects received single subcutaneous doses of 0.4 U/kg
insulin glargine U100 and insulin glargine U300 alternatingly
injected into two opposite sites of the periumbilical area (left,
right, left, right) at four different days. The study medication
was administered with a replicate of treatment R and T in 2
sequences, RTTR or TRRT at P1 to P4. A washout period of 4 to 18
days was separated each dosing day.
R: 0.4 U/kg body weight insulin glargine U100 (commercial
formulation; Reference) T: 0.4 U/kg body weight insulin glargine
U300 (Test)
[0077] P1 must take place no more than 3 to 21 days after SCR. EOS
visit must take place between 4 to 14 days after P4.
[0078] During P1 to P4, subjects have been connected to a Biostator
for measurement of blood glucose and adjustment of glucose infusion
rate. Blood glucose levels and glucose infusion rate (GIR) have
been monitored for 90 minutes (baseline period) before subcutaneous
injection of the study medication and for 30 hours after study
medication administration. Infusion of 20% glucose solution
commenced to maintain blood glucose levels at 5% below the
individual fasting blood glucose level, determined as the mean of
the 3 fasting blood glucose values measured 60, 30 and 5 minutes
before study medication administration. Profiles of GIR have been
obtained. Blood samples have been taken at predetermined times
during the euglycemic clamp period for determination of serum
insulin glargine concentrations. With the exception of tap water,
subjects have been fasting during the glucose clamp period.
[0079] The duration of this study for an individual was expected to
be up to 13 weeks between SCR and EOS visit.
[0080] The protocol was submitted to independent ethics committees
and/or institutional review boards for review and written approval.
The protocol complied with recommendations of the 18th World Health
Congress (Helsinki, 1964) and all applicable amendments. The
protocol also complied with the laws and regulations, as well as
any applicable guidelines, of Germany, where the study was
conducted. Informed consent was obtained prior to the conduct of
any study-related procedures.
Example 2
Selection of Subjects
[0081] Twenty four (24) healthy subjects were planned to be treated
in order to have 20 completers.
[0082] Subjects meeting all of the following criteria have been
considered for enrollment into the study:
Demography
[0083] Subjects of either gender between 18 and 50 years of age;
[0084] Body weight between 50 kg and 110 kg and Body Mass Index
between 18 and 28 kg/m.sup.2;
Health Status
[0084] [0085] Certified as healthy following a comprehensive
clinical assessment (detailed medical history and complete physical
examination); [0086] Non-smoker for at least 3 months; [0087]
12-lead electrocardiogram, and vital signs unless the Investigator
considers an abnormality to be clinically irrelevant [0088] Normal
vital signs after 5 minutes resting in supine position: [0089] 95
mmHg.ltoreq.systolic blood pressure.ltoreq.140 mmHg; [0090] 45
mmHg.ltoreq.diastolic blood pressure.ltoreq.90 mmHg; [0091] 40
bpm.ltoreq.heart rate.ltoreq.100 bpm; [0092] Normal 12-lead ECG;
120 ms<PR<220 ms, QRS<120 ms, QTc.ltoreq.430 ms (for
female: QTc.ltoreq.450 ms); [0093] Laboratory parameters within the
normal range unless the Investigator considers an abnormality to be
clinically irrelevant for healthy subjects; however serum
creatinine and hepatic enzymes (AST, ALT) should be strictly below
the upper laboratory norm; [0094] Normal metabolic control defined
as fasting serum glucose (.ltoreq.100 mg/dL) and glycosylated
hemoglobin (HbA1c.ltoreq.6.1%); [0095] Subjects must be off regular
use of prescription drug therapy, for at least four (4) weeks prior
to participation in the study;
Obligations for Female Subjects
[0095] [0096] Female subjects of childbearing potential (defined as
pre-menopausal and not surgically sterilized or post-menopausal for
less than 2 years) and sexually active must practice adequate birth
control. Adequate birth control is defined as a highly effective
method of contraception (Pearl index <1%) such as implants,
injectables, combined oral contraceptives or hormonal IUDs
(intrauterine devices). Post-menopausal for the purposes of this
clinical trial include: amenorrhea for 2 or more years or
surgically sterile; [0097] Female subjects must have a negative
urine beta-human chorionic gonadotropin (beta-HCG) pregnancy test
during the pre-study screening, and prior to the first clamp;
Regulations
[0097] [0098] Having given written informed consent prior to any
procedure related to the study; [0099] Covered by Health Insurance
System and/or in compliance with the recommendations of National
Law in force relating to biomedical research; [0100] Not under any
administrative or legal supervision.
[0101] Subjects presenting with any of the following have not been
included in the study:
Medical history and clinical status [0102] Any history or presence
of clinically relevant cardiovascular, pulmonary,
gastro-intestinal, hepatic, renal, metabolic, hematological,
neurologic, psychiatric, systemic, ocular or infectious disease;
any acute infectious disease or signs of acute illness; [0103]
Presence or history of drug allergy, or allergic disease diagnosed
and treated by a physician; [0104] Excessive consumption of
beverages with xanthine bases (>4 cups or glasses/day); [0105]
Contraindications from (according to normal ranges--if the value is
outside of the normal range the subject can be included if the
Investigator sees this abnormal value as clinically irrelevant):
[0106] the medical/surgical history and physical examination [0107]
laboratory tests (hematology, clinical chemistry, and urinalysis by
dipstick) [0108] standard 12-lead electrocardiogram [0109] blood
pressure and heart rate [0110] Any ongoing treatment with
prescribed drugs or any regular treatment with prescribed drugs in
the 4 weeks prior to participation in the study [0111] Symptoms of
a clinically significant illness in the 3 months before the study,
or of any major internal medical disease in the 4 weeks before the
study which, according to the Investigator's opinion, could
interfere with the purposes of the study. [0112] Presence or
sequalae of a disease or other conditions known to interfere with
the absorption, distribution, metabolism, or excretion of drugs
[0113] History of drug or alcohol abuse [0114] History of
hypersensitivity to the study medication or to drugs with similar
chemical structures [0115] Progressive fatal disease [0116]
Pre-planned surgery during the study [0117] Blood donation of more
than 500 mL during the previous 3 months
[0118] No subject has been allowed to enroll in this study more
than once.
General Conditions
[0119] Subject who, in the judgment of the Investigator, is likely
to be non-compliant during the study, or unable to cooperate
because of a language problem or poor mental development or due to
a mental condition rendering the subject unable to understand the
nature, scope and possible consequences of the study [0120] Subject
in exclusion period of a previous study according to applicable
regulations; [0121] Subject is the Investigator or any
Sub-Investigator, Research Assistant, Pharmacist, Study
Coordinator, other Staff thereof, directly involved in the conduct
of the protocol; [0122] Receipt of an experimental drug within the
previous 30 days before SCR.
Biological Status
[0122] [0123] Positive reaction to any of the following tests: HBs
antigen, anti-HCV antibodies, anti-HIV1 antibodies, anti-HIV2
antibodies; [0124] Positive results on urine drug screen at SCR
(amphetamines/metamphetamines, barbiturates, benzodiazepines,
cannabinoids, cocaine, opiates); [0125] Positive alcohol breath
test
Example 3
Treatments
TABLE-US-00001 [0126] Details of Study Treatments Drug code: HOE901
(Lantus .RTM. U100 commercial (Insulin glargine U300 formulation)
formulation) INN: Insulin glargine (recombinant Insulin glargine
(recombinant human insulin analogue) human insulin analogue)
Formulation: Cartridges for 3 mL solution U100 Cartridges for 3 mL
solution U300 (1 mL contains 3.637 mg 21.sup.A-Gly- (1 mL contains
10.913 mg 21.sup.A-Gly- 30.sup.Ba-L- Arg-30.sup.Bb-L-Arg human
30.sup.Ba-L-Arg-30.sup.Bb-L-Arg human insulin [equimolar to 100 IU
insulin [equimolar to 300 IU human insulin], 30 .mu.g zinc, 2.7 mg
human insulin], 90 .mu.g zinc, 2.7 mg m-cresol, 20 mg glycerol 85%,
m-cresol, 20 mg glycerol 85%, HCl and NaOH ad pH 4.0; specific HCl
and NaOH ad pH 4.0; specific gravity 1.004 g/mL) gravity 1.006
g/mL) Dose/route of administration 0.4 U/kg body weight; single
s.c. 0.4 U/kg body weight; single s.c. injection into the
periumbilical injection into the periumbilical abdomen after an
overnight fast abdomen after an overnight fast Manufacturer:
Sanofi-Aventis Manufacturer: Sanofi-Aventis Deutschland GmbH
Deutschland GmbH
Calculation of the Dose for Lantus.RTM./Insulin Glargine
Formulation
[0127] To calculate the amount of insulin glargine given for each
subject (0.4 U/kg), the body weight (in kg) has been determined to
one decimal place and the amount of insulin calculated has been
rounded up or down to integer numbers as shown in the following
examples: a subject with a body weight of 75.3 kg has received 30 U
insulin (75.3.times.0.4=30.12 which is rounded down to 30); a
subject with a body weight of 74.4 kg has received 30 U insulin
(74.4.times.0.4=29.76, which is rounded up to 30). The body weight
recorded during Period 1 Day 1 has been used for calculation of
study medication dose for Periods 2, 3 and 4, unless the body
weight changed by more than 2 kg compared to Period 1.
[0128] The amount in Units has been the same for both insulin
glargine U100 and insulin glargine U300. This specific gravity is
the same for both drug products. However, given the three times
higher concentration of insulin glargine in insulin glargine U300
as compared to insulin glargine U100, the to be injected volume and
hence the weight has been 1/3 for insulin glargine U300. The
syringes providing the individual dose have been prepared by
weight. The net weight has been documented only in the
source-documentation of the Investigator.
Calculation and Preparation of the Dose for Infusions
TABLE-US-00002 [0129] TABLE 1 Preparation of infusion Dose/Routeof
Drug Code INN Formulation Manufacturer administration Glucose
Glucose 20% solution Certified, iv infusion for infusion selected
by PROFIL Intramed Heparin Vial containing Certified, iv infusion
Heparin 5 mL solution selected Sodium (5000 IU/mL) by PROFIL 0.9%
Sodium Solution Certified, iv infusion Sodium Chloride selected
Chloride by PROFIL
[0130] Glucose solution: 20% glucose solution has been infused with
the Biostator to keep subjects individual blood glucose at the
determined target level. A second infusion pump (part of the
Biostator) has delivered 0.9% sodium chloride solution to keep the
line patent. In case the amount of 20% glucose solution needed
exceeds the infusion capacity of the Biostator, a second glucose
infusion pump has been engaged.
[0131] Heparin: 10000 IU heparin in 100 mL 0.9% sodium chloride
solution have been infused into the double lumen catheter at a rate
of approximately 2 mL/h to keep it patent for blood glucose
measurement by the Biostator.
Description of Blinding Methods:
[0132] This was a single-blind study. The different volumes of
injection preclude blinding of the medication. Injection has been
done by an authorized medical person otherwise not involved in the
study. The Investigator has access to the randomization code.
Method of Assigning Subjects to Treatment Group
[0133] The study medication has been administered only to subjects
included in this study following the procedures set out in the
clinical study protocol.
[0134] A randomization schedule has been generated, which has
linked the randomization numbers, stratified by gender, to the
treatment sequences of the two Lantus.RTM. formulations to be
injected at P1 to P4.
[0135] In the morning of Day 1 of Period 1, as soon as the
Investigator has confirmed that subjects fulfill the criteria
specified in the protocol, the eligible subjects were randomized by
the site. The randomization number was allocated to the subject
number subsequently in the order in which subjects' eligibility has
been confirmed before P1. The first subject for a gender stratum
qualifying after SCR received the first randomization number for
the appropriate gender stratum. The next subject who qualifies
within a stratum received the next randomization number within the
stratum.
[0136] The randomization number has been used as the treatment kit
number to allocate the treatment kit to the subject. Each subject
were given the study medication carrying the treatment kit number
to which he has been allocated to. The treatment kit containing the
IP carried general information, treatment kit number, period
number, a field to write the subject number on the container-box,
and additional statements as required by local regulations.
[0137] Subjects who permanently discontinue from the study retained
subject number and randomization number, if already given.
Packaging and Labelling
[0138] The study medication has been packed by Sanofi-Aventis
Deutschland GmbH, Frankfurt am Main, Germany according to the
randomization plan. The cartridges containing the study medication
and the cartons they were packed in have been labeled with the
study number, the randomization number, batch number, storage
conditions, Sponsor and the P number.
[0139] Supplies of study medication have been received in one
shipment. All containers had labels of identical format.
Additionally, 1 set of labels for syringes has been supplied. Study
medication and back-up medication were stored in different
refrigerators.
[0140] Before study medication administration, the Pharmacist or
the person designated by him has prepared the syringes with the
appropriate study medication and has labeled the syringe with the
subject number, the randomization number and the appropriate period
according to the study medication containers.
[0141] The content of the labeling was in accordance with the local
regulatory specifications and requirements.
Storage Conditions
[0142] The study medication was stored protected from light at a
temperature of +2.degree. C. to +8.degree. C. The study medication
was prevented from freezing. During preparation it was not
necessary to have the medication protected from light.
[0143] Reserve samples (300 cartridges Lantus.RTM. U100 and 300
cartridges insulin glargine U 300) were stored in the same secure
conditions at the study site level.
Example 4
Assessment of Investigational Product
Activity or Pharmacodynamics
[0144] Stimulation of insulin receptors by insulin glargine is the
mode of action. Subsequent peripheral glucose uptake and
suppression of endogenous glucose production comprise the
glucodynamic effects producing a reduction in blood glucose
concentration. The resulting glucose utilization is best
characterized by the gauge of glucose required to keep the blood
glucose concentration constant.
[0145] The euglycemic clamp technique has been employed to assess
the amount of glucose needed to keep blood glucose concentrations
at 5% below baseline level after injection of insulin glargine.
Clinical Assessment Methods
[0146] Online blood glucose determination has been done by the
Biostator (Life Sciences instruments, Elkhart, Ind., USA) employing
the glucose oxidase method.
[0147] Offsite blood glucose has been determined with a Super GL
glucose analyzer also using the glucose oxidase method.
Pharmacodynamic Variables/Endpoints
[0148] The amount of glucose utilized per unit (dose) of
subcutaneously injected insulin is a measure of the glucodynamic
effect.
[0149] The continuously recorded glucose infusion rate (GIR) is a
reflection of the time action profile of the injected insulin.
Primary Variable/Endpoint
[0150] The primary pharmacodynamic variable is the area under the
glucose infusion rate time curve within 24 hours [GIR-AUC.sub.0-24h
(mgkg.sup.-1)].
Secondary Variable/Endpoint
[0151] The secondary pharmacodynamic variable is the time to 50%
GIR-AUC.sub.0-24h [T.sub.50%-GIR-AUC.sub.(0-24h) (h)].
Pharmacokinetics
Sampling Times
[0152] Blood samples for assessment of serum insulin glargine and
C-peptide concentrations have been taken 1 hour, 30 min and
immediately prior to subcutaneous injection of study medication,
thereafter 30 min, 1 hour, 2 hours and then bi-hourly up to 24
hours, and 30 hours after injection.
[0153] The numbering of insulin glargine samples was P00, P01, P02,
P03, P04, etc., the numbering of C-peptide samples was C00, C01,
C02, C03, C04, etc (see also study flow chart). Number of
Pharmacokinetic Sampling
[0154] A minimum of 18 samples have been taken per clamp visit (P1
to P4). In total 72 samples have been taken per subject.
PK Handling Procedure
[0155] The exact time of sample collection must be recorded on the
CRF. Special procedures for storage and shipping of pharmacokinetic
samples (insulin glargine, C-peptide) have been used.
Bioanalytical Method
[0156] Bioanalysis have been performed using as a basis the Good
Laboratory Practice (GLP) requirements applicable to this type of
study identified in the OECD Principles of Good Laboratory Practice
(as revised in 1997), ENV/MC/CHEM (98)17 and the GLP regulations
applicable to the local country.
[0157] As no back-up samples are available priority is given to
determination of insulin glargine.
Insulin Glargine
[0158] Serum insulin glargine concentrations have been determined
using a radioimmunoassay (RIA) for human insulin (Insulin RIA kit,
ADALTIS, Italy) calibrated for insulin glargine. Kit REF 10624.
[0159] The lower limit of quantification (LLOQ) for this assay was
4.92 .mu.U/mL.
C-Peptide
[0160] Serum C-peptide concentrations have been determined using a
radioimmunoassay (RIA) for C-peptide (C-peptide RIA kit, ADALTIS,
Italy). Kit REF C-peptide 10282.
[0161] The lower limit of quantification (LLOQ) was 0.090
nmol/L.
TABLE-US-00003 Summary of Bioanalytical Method Analyte insulin,
C-peptide Matrix serum Analytical Technique RIA Lower limit of 4.92
.mu.U/mL insulin; 0.090 nmol/L C-peptide quantification Assay
volume 100 .mu.L for insulin; 100 .mu.L for C-peptide Method
Reference Adaltis S.p.A. Italy; Kit REF 10624 Insulin (Method No.
435VAL02) and Kit REF C-peptide 10282 (Method No.
DMPK/FRA/2003-0002)
Pharmacokinetic Variables/Endpoints
[0162] The insulin glargine concentration time curve was a measure
of the systemic insulin exposure of subcutaneously injected IP.
Primary Variable/Endpoint
[0163] The primary pharmacokinetic variable was the area under the
serum insulin glargine concentration time curve [INS-AUC.sub.0-24h
(.mu.UhmL.sup.-1).]
Secondary Variable/Endpoint
[0164] The secondary pharmacokinetic variable was the time to 50%
INS-AUC.sub.0-24h [T.sub.50%-INS-AUC.sub.(0-24h) (h)].
Sampled Blood Volume
TABLE-US-00004 [0165] Sampled blood volume Archival
Blood/Genotyping 0 mL Hematology/Clinical chemistry/Serology (20 +
12 mL) 32 mL RBC, Hb, Hct (2 .times. 2 mL) optional 4 mL Blood
glucose (2 mL/h .times. 32 .times. 4) 256 mL Blood glucose (0.3 mL
.times. 4 .times. 34) 41 mL PK insulin glargine (3.5 mL .times. 18
.times. 4) 252 mL Total 585 mL
Measures to Protect Blinding of the Trial
[0166] This has been a single-blind study. Bioanalytical
determinations have been performed after clinical completion. The
treatment code has been known for reporting of any Serious Adverse
Event (SAE) unexpected and reasonably associated with the use of
the IP according to either the judgment of the Investigator and/or
the Sponsor.
Example 5
Study Procedures
Visit Schedule
Screening Procedures
[0167] The medical records of each potential subject has been
checked before the start of the study to determine eligibility for
participation. The subjects have fasted (except for water) for 10
hours before the screening examination at SCR.
[0168] The following items/examinations have been assessed: [0169]
Age, and race [0170] Physical examination (including cardiovascular
system, chest and lungs, thyroid, abdomen, nervous system, skin and
mucosae, and musculoskeletal system) [0171] Relevant medical and
surgical history (only findings relevant to the study are to be
documented) [0172] Anthropometrics: height and weight, calculation
of BMI [weight in kg(height in m).sup.-2] [0173] Blood pressure and
heart rate (after 5 min in supine and 3 min upright position)
[0174] Core body temperature (tympanic) [0175] Standard 12-lead ECG
[0176] Hematology status, clinical chemistry, and urinalysis (by
dipstick) [0177] Coagulation status (INR, aPPT) [0178] Urine drug
screen [0179] Alcohol screen (breath analyzer) [0180] Normal
metabolic control defined as fasting blood glucose (.ltoreq.100
mgdL.sup.-1) and glycosylated hemoglobin (HbA1c.ltoreq.6.1%) [0181]
Hepatitis B/C and HIV test
[0182] In case the subject is a screening failure, all data
obtained at SCR including laboratory results of screening tests
were available in the subject's medical record.
Description by Type of Visit
Period(s)
[0183] Each study period (P1 to P4) lasted 2 days, Day 1 and Day 2.
Day 1 was the starting day of the euglycemic clamp and
administration of study medication. Day 2 was day of the end of the
euglycemic clamp, which lasted 30 hours after study medication
administration. There was a wash-out period of 4-18 days between
the study periods (P1-P4). No strenuous activity (e.g. mountain
biking, heavy gardening etc.) has been allowed 2 days before each
study medication administration. Consumption of alcoholic
beverages, grapefruit juice, and stimulating beverages containing
xanthine derivatives (tea, chocolate, coffee, Coke.TM.-like drinks,
etc.) and grapefruit has not been permitted from 24 hours before
until completion of the euglycemic clamp. The subjects have fasted
(except for water) for 10 hours before Day 1 of each study period
(P1 to P4) and remained fasting (except for water) until end of the
euglycemic clamp. The subjects had to stay in the clinic for
approximately 32 hours at each clamp visit.
[0184] In the morning of Day 1 of Period 1, the 9-digit subject
number has been allocated to the subject, starting with 276001001.
The next subject who qualifies to enter SCR has received the
subject number 276001002 etc. The first subject has received the
randomization number 101. The next subject who qualifies has
received the randomization number 102.
[0185] Subjects have been asked to ensure that they have had no
clinically significant changes in their physical condition and have
been compliant with the general and dietary restrictions as defined
in the protocol since the previous periods. Violation of the study
criteria has excluded subjects from participation in the study.
Depending on the kind of violation the subject might have been
excluded only from the particular period, allowing a re-scheduling
of the study day. Any protocol violations have been discussed with
the Sponsor on a case-by-case basis in advance.
[0186] Any changes in the health condition of the subjects since
the last period have been reported in the subject's medical records
(source) and the CRF.
[0187] The blood pressure, heart rate and core body temperature
(tympanic) have been recorded in supine position after at least 5
minutes rest in the morning of Day 1, prior to and after completion
of clamp procedures 30 hours after each study medication
administration (Day 2). Body weight, alcohol screen and RBC, Hb,
HcT (only before clamp period of P3 and P4) have been assessed only
before starting the clamp in the morning of Day 1.
[0188] On Day 1 of each period, subjects have been admitted to the
clinic at 6:30 am. After passing the above described examinations,
subjects have been prepared with three venous lines. A dorsal hand
vein or lateral wrist vein of the left arm has been canalized in
retrograde fashion and connected to a Biostator (Life Sciences
instruments, Elkhart, 1N, USA) in order to continuously draw
arterialized venous blood for the determination of blood glucose.
To achieve arterialization the left hand has been placed in a
"Hot-Box" at about 55.degree. C. A second venous line has been
placed into the antecubital vein of the left arm and have been used
to collect samples for serum insulin glargine and reference blood
glucose determination. A third vein has been canalized on the
contralateral forearm allowing the infusion of 20% glucose solution
and 0.9% saline with the Biostator.
[0189] The Biostator determined blood glucose levels and adjusted
the glucose infusion rate to maintain blood glucose levels at 5%
below the individual fasting blood glucose, determined as the mean
of the 3 fasting blood glucose values measured 60, 30 and 5 minutes
before study medication administration. Additional blood samples of
0.3 mL for the determination of blood glucose have been taken 60,
30, and 5 minutes before administration of the study medication to
check against a laboratory reference based on the glucose oxidase
method.
[0190] Approximately at 09:00 am, either insulin glargine U100
(commercial formulation) or insulin glargine U300 have been
injected in the periumbilical area 5 cm lateral to the umbilicus
(left, right, left, right) using a standardized skin fold
technique. U100 insulin syringes (manufacturer: Beckton &
Dickinson) of 0.5 mL volume with a needle of 0.30 mm.times.8 mm
(30G) have been used.
[0191] The study medication was labeled with their respective
treatment kit number, subject number (to be documented on the
container-box after randomization), and Period number (see Section
8.5 Packaging and Labeling).
[0192] After study medication administration, infusion of 20%
glucose solution have commenced at a variable rate once blood
glucose level has fallen by 5% from the individual fasting level to
maintain that level. The duration of the clamp period have been 30
hours. The rate of glucose delivery have been adjusted by the
Biostator in response to changes in blood glucose at 1 minute
intervals using a predefined algorithm. The blood glucose values
from the Biostator have been checked against a laboratory reference
based on the glucose oxidase method at 30 minutes intervals for the
entire clamp. If necessary the Biostator have been re-calibrated
according to results of the laboratory reference method. Subjects
remained in supine position during the period of clamping.
[0193] Blood samples for determination of serum insulin glargine
and C-peptide concentrations have been taken 1 hour, 30 min and
immediately before medication and thereafter 30 min, 1 hour, 2
hours and then bi-hourly up to 24 hours, and 30 hours after
administration of study medication.
[0194] On day 2 of each study period (P1 to P4), a meal have been
served after the euglycemic clamp has been completed. Blood
pressure, heart rate, and core body temperature (tympanic) have
been recorded, and a sample for blood glucose has been taken. The
subjects have been discharged from the clinic after their safety
has been ensured by the Investigator.
[0195] Injection sites have been observed during the entire clamp
period. Any changes in the health condition of the subjects have
been reported in the subject's medical records (source) and the
CRF.
Safety Hematology
[0196] RBC, Hb and Hct at P 3 have been analyzed for incurring
anemia at P 4. If positive, the interval between P 3 and P 4 have
been extended to the maximum allowed 18 days and an additional RBC,
Hb and Hct assessment made prior to P 4.
Discharge Procedures
[0197] Subjects have returned for an EOS visit between 4 to 14 days
after P4. Subjects have fasted (apart from water) for 10 hours. Any
changes in the health condition of the subjects since the last
period have been reported in the subject's medical records (source)
and the CRF.
[0198] The following items/examinations have been assessed: [0199]
Physical examination (including cardiovascular system, chest and
lungs, thyroid, abdomen, nervous system, skin and mucosae, and
musculoskeletal system) [0200] Weight [0201] Blood pressure and
heart rate (after 5 min in supine position) [0202] Core body
temperature (tympanic) [0203] Standard 12-lead ECG [0204]
Hematology status, clinical chemistry, and urinalysis (by dipstick)
[0205] .beta.-HCG test in urine (only for females)
[0206] The subjects have been discharged on Day 2 of each period,
after a complete review by the Investigator of the available safety
data.
Collection Schedule for Biological Samples
Blood
SCR (Screening):
[0207] Hematology, Clinical Chemistry, HbA1c, Serology (Hepatitis
B/C test, HIV test): approximately 20 mL of blood have been
collected.
P1 to P4 (Day 1 and 2):
[0207] [0208] Blood glucose [0209] Biostator has automatically
measured blood glucose at one minute intervals for the entire clamp
period, including the period prior to study medication. The volume
of blood needed by the Biostator have been 2 mLh.sup.-1. An
estimated 252 mL blood volume have been needed for glucose readings
with the Biostator for the four periods. Blood samples (0.3 mL) for
checking blood glucose values from Biostator have been collected
60, 30, 5 and 0 minutes prior to dosing and at 30 minute intervals
after dosing until end of the clamp (30 hours). An estimated 41 mL
blood volume have been collected for the four periods. [0210] Serum
insulin glargine and C-peptide concentrations
[0211] Venous blood samples (3.5 mL) have been collected 1 hour, 30
min and immediately prior to dosing, 30 min, 1 hour, 2 hours and
then bi-hourly up to 24 hours, and 30 hours after dosing. An
estimated 252 mL blood volume have been collected for the four
periods. Determination of insulin glargine has been given priority.
Spare samples only have been used for determination of C-peptide
concentration. [0212] RBC, Hb, Hct [0213] Venous blood have been
collected before commencing clamp period 3 and 4. Approximately 4
mL of blood have been collected for the two periods.
End-of-Study (EOS) Visit:
[0213] [0214] Hematology, Clinical Chemistry: approximately 12 mL
of blood have been collected. [0215] .beta.-HCG test in urine (only
for females)
Total Blood Volume SCR-EOS:
[0216] In total, approximately 585 mL blood have been collected for
each subject during the entire study.
Urine
[0217] Qualitative urine drug screen have been conducted at SCR and
EOS. Urine drug screen consists of amphetamines/metamphetamines,
barbiturates, benzodiazepines, cannabinoids, cocaine, opiates.
Qualitative safety urinalysis with dipsticks have been conducted at
SCR and EOS. Safety urinalysis consists of analysis for: pH,
protein, glucose, blood, erythrocytes, leukocytes, bilirubin,
urobilinogen, ketone, specific gravity, and nitrite.
Measurement Schedule for other Study Variables
[0218] Physical examination have been performed at SCR and EOS.
[0219] Core body temperature (tympanic) have been taken at SCR, P1
to P4 before and after the clamp period, and at EOS.
[0220] Blood pressure and heart rate have been measured after about
5 minutes rest in a supine position, and also after 3 minutes in an
upright position at SCR and EOS. In P1 to P4 blood pressure and
heart rate have been recorded in supine position after at least 5
minutes prior to start of clamp procedures in the morning of day 1,
and after completion of clamp procedures 30 hours after each study
medication administration (day 2).
[0221] Electrocardiograms (standard 12-lead) have been recorded at
SCR and EOS.
[0222] Body weight and height have been measured at SCR. The body
weight have been recorded in the morning of Day 1 of P1 to P4
(prior to administration of study medication) and at EOS. Alcohol
screen (ethanol, breath analyzer) have been conducted at SCR and
EOS, and in the morning of Day 1 of P1 to P4 (prior to
administration of study medication).
Study Restriction(s)
[0223] From Day-1 evening (P1 to P4) and throughout the Periods
(clamp days), the subjects have refrained from drinking alcohol,
tea, coffee, citrus or cola beverages, smoking. Eating citrus
fruits was also prohibited throughout the study. The subjects have
been requested to follow a stable lifestyle throughout the duration
of the trial, until the last control, with no intensive physical
activity.
Definition of Source Data
[0224] All evaluations listed below that are reported in the CRF
were supported by appropriately signed identified source
documentation related to: [0225] subject identification, medical
history; [0226] clinical examination, vital signs, body weight and
height; [0227] laboratory assessments, ECG; [0228] pharmacokinetic
time points; [0229] dates and times of visits and assessments;
[0230] administration dates and times, and site of injection;
[0231] AEs; [0232] duration of clamp (start and end times) [0233]
Other
[0234] The CRF have been considered as source documentation for
other items.
Example 6
Statistical Considerations
[0235] This example provides information for the statistical
analysis plan for the study. A statistical analysis plan have been
drafted prior to inclusion of subjects.
Determination of Sample Size
[0236] INS-AUC.sub.(0-24h) have been the primary parameter for
which therefore the sample size calculation was performed.
[0237] For the purpose of this sample size calculation, several
within-subject SD.sub.within of natural log-transformed
INS-AUC.sub.(0-24h) between 0.125 and 0.225 were considered. A
sample size calculation method for an average bioequivalence
approach was used for a 4-period, 2-treatment, 2-sequence
cross-over design. If the 90% CIs for the formulation ratio have
been wholly contained within [0.80-1.25], then average
bioequivalence have been concluded for the parameter.
[0238] Study HOE901/1022 was the basis for assumptions on
variability. Based on the statistical analysis of study
HOE901/1022, a value of 0.175 could be expected for the within
subject standard deviation (SD.sub.within) on the natural
log-transformed scale.
[0239] The table below indicates the number of subjects required to
demonstrate average bioequivalence of the ratio of adjusted
geometric means (test versus reference formulation) using the
bioequivalence reference interval: [0.80-1.25], assuming a true
ratio between 0.85 and 1.15 with 90% power.
TABLE-US-00005 TABLE 2 Required total number of subjects to achieve
a power of at least 90% SD(within) on natural log-scale 0.125 0.15
0.175 0.2 0.225 Assumed true ratio N N N N N 0.85 38 54 72 94 120
0.90 12 16 20 26 32 0.95 6 8 10 14 16 1.00 6 6 8 10 12 1.05 6 8 10
12 16 1.10 10 14 18 22 28 1.15 20 30 40 50 64 N = total number of
subjects
[0240] With this design, 20 subjects (10 per sequence) are required
to demonstrate equivalence of the two insulin glargine
formulations, with 90% power, allowing true ratio of 0.9, if the
true SD.sub.within on natural log scale is 0.175.
[0241] A number of 24 randomized subjects accounts for potential
cases of withdrawals.
Subject Description
Disposition of Subjects
[0242] A detailed summary of subject accountability including count
of subjects included, randomized, exposed (i.e. received any amount
of study medication), completed (i.e. subjects who completed all
study treatment periods), discontinued along with the main reasons
for discontinuation have been generated for each sequence and for
all subjects in total.
[0243] Subject disposition at the final visit have been presented
in a listing including sequence group, disposition status at the
end of the study with the date of last administration of study
drug, date of final visit, reason for discontinuation. All
withdrawals from the study, taking place on or after the start of
the first study drug administration, have been fully documented in
the body of the clinical study report (CSR).
Protocol Deviations
[0244] Prior to data base lock, the compliance with the protocol
have been examined with regard to inclusion and exclusion criteria,
treatment compliance, prohibited therapies, and timing and
availability of planned assessments. Protocol deviations have been
identified by the study team before database lock and listed in the
Data Review Report, including missing data and IP discontinuations,
and classified as minor or major deviations.
[0245] Individual deviations to inclusion and exclusion criteria as
reported by the Investigator have been listed.
[0246] Other deviations have been listed by and/or described in the
body of the CSR.
Analysis Population
Population to be Analyzed
[0247] Subjects excluded from any analysis population have been
listed with treatment sequence, and with reason for exclusion. Any
relevant information have been fully documented in the CSR.
[0248] In the event of subjects having received treatments that
differed from those assigned according to the randomization
schedule, analyses have been conducted according to the treatment
received rather than according to the randomized treatment.
Pharmacokinetic Population
[0249] All subjects without any major deviations related to study
drug administration, and for whom PK parameters are available, have
been included in the pharmacokinetic population. For subjects with
insufficient PK profiles in some but not all study days, parameters
of the sufficient profiles have been included in the analysis.
Pharmacodynamic Population
[0250] All subjects without any major deviations related to study
drug administration, and for whom PD parameters are available, have
been included in the pharmacodynamic population. For subjects with
insufficient GIR-profiles in some but not all study days,
parameters of the sufficient profiles have been included in the
analysis.
Safety Population
[0251] Safety evaluation have been based on subjects who received a
dose of study drug (exposed population), regardless of the amount
of treatment administered, including subjects prematurely
withdrawn.
Demographic and Baseline Characteristics
Subject Demographic Characteristics, Medical History and
Diagnoses
[0252] The following data have been collected: sex, age at
screening, height, weight, and race. Body mass index (BMI) per
subject have been calculated from body weight and height data:
BMI=body weight[kg](height[m]).sup.-2
[0253] All variables concerning demographic and background
characteristics have been listed individually and summarized.
[0254] Deviations from inclusion criteria related to medical
history and diagnoses have been listed and described
individually.
Baseline Pharmacodynamic Parameters
[0255] Baseline blood glucose levels have been summarized by
sequence.
Baseline Safety Parameters
[0256] For safety variables, the latest scheduled value before
study drug administration within the period or within the study,
whatever is applicable for the variable, have been taken as the
baseline value. If the baseline pre-dosing value is rechecked
before dosing, the rechecked value have been considered as the
baseline and used in statistics.
Extent of Study Treatment Exposure and Compliance
[0257] Details of study drug dosing and complementary information
have been listed individually and summarized if appropriate.
Prior/Concomitant Medication/Therapy
[0258] Prior and concomitant medications/therapies (if any) have
been coded according to the World Health Organization-Drug
Reference List (WHO-DRL) and have been listed individually.
Analysis of Pharmacodynamic Variables
Description of Pharmacodynamic Variable(s)
[0259] In order to achieve comparability between the subjects under
the body weight depending insulin dosing, all values for GIR have
been divided by the subject's body weight in kg for analysis. Thus,
GIR in the below always refers to the body weight standardized
glucose infusion rate.
[0260] Primary PD variable has been: [0261] Area under the body
weight standardized glucose infusion rate time curve
[0261] [GIR-AUC.sub.(0-24h)(mgkg.sup.-1)]
[0262] Secondary PD variable has been: [0263] Time (h) to 50% of
GIR-AUC.sub.(0-24h) [T.sub.50%-GIR-AUC.sub.(0-24h) (h)]
[0264] The following additional PD variables have been derived:
[0265] Area under the body weight standardized glucose infusion
rate time curve up to end of clamp [GIR-AUC.sub.(0-end)
(mgkg.sup.-1)] [0266] Fractional areas under the body weight
standardized glucose infusion rate time curve [GIR-AUC.sub.(4-20h),
GIR-AUC.sub.(0-12h), GIR-AUC.sub.(12-24h) (mgkg.sup.-1)] [0267]
Maximum body weight standardized glucose infusion rate [GIR.sub.max
(mgkg.sup.-1min.sup.-1)] [0268] Time to GIR.sub.max [GIR-t.sub.max
(h)]
[0269] In order to provide meaningful and reliable data, the value
for GIR.sub.max and correspondingly the time to GIR.sub.max have
been derived from a smoothed GIR curve for each subject.
Primary Analysis
[0270] To estimate relative bioefficacy (activity) for
GIR-AUC.sub.(0-24h) (mgkg.sup.-1), the untransformed parameter has
been analyzed with a linear mixed effects model.
[0271] The mixed model includes fixed terms for sequence, period,
formulation, and random terms for subject within sequence, with
formulation specific between-subject and within-subject variances
and subject-by-formulation variance. Point estimate and 90%
confidence interval for the formulation ratio (T/R) have then been
obtained based on Fieller's theorem [Heller, 1954].
[0272] Equivalent bioefficacy (activity) has been concluded if the
confidence interval for the formulation ratio has been placed
within [0.80-1.25].
[0273] Assumptions for the distribution of the variable has been
checked.
[0274] Secondary Analysis/Analysis of Secondary Variables
[0275] Individual and mean body weight standardized GIR-profiles as
well as mean percentage cumulative profiles over time have been
plotted.
[0276] PD parameters have been listed individually, and descriptive
statistics has been generated.
[0277] Formulation ratios (T/R) with confidence limits have been
derived for fractional GIR-AUCs (mgkg.sup.-1) and maximum
standardized glucose infusion rate [GIR.sub.max
(mgkg.sup.-1min.sup.-1)] using the corresponding linear mixed
effects model as described for the primary analysis.
[0278] Time to 50%-GIR-AUC (h) and time to GIR.sub.max
[GIR-t.sub.max (h)] have been analyzed non-parametrically.
Performance of Clamp
[0279] Individual profiles of blood glucose concentration have been
plotted.
Analysis of Safety Data
[0280] All summaries of safety data have been based on the safety
population. The individual on-treatment phase for analysis of
safety data have started with the first administration of study
medication and has ended with the EOS visit.
Adverse Events
[0281] All AEs have been coded using MedDRA (version in use).
Definitions
Treatment Emergent AEs
[0282] All AEs have been classified as follows: [0283]
Treatment-emergent AEs (TEAEs): AEs that occurred during the
on-treatment period for the first time or worsened during the
on-treatment period, if present before; [0284]
Non-treatment-emergent AEs (NTEAEs): AEs that occurred outside the
on-treatment period without worsening during the on-treatment
period;
Assignment to Formulations
[0285] For analysis purposes, each TEAE has been assigned to the
last formulation given before onset and/or worsening of the AE. If
a TEAE develops on one formulation and worsens under a later
formulation, it has been considered a TEAE for both
formulations.
Missing Information
[0286] In case of missing or inconsistent information, an AE has
been counted as a TEAE, unless it can clearly be ruled out that it
is not a TEAE (e.g. by partial dates or other information).
[0287] If the start date of an AE is incomplete or missing, it has
been assumed to have occurred after the first administration of
study medication except if an incomplete date indicated that the AE
started prior to treatment.
Treatment-Emergent Adverse Events
[0288] All AEs have been listed individually. They have been
summarized by formulation, including summary by system organ
class.
Deaths, Serious and other significant Adverse Events
[0289] If any such cases, deaths, serious AEs, and other
significant AEs have been listed individually and described in the
study report in detail.
Adverse Events leading to Treatment Discontinuation
[0290] AEs leading to treatment discontinuation have been listed
individually and described in the study report in detail.
Clinical Laboratory Evaluations
[0291] Potentially clinically significant abnormalities (PCSA) and
out-of-range criteria have been defined in the statistical analysis
plan of this study. Definitions of potentially clinically
significant abnormalities (PCSA) and out-of-range definitions have
been reported by parameter.
[0292] Individual data have been listed by subject and by visit, as
well as complementary information.
[0293] Subjects with values out of normal ranges and subjects with
PCSAs have been analyzed by formulation, and overall for end of
study evaluation. Subjects with post-baseline PCSAs have been
listed.
Vital Signs
[0294] Potentially clinically significant abnormalities (PCSA) and
out-of-range criteria have been defined in the statistical analysis
plan of this study. Definitions of PCSA and out-of-range
definitions have been reported by parameter.
[0295] Subjects with PCSAs have been analyzed by formulation, and
overall for end of study evaluation. Subjects with post-baseline
PCSAs have been listed.
[0296] Raw values and derived parameters have been summarized by
formulation, and overall for end of study evaluation. Individual
data have been listed by subject and by visit with flags for
abnormalities, as well as complementary information.
ECG
[0297] Potentially clinically significant abnormalities (PCSA) and
out-of-range criteria have been defined in the statistical analysis
plan of this study. Definitions of PCSA and out-of-range
definitions have been reported by parameter.
[0298] Subjects with PCSAs at end of study have been analyzed
overall. Subjects with post-baseline PCSAs have been listed.
[0299] Raw values and derived parameters at SCR and at EOS have
been summarized overall. Individual data have been listed by
subject and by visit with flags for abnormalities, as well as
complementary information.
Analysis of Pharmacokinetic Data
Pharmacokinetic Parameters
[0300] Actual relative times have been used to derive PK
parameters.
[0301] Primary variable has been [0302]
INS-AUC.sub.(0-24h)(.mu.UhmL.sup.-1)
[0303] Secondary PK variable has been [0304] Time (h) to 50% of
INS-AUC.sub.(0-24h) [T.sub.50%-INS-AUC.sub.(0-24h) (h)]
[0305] The following additional PK variables have been derived:
[0306] Fractional INS-AUCs [INS-AUC.sub.(4-20h),
INS-AUC.sub.(0-12h), INS-AUC.sub.(12-24h) (.mu.UhmL.sup.-1)] [0307]
INS-AUC up to end of clamp [INS-AUC.sub.0-end) (.mu.UhmL.sup.-1)]
[0308] Maximum serum insulin concentration [INS-C.sub.max
(.mu.UmL.sup.-1)] [0309] Time to INS-C.sub.max [INS-T.sub.max
(h)]
Statistical Analysis
Descriptive Analyses
[0310] Descriptive statistics of concentration data have been
presented by protocol times.
[0311] Individual and mean serum insulin concentration profiles
have been plotted.
[0312] Serum insulin concentrations have been individually listed
and descriptive statistics per time point have been generated.
[0313] Descriptive statistics of PK parameters have been generated
by formulation.
[0314] Profiles of C-peptide have been plotted and characterized
descriptively.
Primary Analysis
[0315] To estimate relative bioavailability for
INS-AUC.sub.(0-24h), the log-transformed parameter has been
analyzed with a linear mixed effects model.
[0316] The mixed model included fixed terms for sequence, period,
formulation, and random terms for subject within sequence, with
formulation specific between-subject and within-subject variances
and subject-by-formulation variance.
For INS-AUC.sub.(0-24), point estimate and 90% confidence intervals
for the formulation ratio (T/R) have been obtained by computing
estimates and 90% confidence intervals for the difference between
formulation means within the mixed effects model framework, and
then converting to the ratio scale by the antilog
transformation.
[0317] Equivalent bioavailability has been concluded if the
confidence interval for the formulation ratio has been placed
within [0.80-1.25].
Analyses of Secondary and Additional PK Parameters
[0318] Time to 50%-INS-AUC (h) and time to maximum concentration
[INS-T.sub.max (h)] have been analyzed non-parametrically.
[0319] Log-transformed fractional INS-AUCs and INS-AUC.sub.(0-end)
(.mu.UhmL.sup.-1) and maximum serum insulin glargine concentration
[INS-C.sub.max (.mu.UmL.sup.-1)] have been analyzed with the
corresponding linear mixed effects model as described for the
primary analysis. Point estimators and confidence intervals have
been reported.
C-Peptide
[0320] As available, profiles of C-peptide have been plotted and
characterized descriptively.
PK/PD Analysis
[0321] PK/PD analyses have been performed in an explorative manner,
if appropriate.
Example 6
Study Results
Subject Disposition
[0322] A total of 35 subjects, 11 women and 24 men, were screened
of which 24 healthy eligible subjects were enrolled, randomized and
received at least one dose of study medication. Of the 24
randomized subjects, 1 subject withdrew from the study on own
request after the first dose treatment period. Twenty-three (23)
subjects completed the study according to the protocol and were
included in the pharmacodynamic (PD) and pharmacokinetic (PK)
analyses. All 24 treated subjects were included in the safety
evaluation.
[0323] There were no major protocol deviations.
Demographics Characteristics
[0324] The following data were collected: sex, age at screening,
height, weight, and race. Body mass indexes (BMI) per subject were
calculated from body weight and height data:
BMI=body weight[kg](height[m]).sup.-2.
TABLE-US-00006 TABLE 3 Summary of Subject Characteristics-Safety
Population Sex Statistics/ Male Female All Category (N = 17) (N =
7) (N = 24) Age N 17 7 24 (years) Mean (SD) 34.8 (6.4) 39.1 (5.6)
36.1 (6.3) (Min, Max) (25, 45) (32, 45) (25, 45) Weight N 17 7 24
(kg) Mean (SD) 80.25 (10.42) 64.17 (5.70) 75.56 (11.82) (Min, Max)
(65.9, 101.2) (57.6, 74.2) (57.6, 101.2) Height N 17 7 24 (cm) Mean
(SD) 180.6 (6.0) 166.3 (5.1) 176.4 (8.7) (Min, Max) (171, 189)
(158, 174) (158, 189) BMI N 17 7 24 (kg/m2) Mean (SD) 24.55 (2.40)
23.19 (1.55) 24.15 (2.24) (Min, Max) (20.5, 28.3) (21.4, 24.6)
(20.5, 28.3) Race Black 1 (5.9) 0 (0) 1 (4.2) [n (%)] Caucasian/ 16
(94.1) 7 (100) 23 (95.8) white
Clamp Performance
[0325] The two treatment groups, Lantus U 100 and Lantus U 300,
were similar regarding the individuals' fasting baseline blood
glucose concentrations, which served to define the individuals'
glucose clamp level. The duration of the clamps after dosing was 30
hours and the same in all treatment periods.
Primary Endpoints
[0326] Equivalence in bio-availability (exposure) for Lantus U 100
and Lantus U 300 was not established. Equivalence in bio-efficacy
(activity) for Lantus U 100 and Lantus U 300 was not
established.
Primary Variables
[0327] The area under the serum insulin glargine concentration time
curve from 0 to 24 hours (INS-AUC.sub.(0-24h)) was not equivalent
for Lantus U 100 and Lantus U 300. The exposure was less by about
40% with U300. The area under the GIR versus time curve from 0 to
24 hours (GIR-AUC.sub.(0-24h)) was not equivalent for Lantus U 100
and Lantus U 300. The activity was less by about 40% with U300.
Secondary Variables
[0328] The time to 50% of INS-AUC.sub.(0-24h) (h) was similar for
Lantus U 100 and Lantus U 300. The time to 50% of
GIR-AUC.sub.(0-24h) (h) was greater by 0.545 (h) (0.158-1.030) for
Lantus U 300, which was statistically significant.
Safety
[0329] No serious adverse events (AEs) were reported. Five (5)
subjects per treatment (test and reference) reported a total 14
TEAEs, all of which were of mild to moderate intensity, and
resolved without sequalae. The most frequently reported event was
headache (4 subjects per treatment) followed by nausea, vomiting
and pyrexia (1 subject each on U 100), and procedural pain (1
subject on U 300). Of note, headache is a common observation for
clamp studies and is related to the infusion of hyper-osmolaric
glucose solutions. However, a link to the investigational products
cannot be excluded. No injection site reactions were reported.
Conclusions
[0330] Insulin glargine U 100 and insulin glargine U 300 are not
equivalent in bio-availability (exposure) and bio-efficacy
(activity). Exposure and activity after insulin glargine U300 were
less by about 40% as compared to exposure and activity after
administration of the same amount (0.4 U/kg) from insulin glargine
U100.
[0331] Insulin glargine U300 did, however, show an even flatter PK
(exposure) and PD (activity) profile than insulin glargine U100, as
would be desired for a basal insulin. These surprising and
unexpected differences in exposure and activity between insulin
glargine U100 and insulin glargine U300 formulations after the same
s.c. dose to healthy subjects are effectively shown in the figures
below. Of note, at the same time blood glucose was constant.
[0332] Administration of insulin glargine U 300 was without safety
and tolerability issues.
Example 7
Study Rationale for Study Comparing the Glucodynamic Activity and
Exposure of Three Different Subcutaneous Doses of Insulin Glargine
U300
[0333] Results from the study in healthy subjects (see examples
1-6) showed the inequivalence in exposure and effectiveness between
Lantus.RTM. U100 and insulin glargine U300. Subjects received the
same dose of insulin glargine (0.4 U/kg) for U100 and U300, but
delivery of the same unit-amount from U300 produced about 40% less
exposure and effect than delivery from U100. Insulin glargine U300
did, however, show an even flatter pharmacodynamic profile than
Lantus.RTM. U100, as would be desired for a basal insulin.
[0334] A new study described in the following examples therefore
compares the glucodynamic activity and exposure of three different
subcutaneous doses of insulin glargine U300 versus a standard dose
of Lantus.RTM. U100 as comparator in a euglycemic clamp setting
with type 1 diabetes patients. This study aims to approximate an
U300 dose that is equieffective to 0.4 U/kg Lantus.RTM. U100 as
assessed by parameters of blood glucose disposal provided by the
clamp technique.
[0335] Insulin glargine exposure is assessed from
concentration-time profiles after subcutaneous administration and
activity as glucose utilization per unit insulin.
[0336] The study is designed to assess the metabolic effect and
exposure of different insulin glargine U300 doses compared to a
standard dose of Lantus.RTM. U100 in a euglycemic clamp setting in
subjects with diabetes mellitus type 1. The study comprises 4
treatments (R, T.sub.1, T.sub.2 and T.sub.3), 4 treatment periods
(TP1-4) and 4 sequences. There is one screening visit (D-28 to
D-3), 4 treatment visits (D1 to D2 in TP1 to TP4), and one
end-of-study visit (between D5 to D14 in after last dosing) with
final assessment of safety parameters.
[0337] Subjects are exposed to each treatment R, T.sub.1, T.sub.2
and T.sub.3 once in a cross-over, double-blind and randomized
manner according to a Latin square design. This design is
considered appropriate to evaluate the pharmacological effect and
exposure of different insulin glargine U300 doses compared to
Lantus.RTM. U100.
[0338] The Lantus.RTM. U100 dose of 0.4 U/kg selected for the study
is well characterized to provide euglycemia in type 1 diabetes
patients and has been readily investigated in other clamp studies
with type 1 diabetes patients.
[0339] Three different doses are tested for insulin glargine U300,
0.4, 0.6 and 0.9 U/kg. This dose range allows interpolating an
approximate dose equieffective to 0.4 U/kg Lantus.RTM. U100. The
dose of 0.4 U/kg of insulin glargine U300 has already been tested
in healthy volunteers (see examples 1-6) and was found to be less
active than 0.4 U/kg Lantus.RTM. U100 within 30 hours, the
predefined end of observation period. Bioactivity of 0.4 U/kg
insulin glargine U300 as measured by the total glucose disposition
was 39.4% lower than that of reference medication (0.4 U/kg
Lantus.RTM. U100). A correspondingly higher dose of insulin
glargine U300, e.g. 0.6 U/kg insulin glargine U300, was expected to
result in an approximately equivalent glucodynamic activity
compared to 0.4 U/kg Lantus.RTM. U100. Moreover, the proportional
dose escalation allows exploring exposure and effect profiles for
dose-proportionality.
[0340] A study in patients with type 1 diabetes avoids confounding
impact of endogenous insulin and better permits assessment of
exposure and duration of action. Furthermore, the lack of an assay
specific for insulin glargine forces to use an assay which reads
all endogenous insulin. Thus, any added source of insulin other
than exogenous insulin glargine would cause falsely too high
insulin concentrations.
[0341] This study has a cross over design; for practical and
ethical reasons not more than 3 U300 doses will be compared to
Lantus.RTM. U100. Assessment of glucodynamic activity of long
acting insulin products requires a euglycemic clamp setting for up
to 36 hours owed to the extended duration of action.
[0342] The active pharmaceutical ingredient, insulin glargine, is
the same in both formulations, U100 and U300. The doses used in
this study are within the range of regular use. Although an overall
risk of hypoglycemia is not completely excluded, it is controlled
by the euglycemic clamp technique.
Pharmacodynamics
[0343] The pharmacodynamic activity of insulin glargine is
evaluated by the euglycemic clamp technique in type 1 diabetes
patients, which is the established standard procedure to evaluate
the effect of exogenous administered insulin products on blood
glucose disposal.
[0344] Parameters specific for assessment of glucose disposition in
a euglycemic clamp setting are the body weight standardized glucose
infusion rate (GIR), total glucose disposed, GIR-AUC.sub.0-36, and
times to a given percentage of GIR-AUC.sub.0-36 such as time to 50%
of GIR-AUC.sub.0-36.
[0345] Ancillary parameters are the maximum smoothed body weight
standardized GIR, GIR.sub.max, and Time to GIR.sub.max,
GIR-T.sub.max.
[0346] Duration of action of insulin glargine is derived from the
time between dosing and pre-specified deviations above the
euglycemic (clamp) level.
[0347] Glucose monitoring is performed for 36 hours due to the long
duration of action of insulin glargine after subcutaneous
administration
Pharmacokinetics
[0348] Due to the sustained release nature of insulin glargine
there is a lack of pronounced peaks in the concentration profile.
Therefore, the time to 50% of INS-AUC (T.sub.50% INS-AUC.sub.0-36)
is calculated as a measure for the time location of the insulin
glargine exposure profile, and INS-C.sub.max and INS-T.sub.max will
serve as additional measures.
Primary Study Objectives
[0349] The primary objective of the study is to assess the
metabolic effect ratios of three different insulin glargine U300
doses versus 0.4 U/kg Lantus.RTM. U100.
Secondary Study Objectives
[0350] The secondary objectives of the study are to assess the
exposure ratios of three different insulin glargine U300 doses
versus 0.4 U/kg Lantus.RTM. U100, to compare the duration of action
of different insulin glargine U300 doses versus 0.4 U/kg
Lantus.RTM. U100, to explore the dose response and dose exposure
relationship of insulin glargine U300, and to asses the safety and
tolerability of insulin glargine U300 in subjects with type 1
diabetes.
Example 8
Study Design, Description of the Protocol
[0351] Phase I, single-center, double-blind, randomized, cross-over
(4 treatments, 4 treatment periods and 4 sequences; Latin square),
active control, with a wash-out duration between treatment periods
(5-18 days, preferred 7 days) in male and female subjects with type
1 diabetes mellitus receiving single-doses of insulin glargine at
[0352] 0.4 U/kg Lantus U100 (=Reference R) [0353] 0.4 U/kg Insulin
glargine U300 (=Test T.sub.1) [0354] 0.6 U/kg Insulin glargine U300
(=Test T.sub.2) [0355] 0.9 U/kg Insulin glargine U300 (=Test
T.sub.3)
[0356] The four treatments R and T.sub.1-3 are given cross-over in
four treatment periods (TP 1 to TP 4) with the four-sequences
[0357] R-T.sub.1-T.sub.2-T.sub.3 [0358] T.sub.3-R-T.sub.1-T.sub.2
[0359] T.sub.2-T.sub.3-R-T.sub.1 [0360] T.sub.1-T.sub.2-T.sub.3-R
randomly assigned to the subjects (1:1:1:1 ratio).
Duration of Study Participation
[0360] [0361] Total study duration for one subject: about 4-11
weeks (min-max duration, depending on wash-out period, excl.
screening) [0362] Duration of each part of the study for one
subject: [0363] Screening: 3 to 28 days (D-28 to D-3) [0364]
Treatment Period 1-4: 2 days (1 overnight stay) [0365] Washout:
5-18 days (preferentially 7 days between consecutive dosings)
[0366] End-of-study visit: 1 day between D5 and D14 after last
study drug administration
Example 9
Selection of Subjects
[0367] Number of subjects planned: At least 24 subjects are to be
enrolled to have 20 evaluable subjects.
Inclusion Criteria
Demography
[0368] I 01. Male or female subjects, between 18 and 65 years of
age, inclusive, with diabetes mellitus type 1 for more than one
year, as defined by the American Diabetes Association (American
Diabetic Association. Report of the Expert Committee on the
Diagnosis and Classification of Diabetes Mellitus. Diabetes Care
1998; 21:5-19) [0369] I 02. Total insulin dose of <1.2 U/kg/day
[0370] I 03. Body weight between 50.0 kg and 95.0 kg inclusive if
male, between 50.0 kg and 85.0 kg inclusive if female, Body Mass
Index between 18.0 and 30.0 kg/m.sup.2 inclusive
Health Status
[0370] [0371] I 04. Fasting negative serum C-peptide (<0.3
nmol/L) [0372] I 05. Glycohemoglobin (HbA1c).ltoreq.9.0% [0373] I
06. Stable insulin regimen for at least 2 months prior to study
(with respect to safety of the subject and scientific integrity of
the study) [0374] I 07. Normal findings in medical history and
physical examination (cardiovascular system, chest and lungs,
thyroid, abdomen, nervous system, skin and mucosae, and
musculo-skeletal system), unless the investigator considers any
abnormality to be clinically irrelevant and not interfering with
the conduct of the study (with respect to safety of the subject and
scientific integrity of the study) [0375] I 08. Normal vital signs
after 10 minutes resting in the supine position: 95
mmHg<systolic blood pressure<140 mmHg; 45 mmHg<diastolic
blood pressure<90 mmHg; 40 bpm<heart rate<100 bpm [0376] I
09. Normal standard 12-lead ECG after 10 minutes resting in the
supine position; 120 ms<PQ<220 ms, QRS<120 ms,
QTc.ltoreq.440 ms if male, .ltoreq.450 ms if female [0377] I 10.
Laboratory parameters within the normal range (or defined screening
threshold for the Investigator site), unless the Investigator
considers an abnormality to be clinically irrelevant for diabetes
patients; however serum creatinine should be strictly below the
upper laboratory norm; hepatic enzymes (AST, ALT) and bilirubin
(unless the subject has documented Gilbert syndrome) should be not
above 1.5 ULN
Female Subjects Only
[0377] [0378] I 11. Women of childbearing potential (less than two
years post-menopausal or not surgically sterile for more than 3
months), must have a negative serum .beta.-HCG pregnancy test at
screening and a negative urine .beta.-HCG pregnancy test at Day 1
on TP1 to TP4 and must use a highly effective method of birth
control, which is defined as those which result in a low failure
rate (i.e. less than 1% per year) according to the Note for
guidance on non-clinical safety studies for the conduct of human
clinical trials for pharmaceuticals (CPMP/ICH/286/95,
modifications). During the entire study female subjects of child
bearing potential must use two independent methods of
contraception, e.g. diaphragm and spermicide-coated condom. The use
of a condom and spermicidal creams is not sufficiently reliable.
[0379] For postmenopausal women with presence of less than two
years post-menopausal, and not surgically sterile for more than 3
months, the hormonal status will be determined (FSH>30 IU/L,
estradiol<20 pg/mL)
Exclusion Criteria
Medical History and Clinical Status
[0379] [0380] E 01. Any history or presence of clinically relevant
cardiovascular, pulmonary, gastro-intestinal, hepatic, renal,
metabolic (apart from diabetes mellitus type 1), hematological,
neurological, psychiatric, systemic (affecting the body as a
whole), ocular, gynecologic (if female), or infectious disease; any
acute infectious disease or signs of acute illness [0381] E 02.
More than one episode of severe hypoglycemia with seizure, coma or
requiring assistance of another person during the past 6 months
[0382] E 03. Frequent severe headaches and/or migraine, recurrent
nausea and/or vomiting (more than twice a month) [0383] E 04. Blood
loss (>300 ml) within 3 months before inclusion [0384] E 05.
Symptomatic hypotension (whatever the decrease in blood pressure),
or asymptomatic postural hypotension defined by a decrease in SBP
equal to or greater than 20 mmHg within three minutes when changing
from the supine to the standing position [0385] E 06. Presence or
history of a drug allergy or clinically significant allergic
disease according to the Investigator's judgment [0386] E 07.
Likelihood of requiring treatment during the study period with
drugs not permitted by the clinical study protocol [0387] E 08.
Participation in a trial with any investigational drug during the
past three months [0388] E 09. Symptoms of a clinically significant
illness in the 3 months before the study, which, according to the
investigator's opinion, could interfere with the purposes of the
study [0389] E 10. Presence of drug or alcohol abuse (alcohol
consumption >40 grams/day) [0390] E 11. Smoking more than 5
cigarettes or equivalent per day, unable to refrain from smoking
during the study [0391] E 12. Excessive consumption of beverages
with xanthine bases (>4 cups or glasses/day) [0392] E 13. If
female, pregnancy (defined as positive .beta.-HCG test),
breast-feeding
Interfering Substance
[0392] [0393] E 14. Any medication (including St John's Wort)
within 14 days before inclusion, or within 5 times the elimination
half-life or pharmacodynamic half-life of that drug, whichever the
longest and regular use of any medication other than insulins in
the last month before study start with the exception of thyroid
hormones, lipid-lowering and antihypertensive drugs, and, if
female, with the exception of hormonal contraception or menopausal
hormone replacement therapy; any vaccination within the last 28
days
General Conditions
[0393] [0394] E 15. Subject who, in the judgment of the
Investigator, is likely to be non-compliant during the study, or
unable to cooperate because of a language problem or poor mental
development [0395] E 16. Subject in exclusion period of a previous
study according to applicable regulations [0396] E 17. Subject who
cannot be contacted in case of emergency [0397] E 18. Subject is
the investigator or any sub-investigator, research assistant,
pharmacist, study coordinator, or other staff thereof, directly
involved in the conduct of the protocol
Biological Status
[0397] [0398] E 19. Positive reaction to any of the following
tests: hepatitis B surface (HBs Ag) antigen, anti-hepatitis B core
antibodies (anti-HBc Ab) if compound having possible immune
activities, anti-hepatitis C virus (anti-HCV2) antibodies,
anti-human immunodeficiency virus 1 and 2 antibodies (anti-HIV1 and
anti HIV2 Ab) [0399] E 20. Positive results on urine drug screen
(amphetamines/methamphetamines, barbiturates, benzodiazepines,
cannabinoids, cocaine, opiates) [0400] E 21. Positive alcohol
test
Specific to the Study
[0400] [0401] E 22. Known hypersensitivity to insulin glargine and
excipients [0402] E 23. Any history or presence of deep leg vein
thrombosis or a frequent appearance of deep leg vein thrombosis in
first degree relatives (parents, siblings or children)
Example 10
Treatments
Investigational Product
[0402] [0403] Insulin glargine [0404] Two different formulations of
insulin glargine are used: [0405] Lantus.RTM. U100 solution for
injection containing 100 U/mL insulin glargine (marketed product)
[0406] Insulin glargine U300 solution for injection containing 300
U/mL insulin glargine [0407] Dose: [0408] Lantus.RTM. U100:0.4 U/kg
(=Reference R) [0409] Insulin glargine U300: 0.4, 0.6 and 0.9 U/kg
(=Test T.sub.1-T.sub.3) [0410] Container: 3 mL glass cartridges
[0411] Route of application: Subcutaneously horizontally 5 cm right
and left of the umbilicus [0412] Conditions: Fasted [0413] Duration
of treatment: 1 day at each period, single dose [0414] Start: 09:00
on Day 1 (D1) in Treatment Periods 1 to 4 (TP1-4) [0415] Additional
treatments for 100% of included subjects are provided
TABLE-US-00007 [0415] TABLE 4 Treatments Reference treatment Test
treatment Lantus .RTM. U100 Insulin glargine U300 INN Insulin
glargine Insulin glargine (recombinant (recombinant human insulin
human insulin analogue) analogue) Formulation Cartridges for 3 mL
Cartridges for 3 mL solution U100 solution U300 1 mL contains: 1 mL
contains: 3.637 mg 21A-Gly-30Ba-L- 10.913 mg 21A-Gly-30Ba-
Arg-30Bb-L-Arg human L- Arg-30Bb-L-Arg human insulin [equimolar to
insulin [equimolar to 100 IU human insulin] 300 IU human insulin]
30 .mu.g zinc 90 .mu.g zinc 2.7 mg m-cresol 2.7 mg m-cresol 20 mg
glycerol 85% 20 mg glycerol 85% HCl and NaOH, pH 4.0 HCl and NaOH,
pH 4.0 specific gravity 1.004 g/mL specific gravity 1.006 g/mL Dose
0.4 U/kg 0.4 U/kg 0.6 U/kg 0.9 U/kg Manufacturer sanofi-aventis
sanofi-aventis Deutschland GmbH Recherche & Development,
Montpelier, France Batch commercial formulation, tbd number
purchased through CRO INN = international nonproprietary name
Dosing
[0416] This is a single dose study with in total 4 administrations
of study medication. Subjects are randomized to different sequences
of the reference and test treatment such that each subject receives
the reference treatment (R) and each of the test treatments
(T.sub.1-3) once.
[0417] Injections are given left or right of the umbilicus, with
both sites being used for separate injections. A washout period of
5 to 18 days separates consecutive dosing days, the preference is 7
days (7 days between consecutive dosing). The length of the
wash-out period varies individually allowing both the participant
and the investigator to adjust to their needs. By experience, 5
days comprise a minimum period for recovery enabling 1 clamp per
week for a participant, while 18 days represent a break of 3 weeks
between dosing days, allowing subjects the freedom to fulfill
non-study related obligations, if unavoidable.
[0418] IP administration is administered under fasting conditions;
subject continues to fasten throughout the whole clamp period.
[0419] The blood glucose concentration is within a range of 5.5
mmol/L (100 mg/dL).+-.20% without any glucose infusion for the last
hour prior to dosing during pre-clamp. When blood glucose has been
stable for at least 1 hour without any glucose infusion, IP is
administered. IP administration does not occur earlier than 09:00
clock time in the morning and not later than 14:00 clock time on
Day 1 in Treatment Periods 1 to 4. If blood glucose is not
stabilized before 14:00 hours, dosing does not occur. The visit is
terminated and the subject is scheduled for a new dosing visit 1-7
days later.
[0420] Per subject and dosing a new cartridge is used.
[0421] IP administration is done by a person who is not otherwise
involved in the study or part of the study team at the CRO. This
person gets the random code to prepare IP administration in
accordance to the open random list and doses subjects accordingly.
The preparation and dosing is followed and checked by a second
independent person. Respective documents of dose preparation and
treatment sequence is kept strictly confidential and is not being
disclosed to any other person.
Calculation of Dose of Ip (Insulin Glargine)
[0422] To calculate the amount of insulin glargine given for each
subject, the body weight (in kg) is determined to one decimal place
and the amount of insulin calculated is rounded up or down to
integer numbers as shown in the following examples for a dose of
0.6 U/kg insulin glargine: [0423] a subject with a body weight of
75.3 kg receives 45 U insulin (75.3.times.0.6=45.18 which is
rounded down to 45); [0424] a subject with a body weight of 74.4 kg
receives 45 U insulin (74.4.times.0.6=44.64, which is rounded up to
45).
[0425] The body weight recorded during TP1D1 is used for
calculation of study medication dose for all treatment periods. The
study medication dose is not to be changed if a subject's weight
changes by less than or equal to 2 kg between TP1 and one of the
subsequent TPs. If a subject's body weight changes by more than 2
kg between TP1 and one of the subsequent TPs, the study medication
dose is re-calculated based on the weight at D1 of the respective
treatment period.
Syringes and Needles
[0426] Syringes with needles attached appropriate to accurately
administer small amounts of injection solution are used only (e.g.
Becton Dickinson, Ref 305502, Dimensions: 1 mL 27G 3/8
0.40.times.10). The syringes are supplied by the investigator.
Other Products
[0427] Other products used during the clamp procedure are described
in Table 5.
TABLE-US-00008 TABLE 5 Preparation of infusion Drug Dose/Routeof
Code INN Formulation Manufacturer administration Glucose Glucose
20% solution Certified, iv infusion for infusion selected by PROFIL
Intramed Heparin Vial containing Certified, iv infusion Heparin 5
mL solution selected by Sodium (5000 IU/mL) PROFIL 0.9% Sodium
Solution Certified, iv infusion Sodium Chloride selected by
Chloride PROFIL Apidra .RTM. Insulin 100 U/mL for sanofi- iv
infusion glulisine injection aventis
[0428] Glucose solution, sodium chloride solution, heparin and
insulin glulisine is provided by the Investigator.
[0429] Glucose solution: 20% glucose solution is infused with the
Biostator.TM. to keep subjects individual blood glucose at the
determined target level. A second infusion pump (part of the
Biostator.TM.) delivers 0.9% sodium chloride solution to keep the
line patent. In case the amount of 20% glucose solution needed
exceeds the infusion capacity of the Biostator.TM., a second
glucose infusion pump is engaged.
[0430] Heparin: A low dose heparin solution (10.000 Units
heparin/100 mL saline) is infused via a double lumen catheter. The
heparin solution is taken up together with blood used for the
Biostator's.TM. blood glucose measurement in the other lumen of the
catheter and is aimed to prevent blood clotting in the system.
[0431] Insulin glulisine: 15 U Apidra.RTM. [100 U/mL] is given to
49 mL of saline solution, to which 1 mL of the subject's own blood
is added to prevent adhesion, producing a concentration of 0.3
U/mL, which is infused at an individual rate to achieve
euglycemia.
Description of Blinding Methods
[0432] Subjects receive four different treatments (R, T.sub.1,
T.sub.2 and T.sub.3) in a randomized, blinded and crossover
design.
[0433] In order to maintain the blinding, a third party un-blinded
person is involved for IP dispensing and administration. This
person is not otherwise involved in the study and/or part of the
study team at the CRO, does not disclose any information to anyone
and ensures to maintain blinding condition of the study. He/she
gets the random code and doses subjects accordingly. The
preparation of IP and dosing is followed and checked by a second
independent person who has also access to the random code but is
equally bound to confidentiality.
Method of Assigning Subjects to Treatment Group
[0434] IPs are administered according to the Clinical Study
Protocol only to subjects who have given written informed
consent.
[0435] Subjects who comply with all inclusion/exclusion criteria
are assigned just before the Investigational Product administration
on Day 1 in Treatment Period 1: [0436] an incremental subject
number according to the chronological order of inclusion on the
morning of D1 in Treatment Period 1. The 9 digit subject number
consists of 3 components (e.g. 276 001 001, 276 001 002, 276 001
003, etc.), of which the first 3 digits (276) are the country
number, the middle 3 digits are the site number and the last 3
digits are the subject incremental number within the site. The
subject number remains unchanged and allows the subject to be
identified during the whole study [0437] a treatment number in a
pre-planned order following the randomized list with the next
eligible subject always receiving the next treatment number
according to the randomization list
[0438] IP administration is in accordance with the randomized
treatment sequence.
[0439] Subjects withdrawn from the study retain their subject
number and their treatment number, if already assigned. Replacement
subjects have a different identification number (i.e., 500+the
number of the subject who discontinued the study). Each subject
receives the same treatment sequence as the subject, who
discontinued the trial
[0440] Screen Failed subjects are assigned a different number,
e.g., 901, 902 (to be recorded in the CRF only in case of AE
occurring during screening period after signing of informed
consent).
[0441] Notes: The randomization of a subject occurs after
Investigators confirmation of subject's eligibility for this study.
Baseline parameters are the parameters available the closest before
the dosing.
Packaging and Labeling
[0442] Insulin glargine U300 solution is provided by sanofi-aventis
in regrouping boxes of 3 mL cartridges.
[0443] The respective number of IP is packaged under the
responsibility of sanofi-aventis according to good manufacturing
practice and local regulatory requirement and provided to CRO.
[0444] The content of the labeling is in accordance with the local
regulatory specifications and requirements.
[0445] Lantus.RTM. U100 is commercially available and will be
ordered by the CRO.
Storage Conditions
[0446] All IP is stored in an appropriate locked room under the
responsibility of the Investigator, and must be accessible to
authorized personnel only.
[0447] The IP has to be stored at .+-.2.degree. C. to .+-.8.degree.
C., protected from light, and must not be frozen.
Access to the Randomization Code During the Study
[0448] In order to maintain the blinding, a third party un-blinded
person is responsible for IP dispensing and administration. This
person is not otherwise involved in the study and/or part of the
study team at the CRO, does not disclose any information to anyone
and ensures to maintain blinding condition of the study. He/she
gets the random code and doses subjects accordingly. The
preparation of IP and dosing is followed and checked by a second
independent person who has also access to the random code but is
equally bound to confidentiality.
[0449] In case of an Adverse Event, the code is not being broken
except in the circumstances when knowledge of the Investigational
Product is essential for treating the subject. For each subject,
code-breaking material which contains the name of the treatment is
supplied as envelopes. It is kept in a safe place on site
throughout the Clinical Trial. The Sponsor retrieves all
code-breaking material (opened or sealed) on completion of the
Clinical Trial.
[0450] If the blind is broken, the Investigator documents the date
of opening and reason for code breaking in the source data.
[0451] The Investigator, the clinical site pharmacist, or other
personnel allowed to store and dispense IP is responsible for
ensuring that the IP used in the study is securely maintained as
specified by the Sponsor and in accordance with the applicable
regulatory requirements.
[0452] All IP is dispensed in accordance with the Clinical Trial
Protocol and it is the Investigator's responsibility to ensure that
an accurate record of IP issued and returned is maintained.
Concomitant Treatment
[0453] The use of concomitant medication is not allowed during the
study as specified in Exclusion Criteria No. E14, with the
exception of drugs mentioned there under, and is stopped within a
given time frame (see E14) before inclusion of the subject on Day 1
of Treatment Period 1.
[0454] To prevent interference of subjects' standard insulin
treatment with the clamp measurement, subjects have to abstain from
using basal insulins and switch to [0455] intermediate- or
short-acting insulin products from 48 hours prior to dosing at D1
of TP1 to TP4, if on long-acting insulin products, i.e. Lantus.RTM.
(insulin glargine), Levemir.RTM. (detemir) or ultralente insulins,
[0456] short-acting insulins from 24 hours prior to dosing at D1 of
TP1 to TP4 if on intermediate acting insulin products, i.e.
NPH-insulin
[0457] The last subcutaneous injection of short-acting insulin is
no later than 9 hours before study drug administration. Subjects on
pump therapy discontinues the insulin infusion in the morning of
Day 1, at least 6 hours prior to each IP administration (around
03:00 clock time assuming start of IP administration at 09:00).
[0458] For symptomatic adverse events which are not jeopardizing
the subjects' safety (e.g. headache) concomitant medication is
reserved for adverse events of severe intensity or of moderate
intensity which persist for a long duration. In particular, the use
of acetaminophen/paracematol is prohibited if there is a known risk
of hepatotoxicity, or as soon as abnormalities of liver enzymes
occur.
[0459] However, if a specific treatment is required for any reason,
an accurate record must be kept on the appropriate record form,
including the name of the medication (international nonproprietary
name), daily dosage and duration for such use. The Sponsor must be
informed within 48 h via e-mail or fax, with the exception of
treatment of headache.
[0460] Treatment of potential allergic reactions will be in
compliance with the recommendations as published elsewhere (Samspon
H A, Munoz-Furlong A, Campbell R L et al. Second symposium on the
definition and management of anaphylaxis: summary report--Second
National Institute of Allergy and Infectious Disease/Food Allergy
and Anaphylaxis Network symposium. Journal of Allergy and Clinical
Immunology 2006; 117(2):391-397). Dependent on the severity of the
allergic reaction treatment with antihistamines, corticosteroids
and epinephrine may be considered.
Treatment Accountability and Compliance
[0461] IP compliance: [0462] IP is administered under direct
medical supervision, and an appropriate record is completed by the
person responsible for dispensing and administration of IP or
his/her delegate; any information on treatment sequence or dose is
not disclosed and documents are locked with no access by other
persons involved in the study [0463] IP intake is confirmed by
measurable drug assay results [0464] IP accountability: [0465] The
person responsible for dispensing and administration of IP or
his/her delegate counts the number of cartridges remaining in the
returned packs, then fills in the Treatment Log Form [0466] The
Investigator records the information about day and time of dosing
on the appropriate page(s) of the Case Report Form (CRF) [0467] The
Monitor Team in charge of the study then checks the CRF data by
comparing them with the IP and appropriate accountability forms
after data base lock (to prevent unblinding of the study)
[0468] Used cartridges are kept by the Investigator up to the fully
documented reconciliation performed with the Sponsor at the end of
the study after data base lock.
Example 11
Assessment of Investigational Product
[0469] The present study is designed to assess the metabolic effect
and exposure ratios of three different insulin glargine U300 doses
versus 0.4 U/kg Lantus.RTM. U100, to compare the duration of action
of different insulin glargine U300 doses versus 0.4 U/kg
Lantus.RTM. U100, to explore the dose response and dose exposure
relationship of insulin glargine U300, and to asses--the safety and
tolerability of insulin glargine U300 in an euglycemic clamp
setting in subjects with diabetes mellitus type 1.
Pharmacodynamics
Euglycaemic Clamp
[0470] The pharmacodynamic effect of insulin glargine, mainly the
total glucose disposal and duration of insulin action, is evaluated
by the euglycemic clamp technique.
[0471] During the euglycemic clamp, arterialized venous blood
glucose concentration, which reflects the supply for total glucose
utilization of all tissues, and the glucose infusion rate (GIR)
needed to keep a subject's blood glucose concentration at its
target level (clamp level) is continuously measured and recorded
using the Biostator.TM. device (continuous glucose monitoring
system, Life Sciences Instruments, Elkhart, Ind., USA).
[0472] The amount of glucose required (GIR-AUC) is a measure of the
glucose uptake into tissues (glucose disposal or glucose lowering
activity) mediated by the exogenous insulin excess. The
Biostator.TM. determines blood glucose levels in 1 min intervals
and adjusts the glucose infusion rate in response to changes in
blood glucose using a predefined algorithm.
Clamp Procedure
[0473] To prevent interference of subjects' standard insulin
treatment with the clamp measurement, subjects have to abstain from
using basal insulins and switch to [0474] intermediate- or
short-acting insulin products from 48 hours prior to dosing at D1
of TP1 to TP4, if on long-acting insulin products, i.e. Lantus.RTM.
(insulin glargine), Levemir.RTM. (detemir) or ultralente insulins,
[0475] short-acting insulins from 24 hours prior to dosing at D1 of
TP1 to TP4 if on intermediate acting insulin products, i.e.
NPH-insulin
[0476] The last subcutaneous injection of short-acting insulin is
no later than 9 hours before IP administration. Subjects on pump
therapy discontinue the insulin infusion in the morning of Day 1,
at least 6 hours prior to each IP administration (around 03:00
clock time assuming start of IP administration at 09:00).
[0477] During Treatment Periods 1 to 4 (TP1-TP4), subjects are
admitted to the clinic in the morning of D1 after an overnight fast
of at least 10 hours.
[0478] In the morning of Day 1 the pre-clamp procedure starts and
subjects are linked to the Biostator.TM.. Blood glucose
concentration is adjusted to 4.4-6.6 mmol/L (80-120 mg/dL) and
maintained within these limits by means of iv bolus-administrations
of a rapid acting insulin analog (e.g. insulin glulisine) and
subsequent individual infusions of glucose as needed.
[0479] 60 min before study medication administration blood glucose
is then adjusted to 5.5 mmol/L (100 mg/dL).+-.20% (euglycemic clamp
level) without any glucose infusion for the last hour prior to
dosing. The insulin glulisine infusion is discontinued immediately
prior to the administration of the study medication.
[0480] When blood glucose has been stable for at least 1 hour
within a range of 5.5 mmol/L (100 mg/dL).+-.20% without any glucose
infusion, IP is administered (=TO on D1 in TP1 to TP4, around
09:00). Subjects receive reference or test medication (R,
T.sub.1-3, see Table 4) as assigned by randomization. Injections is
given left or right of the umbilicus. IP administration does not
occur earlier than 09:00 clock time in the morning and not later
than 14:00 clock time on Day 1 in Treatment Periods 1 to 4. If
blood glucose is not stabilized during pre-clamp before 14:00 clock
time, dosing does not occur. The visit is terminated and the
subject is scheduled for a new dosing visit 1-7 days later.
[0481] IP administration is administered under fasting conditions;
subject continues to fasten throughout the whole clamp period.
[0482] The euglycemic clamp blood glucose level is continuously
maintained by means of iv infusion of glucose solution until clamp
end.
[0483] The goal of any basal insulin supplementation is to add to
or even to substitute endogenous insulin secretion between meals.
In subjects without endogenous insulin secretion, as invited to
participate in this study, exogenous insulin should provide for
just the amount of insulin required to dispose hepatic glucose
production. If perfectly matched, there is no need for extra
glucose to compensate for excess insulin. The resulting glucose
infusion rate approximates zero. Once insulin action ceases, blood
glucose concentration rises. The times to onset of rise and to
times blood glucose concentrations exceeding predefined thresholds
are read by the Biostator.TM..
[0484] Selected doses of Lantus.RTM. U100 and insulin glargine U300
are above the average basal need which in turn produce some glucose
demand reflected in a sizeable GIR up to 36 hours.
[0485] The corresponding parameter indicative of the clamp
performance, i.e. the precision for keeping blood glucose at clamp
baseline level, is the blood glucose variability over the clamp
period. A measure for blood glucose variability is the coefficient
of variation (CV %) per individual clamp.
[0486] A low coefficient of variation in blood glucose is a
prerequisite to properly assess the insulin effect in clamp
settings.
[0487] The clamp period is not to exceed 36 hours post study
medication injection, the predefined clamp end.
[0488] Subjects continue fasting during the whole glucose clamp
(pre-clamp and clamp) period while having access to water ad
libitum.
[0489] In case blood glucose passes 11.1 mmol/L (200 mg/dL) prior
to the clamp end for 30 minutes after cessation of glucose infusion
and the investigator confirms that any possible errors leading to
false blood glucose levels above 11.1 mmol/L (200 mg/dL) have been
excluded, insulin glulisine used in the pre-IP administration time
of the clamp is given to extend the observation period to 36 hours.
In that case, the sponsor has to be informed.
[0490] The subjects are delinked from the clamp setting when blood
glucose is well within the isoglycemic range.
[0491] Participants resume their pre-study medication on the day of
discharge at TP1 to TP4, i.e. Day 2.
[0492] The effect of the IPs is to last about 24-36 hours, which is
why the participants is confined to the institute for 2 days.
[0493] A washout period of 5 to 18 days separates consecutive clamp
period days, the preference is 7 days (7 days between consecutive
dosing). The length of the wash-out period varies individually
allowing both the participant and the investigator to adjust to
their needs. By experience, 5 days comprise a minimum period for
recovery enabling 1 clamp per week for a participant, while 18 days
represent a break of 3 weeks between dosing days, allowing subjects
the freedom to fulfill non-study related obligations, if
unavoidable.
[0494] Screening and D1 of TP1 is not separated by more than 28
days, while the EOS occurs no earlier than D5 or no later than D14
after last dosing, respectively.
Pharmacodynamic Sampling Times
[0495] Arterialized venous blood is continuously drawn at a rate of
2 mL/h for determination of arterial blood glucose concentration
every minute during pre-clamp (prior to IP administration) and
clamp period (up to 36 hours after IP administration).
[0496] Arterialized venous blood samples (0.2 mL) for concurrent
Biostator.TM. calibration, which is a technical requirement, is
collected at least in 30 minute intervals after connection to the
Biostator.TM. up to 36 hours after medication.
Number of Pharmacodynamic Samples
[0497] Blood glucose is continuously measured during the clamp
procedure. In addition, at least 74 samples per subject and
treatment period will be collected for calibration of the
Biostator.TM. after IP administration. In total 74*4*24 samples or
7104 samples are collected (see table below).
TABLE-US-00009 TABLE 6 Number of blood samples and aliquots per
subject during clamp Periods Glucose .sup.a Glucose .sup.b TP1
Continuously 74 TP2 Continuously 74 TP3 Continuously 74 TP4
Continuously 74 Total number of Continuously 296 samples per
subject .sup.a continuous glucose monitoring at 2 mL/h for PD
.sup.b calibration
Pharmacodynamic Handling Procedure
TABLE-US-00010 [0498] TABLE 7 Sample Handling Procedures Blood
Sample Analyte Volume Handling Procedures Glucose for PD 2 mL/h
none Glucose for 200 .mu.L Blood to be filled into calibration
capillary and then into sample cup for immediate analysis
Pharmacodynamic Parameters
[0499] The area under the body weight standardized GIR within 36
hours (GIR-AUC.sub.0-36) and the time to 50% of the total GIR-AUC
within 36 hours (T.sub.50%-GIR-AUC.sub.0-36) is calculated.
[0500] Duration of blood glucose control is taken as the time in
euglycemia from dosing to deviation above clamp glucose level (100
mg/dL). Times of controlled blood glucose within predefined margins
is taken from dosing to specified thresholds, e.g. blood glucose
levels at 110, 130 and 150 mg/dL.
[0501] In addition, the maximum smoothed body weight corrected GIR
(GIR.sub.max) and the time to GIR.sub.max, GIR-T.sub.max, is
assessed.
[0502] Further supplemental parameters is derived as
appropriate.
Safety
Baseline Demographic Characteristics
[0503] The baseline demographic characteristics consists of: [0504]
Age (years) [0505] Body weight (kg) [0506] Height (cm) [0507] Body
Mass Index (BMI) (kg/m.sup.2)
Safety Assessment at Baseline and During the Study
[0507] [0508] Physical examination at screening: cardiovascular
system, chest and lungs, thyroid, abdomen, nervous system, skin and
mucosae, and musculo-skeletal system and relevant medical and
surgical history, diabetes history (diagnosis of diabetes, onset of
insulin treatment, late complications); only findings relevant to
the study are documented [0509] Past and current smoking status
[0510] Physical examination at pre-dose and during the study:
cardiovascular system, abdomen and lungs; only findings relevant to
the study are documented [0511] Body temperature (aural) [0512]
Vital signs: Heart rate, respiratory rate and systolic and
diastolic blood pressure measured after 10 minutes in supine
resting position, heart rate and systolic and diastolic blood
pressure-also after 3 minutes in standing position (except for
unscheduled measurements when connected to Biostator.TM.)
[0513] Laboratory tests (in fasted conditions for blood samples):
[0514] Hematology: Red blood cell count (RBC), hematocrit (Hct),
hemoglobin (Hb), white blood cell count (WBC) with differential
(neutrophils, eosinophils, basophils, monocytes and lymphocytes),
platelets, INR and aPTT [0515] Biochemistry: [0516] Electrolytes:
Sodium, potassium, bicarbonate, chloride, calcium [0517] Liver
function: AST, ALT, alkaline phosphatase, gamma-glutamyl
transferase (.gamma.GT), total and conjugated bilirubin [0518]
Renal function: creatinine, BUN [0519] Metabolism: Glucose,
albumin, total proteins, total cholesterol, triglycerides, HbA1c
(at screening, D1 TP1 and EOS), LDH, amylase, lipase, C-peptide
(screening only) [0520] Potential muscle toxicity: Creatinine
phosphokinase (CPK) [0521] Serology: Hepatitis B antigen (HBs Ag),
anti-hepatitis B core antibodies (anti-HBc Ab), anti-hepatitis C
antibodies (anti-HCV2), anti-HIV1 and anti-HIV2 antibodies [0522]
Archival blood sample: a 5 mL blood sample is collected into a dry,
red topped tube, centrifuged at approximately 1500 g for 10 minutes
at 4.degree. C.; the serum is then transferred into three storage
tubes, which are immediately capped and frozen in an upright
position at -20.degree. C. This sample is used if any unexpected
safety issue occurs to ensure that a pre drug baseline value is
available for previously non-assessed parameters (e.g., serology).
If this sample is not used, the Investigator destroys it after the
Sponsor's approval [0523] Urinalysis: Proteins, glucose, blood,
ketone bodies, pH [0524] Qualitative: A dipstick is performed on a
freshly voided specimen for qualitative detection using a reagent
strip; [0525] Quantitative: A quantitative measurement for glucose,
protein, erythrocytes and leucocytes count is required in the event
that the urine sample test is positive for any of the above
parameters by urine dipstick (e.g., to confirm any positive
dipstick parameter by a quantitative measurement). [0526] Urine
drug screen: Amphetamines/metamphetamines, barbiturates,
benzodiazepines, cannabinoids, cocaine, opiates [0527] Alcohol
breath test [0528] Pregnancy/hormone test (if female): [0529]
.beta.-HCG in blood at screening [0530] urine .beta.-HCG at TP1 to
TP4, Day 1 [0531] FSH/estradiol, if postmenopausal less than 2
years, at screening only [0532] Adverse Events: Spontaneously
reported by the subject or observed by the Investigator [0533] ECG
telemetry (single lead) [0534] 12-lead ECG (automatic) [0535]
Anti-insulin antibodies
[0536] Blood samples for laboratory tests are taken under fasted
conditions.
ECG Methodology
ECG Telemetry
[0537] ECG telemetry is continuously monitored by medical
personnel. All arrhythmic events will be documented by printing and
included in the subject's CRF. This documentation allows for
diagnosis of the event, time of occurrence, and duration, and is
signed by the Investigator or delegate. The ECG telemetry records
is kept for a potential re-analyze taking account the
Investigational Product exposure.
Twelve-Lead ECGs
[0537] [0538] Twelve-lead ECGs are recorded after at least 10
minutes in supine position using an electrocardiographic device
(MAC 5500.TM.). The electrodes are positioned at the same place for
each ECG recording throughout the study (attachment sites of the
leads are marked with an indelible pen). [0539] ECGs is always
recorded before the PK sampling (if any). PK samples are drawn as
soon as possible (within 15 minutes) after ECG. [0540] Each ECG
consists of a 10 second recording of the 12 leads simultaneously,
leading to: [0541] a single 12-lead ECG (25 mm/s, 10 mm/mV)
print-out with HR, PR, QRS, QT, QTc automatic correction
evaluation, including date, time, initials and number of the
subject, signature of the investigator, and at least 3 complexes
for each lead. The Investigator medical opinion and automatic
values is recorded in the CRF. This print-out is retained at the
site level [0542] a digital storage that enables eventual further
reading by an ECG central lab: each digital file is identified by
theoretical time (day and time DxxTxxHxx), real date and real time
(recorder time), Sponsor study code, subject number (i.e., 3
digits) and site and country numbers if relevant. [0543] The
digital recording, data storage and transmission (whenever
requested) comply with all the applicable regulatory requirements
(i.e., FDA 21 CFR, part 11).
[0544] When vital signs, ECG, and blood samples are scheduled at
the same time as an Investigational Product administration and/or a
meal, they are done prior to Investigational Product administration
and/or meal. Whenever measurements of vital signs, ECG, and blood
samples for PK, PD, or safety coincide, the following order is
respected: ECG, vital signs, PD, PK, and safety samples; in order
to respect exact timing of PK samples (refer to flow-chart for time
window allowance for PK samples), the other measures are done ahead
of the scheduled time. The assessment schedule is adapted to the
design of the study
Local Tolerability at Injection Site
[0545] Findings at the site of injection (such as erythema, edema,
papules, induration, vesicles, blisters) are graded mainly
according to a Global Irritation Score. A local injection site
reaction with a score of .gtoreq.3 according to the rating scale is
documented additionally as an adverse event.
[0546] The subjects are asked to report sensations at the injection
site.
Pharmacokinetics
[0547] For the assessment of insulin glargine pharmacokinetics, the
area under the insulin concentration curve (INS-AUC) up to 36
hours, INS-AUC.sub.0-36 and the time to 50% of INS-AUC.sub.0-36 is
derived. In addition, the maximum insulin concentration
INS-C.sub.max, and time to C.sub.max (INS-T.sub.max) is
obtained.
Sampling Times
[0548] Blood is collected for the determination of insulin glargine
concentrations at time points OH, 1H, 2H, 4H, 6H, 8H, 12H, 16H,
20H, 24H, 28H, 32H and 36H after injection of study medication.
Number of Pharmacokinetic Samples
TABLE-US-00011 [0549] TABLE 8 Number of blood samples per subject
Periods Insulin (glargine) Treatment Period 1 13 Treatment Period 2
13 Treatment Period 3 13 Treatment Period 4 13 Total number of 52
samples per subject Total number of samples .sup.a 52*24 = 1248
.sup.a assuming 24 subjects completed the study
Pharmacokinetic Handling Procedure
[0550] The exact time of IP administration and sample collection
must be recorded in the CRF.
Pharmacokinetic Parameters
[0551] The following pharmacokinetic parameters are calculated,
using non-compartmental methods for insulin glargine concentrations
after single dose. The parameters include, but are not be limited
to the following.
TABLE-US-00012 TABLE 9 List of pharmacokinetic parameters and
definitions Drug/ Parameters Analyte Definition/Calculation
C.sub.max Insulin Maximum concentration observed T.sub.max Insulin
First time to reach C.sub.max AUC.sub.0-36 Insulin Area under the
concentration versus time curve calculated using the trapezoidal
method from time zero to 36 hours post dosing T.sub.50%-AUC Insulin
Time to 50% of AUC.sub.0-36
Sampled Blood Volume
TABLE-US-00013 [0552] TABLE 10 Sampled Blood Volume Volume per
Sample Type Sample Number Total Serology 2 mL 1 2 mL Hematology 2.7
mL 5 13.5 mL Coagulation 2 mL 3 6 mL Biochemistry 5 mL 3 15 mL
Archival Sample 5 mL 1 5 mL Insulin 3 mL 13*4 156 mL Glucose
calibration 0.2 mL 74*4 59.2 mL Glucose continuously 2 mL/h 40*4
320 mL .beta.-HCG (if female) .sup.a 0 mL 1 0 mL FSH/estradiol (if
female) .sup.a,b 0 mL 1 0 mL Anti-insulin antibodies 3 mL 2 6 mL
Total 582.7 mL .sup.a included in serology .sup.b if postmenopausal
less than 2 years
Measures to Protect Blinding of the Trial
[0553] In order to maintain the blinding, a third party un-blinded
person is involved for IP dispensing and administration. This
person is not otherwise involved in the study and/or part of the
study team at the CRO or sponsor. He/she gets the random code
provided by sanofi-aventis and does not disclose the random code or
any other information to any other person. For safety reason, the
treatment randomization code is unblinded for reporting to the
Health Authority of any Suspected Unexpected Adverse Drug Reaction
(SUSAR) and reasonably associated with the use of the IP according
to either the judgment of the Investigator and/or the Sponsor.
Subject Safety
[0554] The Investigator is the primary person responsible for
taking all clinically relevant decisions in case of safety
issues.
[0555] If judged necessary, the opinion of a specialist should be
envisaged in a timely manner (e.g. acute kidney failure,
convulsions, skin rashes, angioedema, cardiac arrest,
electrocardiographic modifications, etc).
Example 12
Study Procedures
Visit Schedule
Screening Procedures
[0556] Screening procedures are carried out within 28 days up to 3
days prior to inclusion to determine subject's eligibility for
participation. The subject receives information on the study
objectives and procedures from the Investigator. The subject signs
the informed consent prior to any action related to the study.
Recording of adverse events starts thereafter.
[0557] Prior to screening, subjects have fasted (apart from water)
for 10 hours (excluding a small amount of carbohydrates as
countermeasure for hypoglycemia, if necessary).
[0558] The screening visit includes the following investigations:
[0559] 1 Demographics (age, sex, race, past and current smoking
status, height, body weight, BMI) [0560] 2 Physical examination
(cardiovascular system, chest and lungs, thyroid, abdomen, nervous
system, skin and mucosae, and musculo-skeletal system) and relevant
medical and surgical history, diabetes history (diagnosis of
diabetes, onset of insulin treatment, late complications); only
findings relevant to the study are documented [0561] 3 Relevant
previous and all concomitant treatments, average insulin regimen in
the last 2 months prior to study entry [0562] 4 ECG (standard 12
lead), vital signs measurements (pulse rate, systolic and diastolic
blood pressure measured after 10 minutes in supine resting
position, and after 3 minutes in standing position), and core body
temperature (aural) [0563] 5 Laboratory tests with hematology, HbA
1c, C-peptide, clinical chemistry, serology, urinalysis, urine drug
screen, alcohol breath test, .beta.-HCG and FSH/estradiol blood
test (female only, if applicable)
[0564] One retest within a week is permitted with the result of the
last test being conclusive.
[0565] Subjects who meet all the inclusion criteria, and none of
the exclusion criteria, are eligible for the inclusion visit.
[0566] In case of screening failures the basic results of the
screening examination are recorded in the source documents.
[0567] Inclusion Procedures (Day 1 of Treatment Period 1)
[0568] Subjects, who qualify for enrollment into the study, are
admitted to the clinic in the fasted state in the morning of D1 of
TP1 at approximately 07:00.
[0569] The inclusion examination is carried out on the first dosing
day (D1, TP1) and includes the following investigations: [0570]
Physical examination with updated medical history (AEs),
previous/concomitant medication and aural body temperature
Body Weight, BMI (Height Measured at Screening)
[0570] [0571] ECG (standard 12 lead), vital signs measurements
(heart rate, respiratory rate, systolic and diastolic blood
pressure measured after 10 minutes in the supine resting position,
and after 3 minutes in the standing position) [0572] Laboratory
tests with hematology, clinical chemistry, urinalysis, urine drug
screen, alcohol breath test, .beta.-HCG urine test (female only, if
applicable).
[0573] Each subject receives an incremental identification number
according to the chronological order of his/her inclusion in the
study.
[0574] Randomization occurs on D1/TP1 after confirmation of
subject's eligibility by the Investigator. If more than one subject
is randomized at the same time, subjects are randomized
consecutively according to the chronological order of inclusion on
the morning of Day 1/TP1, i.e. the subject with the lowest subject
number receives the next available randomization number.
[0575] Results of laboratory tests of D1/TP1 are baseline values
and considered confirmatory, with the exception of the .beta.-HCG
urine test (based on sample collected during screening visit),
which must be negative.
[0576] If a subject is finally enrolled, a blood sample is taken
for archiving and for determination of anti-insulin antibodies (on
D1/TP1 only).
Description by Type of Visit
[0577] Treatment Periods 1-4 (D1 to D2)
[0578] To prevent interference of subjects' standard insulin
treatment with the clamp measurement, subjects abstain from using
basal insulins and switch to [0579] intermediate- or short-acting
insulin products from 48 hours prior to dosing at D1 of TP1 to TP4,
if on long-acting insulin products, i.e. Lantus.RTM. (insulin
glargine), Levemir.RTM. (detemir) or ultralente insulins, [0580]
short-acting insulins from 24 hours prior to dosing at D1 of TP1 to
TP4 if on intermediate acting insulin products, i.e.
NPH-insulin
[0581] The last subcutaneous injection of short-acting insulin is
no later than 9 hours before IP administration. Subjects on pump
therapy discontinues the insulin infusion in the morning of Day 1,
at least 6 hours prior to each IP administration (around 03:00
clock time assuming start of IP administration at 09:00).
[0582] Upon arrival at the clinic, subjects are asked to ensure
that they have had no clinically relevant changes in their physical
condition since the previous visit, that they have been compliant
with the general and dietary restrictions as defined in the
protocol and that they changed their insulin treatment, if
required. Violation of the study criteria excludes the subject from
further participation in the study. Depending on the kind of
violation, a subject may be excluded only from the particular study
day, allowing a re-scheduling of the study day once, or for the
entire study.
[0583] Any changes in the health condition and the concomitant
medication of the subjects since the last visit are reported in the
subject's medical records (source) and the CRF.
[0584] In the morning shortly before administration of study
medication (D1 of each TP) body weight, vital signs, 12-lead ECG,
ECG monitoring and core body temperature are recorded, an
urinalysis and a urine drug and alcohol screen are performed.
[0585] The amount of insulin glargine required for injection will
be calculated according to subject's body weight.
[0586] Hematology is analyzed for incurring anemia on Day 1 of
Treatment Period 3. If positive, the wash-out interval between
Treatment Periods 3 and 4 is extended to the maximum allowed 18
days or start of TP4 will be postponed until hematological
parameters have been normalized. An additional hematological
assessment is made on Day 1 of Treatment Period 4. Subjects remains
fasting (apart from water) until the end of the euglycemic
clamp.
[0587] Subjects are then be prepared for the start of the pre-clamp
procedure with three venous lines connected to an automatic glucose
reading device (Biostator.TM.) and remain in semi-recumbent
position for the entire duration of the sampling period. At
approximately 07:30a dorsal hand vein or lateral wrist vein of the
left arm is cannulated and connected to the Biostator.TM. in order
to continuously draw arterialized venous blood for the
determination of blood glucose concentration. The left hand is
placed into a heated box ("Hot-Box"), which provides for an air
temperature of about 55.degree. C., allowing arterialization of
venous blood. A second venous line is placed into the antecubital
vein of the left arm and is used to collect samples for insulin and
reference blood glucose determination. A third vein is cannulated
on the contralateral forearm allowing the infusion of 0.9% saline
and 20% glucose solution with a pump in the Biostator.TM. or
insulin glulisine with an external pump.
[0588] From insertion of the vascular catheters until 60 min before
study medication administration at approximately 09:00 on DE the
blood glucose level is maintained within 4.4 to 6.6 mmol/L (80-120
mg/dL, pre-clamp). Depending on the blood glucose level, additional
intravenous bolus injection of insulin glulisine is given to keep
the blood glucose within the target range. In the 1 hour before
study medication administration no intravenous bolus injections are
given until clamp end.
[0589] Additional blood samples for the determination of blood
glucose are taken in at least 30 min intervals to check against a
laboratory reference based on the glucose oxidase method. If
necessary the Biostator.TM. is re-calibrated according to results
of the laboratory reference method.
[0590] Insulin infusion rates are adjusted individually. While
keeping blood glucose at the target level both, insulin and glucose
infusion rate are minimized during the clamp run-in phase. Insulin
glulisine solution is infused by means of a high precision infusion
pump (Terumo Spritzenpumpe TE 311.TM.), 20% glucose solution is be
applied by a high precision infusion pump (Terumo Infusionspumpe TE
171.TM.)
[0591] The clamp level is adjusted 60 min before study medication
administration to maintain the blood glucose at about 5.5 mmol/L
(100 mg/dL) until the end of the clamp period. The pre-clamp is
prolonged and IP administration postponed until 14:00 clock time in
case the target glucose level has not been met during the run-in
phase (pre-clamp). If the target glucose level cannot be
established within until 14:00 clock time, the visit is terminated
and the subject may be scheduled for a new dosing visit 1-7 days
later.
[0592] The insulin glulisine infusion is discontinued immediately
before study medication administration. The first insulin sample
for PK is taken immediately thereafter. At about 09:00 the study
medication is administered (Table 4), either [0593] the Reference
treatment (R, 0.4 U/kg Lantus.RTM. U100) [0594] or the Test
treatment (T.sub.1-3) at one peri-umbilical site according to the
randomization plan, using a standardized skin-fold technique.
[0595] During the clamp 12-lead ECGs are taken 2 and 12 hours after
injection of IP and at clamp end.
[0596] The study medication is administered preferably by the same
person at during the whole study. The end of the injection defines
time zero (T0), which defines the starting time of the subsequent
clamp period and PK sampling.
[0597] Every clamp observation period lasts 36 hours and thus ends
at approximately at 21:00 on D2, the predefined end-of-clamp.
Thereafter the subjects are delinked from the euglycemic clamp
setting when blood glucose is well within the isoglycemic range,
receive a meal and their usual insulin treatment.
[0598] In case blood glucose passes 11.1 mmol/L (200 mg/dL) during
the clamp period for 30 minutes after cessation of glucose infusion
and the investigator confirms that any possible errors leading to
false blood glucose levels above 11.1 mmol/L (200 mg/dL) have been
excluded, the rapid acting insulin analog (e.g. insulin glulisine)
used in the pre-IP administration time of the clamp is given to
extend the clamp period to 36 hours for pharmacokinetic blood
sampling. In that case, the sponsor has to be informed. Thereafter
the subjects are delinked from the euglycemic clamp setting when
blood glucose is well within the isoglycemic range, receive a meal
and their usual insulin treatment.
[0599] The injection site reaction is assessed 15 minutes as well
as one hour after injection of the study medication and documented
as an AE if a score of >3 is observed according to the rating
scale.
[0600] Prior to discharge, a meal ad libitum is served and the
usual insulin-treatment will be resumed. Vital signs (heart rate;
systolic and diastolic blood pressure measured after 10 minutes in
the supine resting position, and after 3 minutes in the standing
position) are repeated and blood glucose is measured (the blood
glucose reading must be above 80 mg/dL). Subjects are discharged on
D2 of TP1 to TP4 after their well-being is ensured by the
investigator.
End-of-Study Visit
[0601] Subjects return for an end-of-study (EOS) visit between D5
and D14 after last dosing in TP4.
[0602] Subjects have fasted (apart from water) for 10 hours. The
EOS includes the following investigations:
Physical Examination (Weight, Body Temperature) with Updated
Medical History
ECG, Vital Signs Measurement
[0603] Laboratory tests with hematology, HbA1c, biochemistry,
urinalysis, and if female a .beta.-HCG blood test
[0604] Any AE occurred or concomitant medication taken since
TP4
[0605] Blood sample for anti-insulin antibody determination.
[0606] The Investigator ensures that based on all available
clinical results, the subject can be safely released from the
study.
Study Restriction(s)
[0607] Subjects ceases their usual insulin treatment on Days-2 to
-1, depending on the type of insulin used (long acting, NPH,
intermediate). Thereafter, the blood glucose levels are controlled
solely by multiple subcutaneous injections of the usual
short-acting insulin.
[0608] The usual insulin treatment is resumed after discharge on
Day 2 in TP1 to TP4.
[0609] The subjects do not take any concomitant medication, which
will interfere with the metabolic control or the insulin
sensitivity of subjects throughout the study and in the two weeks
before the study.
[0610] Consumption of alcoholic beverages, grapefruit juice, and
stimulating beverages containing xanthine derivatives (tea, coffee,
Coca Cola-like drinks, chocolate) is not permitted 24 hours before
administration of each study medication until the end of the clamp.
Orange juice or similar carbohydrates are given as corrective
measures for hypoglycemia during clamp if not adequately
counteracted by intravenous glucose infusion when connected to the
Biostator.TM..
[0611] No strenuous physical activity is allowed within 2 days
before each study medication administration.
[0612] Subjects who smoke 5 or less cigarettes per day are included
in the study and subjects may smoke during the study, except on D1
and D2 of TP1 to TP4.
[0613] On the screening day, subjects come to the unit after an
overnight fast of at least 10 hours (excluding a small amount of
carbohydrates as countermeasure for hypoglycemia, if
necessary).
[0614] In the morning of Day 1 in TP1 to TP4, subjects are admitted
to the clinic after an overnight fast of at least 10 hours and
remain fasting until end of clamp period in Day 2. A meal ad
libitum is served after the end of the clamp.
[0615] Fluid supply is at least 2500 mL for each 36-hour
period.
Definition of Source Data
[0616] All evaluations listed below that are reported in the CRF
are supported by appropriately signed identified source
documentation related to: [0617] subject identification [0618]
medical history (in case of allergic reaction) [0619] clinical
examination, vital signs, body weight and height, body temperature;
[0620] laboratory assessments, ECG [0621] pharmacokinetic time
points [0622] dates and times of visits and assessments [0623]
adverse events [0624] IP administration [0625] previous/concomitant
medication [0626] start/end of clamp procedure, clamp data
Example 13
Statistical Considerations
Determination of Sample Size
[0627] The primary objective of the study is to assess the relative
metabolic effect for insulin glargine given as one dose of U100 (R)
and three different doses of U300 (T.sub.1 to T.sub.3).
[0628] Based on the data of study PKD10086, a value of
approximately 0.375 can be expected for the SD.sub.within of
GIR-AUC.sub.end of clamp on the natural log-transformed scale.
[0629] For the purpose of the sample size calculation
within-subject SDs between 0.325 and 0.425 were used.
[0630] Table 11 shows the maximum imprecision (in terms of the 90%
confidence interval) for a pairwise treatment ratio of adjusted
geometric means that will be obtained with 90% assurance, for total
number of subject N between 16 and 24, assuming a true
within-subject SD of values between 0.325 and 0.425 for log
GIR-AUC.sub.0-36.
TABLE-US-00014 TABLE 11 Maximum imprecision for any pairwise ratio
Confidence level: 90% Assurance: 90% Within- Total subject number
Maximum Maximum width 90% CI SD on of imprecision for an observed
ratio equal to log scale subjects (%) 0.6 0.8 1 0.325 16 19.7
(0.48; 0.75) (0.64; 1.00) (0.80; 1.25) 20 17.5 (0.49; 0.73) (0.66;
0.97) (0.82; 1.21) 24 15.9 (0.50; 0.71) (0.67; 0.95) (0.84; 1.19)
0.350 16 21.0 (0.47; 0.76) (0.63; 1.01) (0.79; 1.27) 20 18.7 (0.49;
0.74) (0.65; 0.98) (0.81; 1.23) 24 17.0 (0.50; 0.72) (0.66; 0.96)
(0.83; 1.21) 0.375 16 22.4 (0.47; 0.77) (0.62; 1.03) (0.78; 1.29)
20 19.9 (0.48; 0.75) (0.64; 1.00) (0.80; 1.25) 24 18.1 (0.49; 0.73)
(0.65; 0.98) (0.82; 1.22) 0.400 16 23.7 (0.46; 0.79) (0.61; 1.05)
(0.76; 1.31) 20 21.1 (0.47; 0.76) (0.63; 1.01) (0.79; 1.27) 24 19.2
(0.48; 0.74) (0.65; 0.99) (0.81; 1.24) 0.425 16 24.9 (0.45; 0.80)
(0.60; 1.07) (0.75; 1.33) 20 22.3 (0.47; 0.77) (0.62; 1.03) (0.78;
1.29) 24 20.3 (0.48; 0.75) (0.64; 1.00) (0.80; 1.25)
[0631] With 20 subjects, if the true within-subject SD of
GIR-AUC.sub.0-36 is as much as 0.375, the treatment ratio will be
estimated with a maximum imprecision of 19.9% (i.e. the 90% CI will
be 0.80 and 1/0.80=1.25 times the observed ratio), with 90%
assurance.
24 subjects will be included in order to have 20 completed
subjects
Subject Description
Disposition of Subjects
[0632] A detailed summary of subject accountability including count
of subjects included, randomized, exposed (i.e. received any amount
of study medication), completed (i.e. subjects who completed all
study treatment periods), discontinued along with the main reasons
for discontinuation is generated.
[0633] Subject disposition at the final visit is presented in a
listing including sequence group, disposition status at the end of
the study with the date of last administration of study drug, date
of final visit, reason for discontinuation. All withdrawals from
the study, taking place on or after the start of the first study
drug administration, are fully documented in the body of the
clinical study report (CSR).
Protocol Deviations
[0634] Prior to data lock of the study, Clinical Trial Protocol
deviations are examined relative to criteria defined for definition
of populations and other study criteria including: [0635] Inclusion
and exclusion criteria; [0636] Treatment compliance; [0637]
Compliance with the Clinical Trial Protocol with regard to
prohibited therapies; [0638] Compliance with the Clinical Trial
Protocol with regard to intervals between visits and total
treatment duration; and [0639] Whether planned activity and safety
evaluation were performed, etc.
[0640] Deviations covered include but not be limited to: [0641]
Subjects without any evaluation (of any variables) after
randomization; [0642] Subjects not exposed; [0643] Subject without
any evaluation of the primary variable (if relevant); [0644]
Subjects who entered the study even though they did not satisfy the
inclusion criteria; [0645] Subjects who developed withdrawal
criteria during the study but were not withdrawn; [0646] Subjects
who received the wrong treatment or incorrect dose; [0647] Subjects
who received a prohibited concomitant medication.
[0648] Major deviations are listed and summarized.
Analysis Population
[0649] All exclusions from any analysis populations
(pharmacodynamic, pharmacokinetic and/or safety) are fully
documented in the CSR.
[0650] Subjects excluded from any analysis population are listed
with treatment sequence, and with reason for exclusion. Any
relevant information is fully documented in the CSR. Frequencies of
subjects, overall and per treatment, for the analysis populations
are tabulated.
[0651] For the event of subjects having received treatments that
differed from those assigned according to the randomization
schedule, analyses are conducted according to the treatment
received rather than according to the randomized treatment.
Pharmacodynamic Population
[0652] All subjects without any major deviations related to study
drug administration, and for whom PD parameters are available, are
included in the pharmacodynamic population. For subjects with
insufficient PD profiles in one but not both treatment periods,
parameters of the sufficient profiles are included in the
analysis.
[0653] For subjects, who receive (for safety reasons) insulin
glulisine within the observation period of 36 hours after dosing of
IP, pharmacodynamic data are only taken into account up to the time
of administration of insulin glulisine.
Exclusions from Pharmacodynamic Analysis
[0654] All exclusions form the pharmacodynamic analysis are listed
together with the reason. Exclusions are decided and documented
based on the review of the data prior to database lock and
unblinding.
Safety Population
[0655] All subjects who were exposed to any comparative study
treatment, regardless of the amount of treatment administered, are
included in the safety population.
Pharmacokinetic Populations
[0656] All subjects without any major deviations related to study
drug administration, and for whom insulin PK parameters are
available, are included in the pharmacokinetic population. For
subjects with insufficient insulin PK profiles at one but not all
treatment periods, parameters of the sufficient profiles are
included in the analysis.
[0657] The bioanalytical assay for insulin glargine is interfered
by other insulins like insulin glulisine. Therefore, the
pharmacokinetic data for insulin glargine of those subjects are
excluded from evaluation, who have received (for safety reasons)
insulin glulisine within the clamp observation period of 36 hours
after IP administration.
Demographic and Baseline Characteristics
Subject Demographic Characteristics, Medical History and
Diagnoses
[0658] The following data are collected: sex, age, height, weight,
and race. Baseline body mass index (BMI) per subject is calculated
from pre-dose body weight and height data:
BMI=body weight[kg]/(height[m]).sup.2
[0659] All variables concerning demographic and background
characteristics are listed individually and summarized for the
safety population.
[0660] Deviations from inclusion criteria related to medical
history and diagnoses are listed and described individually.
Baseline Safety Parameters
[0661] For safety variables, the latest scheduled value before
study drug administration within the period or within the study,
whatever is applicable for the variable, is taken as the baseline
value. If the baseline pre-dosing value is rechecked before dosing,
the rechecked value is considered as the baseline and used in
statistics.
Extent of Study Treatment Exposure and Compliance
[0662] Details of study drug dosing and complementary information
are listed individually and summarized if appropriate.
[0663] Individual total doses of insulin glargine are summarized by
treatment.
Prior/Concomitant Medication/Therapy
[0664] Prior and concomitant medications/therapies (if any) are
coded according to the World Health Organization-Drug Reference
List (WHO-DRL, latest version in use at time of database lock) and
are listed individually.
[0665] Concomitant insulin medication (subcutaneous) is listed
separately.
[0666] Insulin infusion or bolus given at any time during the clamp
procedure is listed or plotted over time on an individual basis.
Insulin infusion or bolus given after dosing during the clamp
procedure is listed on an individual basis.
Analysis of Pharmacodynamic Variables
[0667] All pharmacodynamic analyses encompass data of the
pharmacodynamic population. No adjustment of the alpha-level is
made for multiple analyses.
[0668] For pharmacodynamics of insulin glargine, the blood glucose
concentration and glucose infusion rate (GIR) is continuously
recorded during the clamp procedure.
[0669] Statistical analyses compare test treatments (T.sub.1 to
T.sub.3) with the reference treatment (R)
Description of Pharmacodynamic Variables
[0670] In order to achieve comparability between the subjects body
weight adjusted insulin dosing, all values for GIR are divided by
the subject's body weight in kg for analysis. Thus in the below, if
not stated otherwise, GIR always refers to the body weight
standardized glucose infusion rate.
Primary PD Variable
[0671] The following PD variable is considered primary. [0672] Area
under the body weight standardized glucose infusion rate time curve
[GIR-AUC.sub.0-36 (Mg/kg)]
[0673] GIR-AUC.sub.0-36 is calculated according to the rectangular
rule for the stepwise constant function with timescale in
minutes.
Secondary PD Variables
[0674] The following PD variables are derived and considered
secondary: [0675] Time (h) to 50% of GIR-AUC.sub.0-36
[T.sub.50%-GIR-AUC.sub.0-36 (h)] [0676] Maximum smoothed body
weight standardized glucose infusion rate [GIR.sub.max (mg*min/kg)]
[0677] First time after dosing to reach GIR.sub.max [GIR-T.sub.max
(h)] [0678] Duration of euglycemia (time to elevation of smoothed
blood glucose profile above clamp level) is calculated as the time
from dosing to the last value of the smoothed blood glucose
concentration curve at or below 105 mg/dL [0679] Durations of
controlled blood glucose within predefined margins are defined as
the time from dosing to the last value of the smoothed blood
glucose concentration curve at or below [0680] 110 mg/dL [0681] 130
mg/dL [0682] 150 mg/dL
Smoothing
[0683] The maximum of the raw body weight standardized GIR is
subject to the noise in the GIR adjustment. Thus, the derivation of
GIR.sub.max and the time to GIR.sub.max, is based upon a LOESS
(locally weighted regression in smoothing scatterplots) smoothing
technique for the raw body weight standardized GIR data. Due to the
expected morphology of the GIR-profiles as known under Lantus.RTM.,
a smoothing factor of 6% is used (SAS.RTM., PROC LOESS, factor
0.06).
[0684] Blood glucose levels are well be subject to noise.
Therefore, the duration of euglycemia and the duration of blood
glucose control are based upon a LOESS (locally weighted regression
in smoothing scatterplots) smoothing technique for the raw blood
glucose levels. Due to the expected morphology, a smoothing factor
of 6% is used (SAS.RTM., PROC LOESS, factor 0.06).
[0685] In case of inadequate smoothing a different smoothing factor
is used for an additional analysis.
Additional PD Variables
[0686] Further parameters are derived, as: [0687] Time to end of
glucose infusion, as the latest time after dosing with GIR above
zero
[0688] Additional PD variables are derived if deemed necessary for
interpretation of results.
Primary PD Analysis
[0689] Prior to the analysis described below, GIR-AUC.sub.0-36 is
log-transformed (natural log).
[0690] Log-transformed GIR-AUC.sub.0-36 is analyzed with a linear
mixed effects model with fixed terms for sequence, period and
treatment
log(parameter)=sequence+period+treatment+error
and with an unstructured R matrix of treatment (i, i) variances and
covariances for subject within sequence blocks, using SAS PROC
MIXED.
[0691] 90% confidence interval (CI) for the ratio of treatments
geometric means (T.sub.1/R, T.sub.2/R, T.sub.3/R) is obtained by
computing estimate and 90% CI for the difference between treatment
means within the linear mixed effects model framework, and then
converting to ratio of geometric means by the antilog
transformation. Equivalence is concluded if the 90% CI for the
ratio is entirely within the 0.80 to 1.25 equivalence reference
interval.
[0692] Listings of individual ratios (test treatments versus
reference treatment) are provided with the corresponding
descriptive statistics.
Secondary Analysis/Analysis of Secondary Variables
Descriptive Presentations for GIR Profiles
[0693] Individual body weight standardized GIR (mg*min/kg) is
plotted for raw, smoothed and cumulative raw values.
[0694] Mean and median body weight standardized GIR-profiles as
well as median percentage cumulative profiles over time are plotted
by treatment.
[0695] Cumulative plots cover the time between dosing to end of
clamp.
Descriptive Presentations for Derived PD Parameters
[0696] PD parameters are listed individually, and descriptive
statistics are generated by treatment.
Treatment Ratios for Secondary Pd Parameters
[0697] Treatment ratios (T.sub.1/R, T.sub.2/R, T.sub.3/R) with
confidence limits are derived for maximum standardized glucose
infusion rate [GIR.sub.max(mg*min/kg)] using the corresponding
linear mixed effects model as described above for the primary
analysis. Exploratory comparisons between treatments are based on
conventional bioequivalence criteria (90% confidence limits 0.80 to
1.25).
[0698] The distribution of GIR-T.sub.max values is represented by
histogram plots for each treatment. In addition, a histogram of
differences in GIR-T.sub.max between test treatments and reference
is provided.
Treatment Differences for Secondary PD Parameters
[0699] T.sub.50%-GIR-AUC.sub.0-36 (h) is analyzed
non-parametrically based on Hodges-Lehmann method for paired
treatment comparisons. CIs for pair-wise treatment differences
(T1-R, T2-R, T3-R) in medians are derived. The distribution of
T.sub.50%-GIR-AUC.sub.0-36 values is represented by histogram plots
for each treatment. In addition, a histogram of differences in
T.sub.50%-GIR-AUC.sub.0-36 between treatments (T1-R, T2-R, T3-R) is
provided.
[0700] The distribution of GIR-T.sub.max values is represented by
histogram plots for each treatment. In addition, a histogram of
differences in GIR-T.sub.max between test treatments and reference
is provided.
[0701] Duration of euglycemia and of blood glucose control are
presented by histogram plots. Treatment comparisons are performed
non-parametrically.
Performance of Clamp
[0702] Individual profiles of blood glucose concentration are
plotted.
[0703] Duration of clamp is derived per clamp as the time between
dosing and end of clamp in hours.
[0704] Individual variability of blood glucose per clamp is derived
as the coefficient of variation (CV %) of blood glucose values
between individual start and individual end of clamp (or first
administration of insulin glulisine during clamp). Individual
average blood glucose level per clamp is derived as the arithmetic
mean of blood glucose values between individual start and
individual end of clamp (or first administration of insulin
glulisine during clamp).
[0705] Parameters are listed individually and summarized
descriptively within treatment.
Analysis of Safety Data
[0706] The safety evaluation is based upon the review of the
individual values (potentially clinically significant
abnormalities), descriptive statistics (summary tables, graphics)
and if needed on statistical analysis (appropriate estimations,
confidence intervals). "Potentially Clinically Significant
Abnormalities" (PCSA) criteria are used according to standard
criteria of sanofi-aventis. Criteria are documented in the
statistical analysis plan of this study. The safety analysis is
conducted according to the sanofi-aventis standards related to
analysis and reporting of safety data from clinical trials.
[0707] All safety analyses encompass data of the safety
population.
[0708] For all safety data, the observation period is divided into
segments of three different types: [0709] the pre-treatment period
is defined as the time between when the subject gives informed
consent and the first administration of study medication. [0710]
the on-treatment period is defined as the time from (first) study
medication administration up to 72 hours later. [0711] the
post-treatment period is defined as the time after on-treatment
period to either the (first) administration of study medication in
the next period or the end of the follow-up period.
Adverse Events
[0712] All AEs are coded using MedDRA (latest version in use at
time of database lock).
[0713] The following listings are provided for all adverse events:
[0714] Listing of all adverse events (by subject) [0715] Listing of
comments related to adverse events
DEFINITIONS
[0716] For safety data, the observation period is divided into
segments of three different types: [0717] the pre-treatment period
is defined as the time between when the subject gives informed
consent and the first administration of comparative study
medication. [0718] the on-treatment period per period is defined as
the time from (first) study medication administration up to 72
hours later. [0719] the post-treatment period is defined as the
time after on-treatment period to either the (first) administration
of study medication in the next period or the end of the follow-up
period.
Treatment Emergent Adverse Events
[0720] All AEs are classified as follows: [0721] Treatment-emergent
adverse events (TEAEs) are any AEs with an onset (incl. worsening)
during an on-treatment period [0722] Non-treatment-emergent adverse
events (NTEAEs) are any AEs not classified as TEAE: [0723]
Pre-treatment AEs, defined as AEs that developed (or worsened)
during the pre-treatment period before the first dose of study
medication [0724] Post-treatment AEs, defined as AEs that developed
during a post-treatment period without worsening during an
on-treatment phase.
Assignment to Treatments
[0725] For analysis purposes, each TEAE is assigned to the last
treatment given before onset (or worsening) of the AE. If a TEAE
develops on one treatment and worsens under a later treatment, it
is considered treatment emergent for both treatments.
Missing Information
[0726] In case of missing or inconsistent information, an AE is
counted as a TEAE, unless it can clearly be ruled out that it is
not a TEAE (e.g. by partial dates or other information).
[0727] If the start date of an AE is incomplete or missing, it is
assumed to have occurred after the first administration of study
medication except if an incomplete date indicates that the AE
started prior to treatment.
Treatment-Emergent Adverse Events
[0728] Treatment emergent adverse events are listed and summarized
by treatment: [0729] Overview of TEAEs (number and percentage of
subjects with at least one TEAE, severe TEAE, TEAE leading to
discontinuations, death (if any)) [0730] Summary of all
treatment-emergent adverse events by primary system organ class and
preferred term (number and percentage of subjects with at least one
TEAE) ("in-text table") [0731] Table without number of events (for
body of the clinical study report) [0732] Table with number of
events (for appendix of the clinical study report) [0733] Table
with number of subjects per formulation (U100, U300) and of
subjects overall (for appendix of the clinical study report) [0734]
Listing of subjects presenting treatment emergent adverse events by
treatment, system organ class and preferred term
Deaths, Serious and Other Significant Adverse Events
[0735] In case of any occurrences, deaths, serious AEs, and other
significant AEs are listed individually and described in the study
report in detail.
Adverse Events Leading to Treatment Discontinuation
[0736] In case of any occurrences, individual subject listings are
generated for all adverse events leading to treatment
discontinuation.
Clinical Laboratory Evaluations
Hematology and Biochemistry Data
[0737] Laboratory safety parameters are measured on D1 of treatment
period 1 and at EOS. Per schedule, these safety parameters are
assessed during the on-treatment period (except hematology at TP3
and TP4).
[0738] The values to be used as baseline (hematology and
biochemistry) are the values collected on D1 predose in the first
treatment period. If any of the scheduled baseline tests are
repeated for any subject, the last rechecked values are considered
as baselines, provided they were done before the first IP
administration.
[0739] The following tables and listings are provided: [0740]
Descriptive statistics for raw data and changes from baseline
(including % change for creatinine) [0741] A specific listing of
individual data from subjects with post-baseline PCSAs will be
provided, sorted by function and time of measurement [0742] All
individual data, including rechecked values, for planned hematology
and biochemistry, are listed by biological function and time of
measurement. If any, data from unscheduled laboratory tests are
included in this listing. In these listings, individual data are
flagged when lower or higher than the lower or upper laboratory
limits and/or when reaching the absolute limit of PCSA criteria,
when defined [0743] A listing of liver function data for subjects,
who experienced at least one of the following: [0744] at least one
occurrence of ALT>3ULN and at least one occurrence of total
bilirubin [0745] >2 ULN during the study with at least one of
them being post first dose [0746] conjugated bilirubin >35%
total bilirubin and total bilirubin >1.5 ULN will be provided on
the same sample post first dose, irrespective of the definition for
the on-treatment phase. [0747] A listing related to increase in
ALT.gtoreq.2 ULN is provided, including notably the information on
drug intake, medical and surgical history, alcohol habits, trigger
factors, event details with ALT values, associated signs and
symptoms. [0748] A listing of out-of-range definitions is
provided.
[0749] In the listings of subjects with PCSAs, liver function data,
CPK, and eosinophils are expressed as multiple of the corresponding
ULN.
Urinalysis Data
[0750] All qualitative urinary test results (dipstick), including
rechecked values, are listed.
Vital Signs
Blood Pressure and Heart Rate
[0751] Heart rate and systolic and diastolic blood pressure (SBP
and DBP) are measured after 10 minutes in supine resting position
and also after 3 minutes in standing position, except when
connected to the Biostator.TM..
[0752] The values to be used as the baselines are the D1 pre-dose
assessment value of each treatment period. If any of the scheduled
baseline tests are repeated for any subject, the last rechecked
values are considered as baselines, provided they were done before
the IP administration.
[0753] For heart rate and blood pressures, orthostatic differences
are calculated as the change from supine to standing position.
[0754] For all parameters, an "On-Treatment" analysis will be
performed including all unplanned values and rechecked values.
[0755] The following tables and listings are provided: [0756]
Summary tables of counts of subjects with PCSAs are provided as
incidence tables of post-baseline PCSAs, regardless of the normal
or abnormal status of the baseline [0757] For heart rate and blood
pressures (supine and standing positions), raw data and changes
from baseline (supine position only) are summarized in descriptive
statistics, for type of measurement (position) each parameter and
time point, based on planned pre-dose measurements and the baseline
defined [0758] All individual data, including unplanned and
rechecked values, are listed (supine, standing, orthostatic
difference). In the listings, values are flagged when reaching the
limits of the PCSA criteria when defined [0759] A data listing of
individual post-baseline PCSAs is provided [0760] Comments related
to vital sign evaluations are also listed in the Appendix, if
any.
Body Weight, Body Mass Index, and Body Temperature
[0761] The values to be used as baselines for body weight and BMI
are the values collected on D1 of TP1.
[0762] The values to be used as baselines for body temperature are
the values collected on D1 of each TP.
[0763] Individual data are listed including flags (weight only) for
values when reaching the limits of the PCSA criteria.
ECG
[0764] Heart rate, PQ-, QRS-, and QT-intervals and corrected QT
(QTc) from automatic reading are analyzed as raw parameter value
and change from baseline.
[0765] The values to be used as the baseline are the Day 1 predose
value of each period. If any of the scheduled baseline tests are
repeated for any subject, the rechecked values are considered as
baselines, provided they were done before the drug administration
of the period.
[0766] For all parameters, an on-treatment analysis is performed
using all post-baseline assessments done during the on-treatment
period, including rechecked values. Counts of subjects with
postbaseline PCSAs are provided in summary tables regardless of the
normal or abnormal status of the baseline, by treatment group.
[0767] Raw data for all parameters and change from baseline are
summarized in descriptive statistics by parameter, treatment, and
time of measurement.
[0768] Individual data, including rechecked values, are listed,
sorted by treatment, subject, visit and time of measurement. In the
listings, values reaching the limits of the PCSA criteria are
flagged.
[0769] A listing of individual data from subjects with
post-baseline PCSAs is provided, sorted by type of measurement and
sorted by subject, period, and time of measurement.
[0770] Additionally, a separate listing of the cardiac profile for
subjects with prolonged QTc (>450 ms for Males and >470 ms
for Females) or changes from baseline in QTc>60 ms (for males
and females) and a listing of subjects with at least one
abnormality in qualitative assessment (i.e., abnormal ECG) after
the 1st dosing are also provided.
Other Related Safety Parameters
Physical Examination
[0771] Listing of comments related to physical examination is
provided, if any.
Local Tolerability at Injection Site
[0772] Frequency distributions by treatment are provided for levels
of local tolerability at injection site. Individual data are
listed. Within each criterion and treatment, a subject is counted
with their most severe result.
Allergic Reactions
Listings for Allergic Reactions
[0773] Any cases of allergic reaction are documented as adverse
events with detailed complementary information. All cases are
described in detail in the clinical study report.
[0774] Individual cases and all complementary data are listed.
Allergic Medical History and Family Medical History
[0775] Allergic medical history and family medical history is
documented for subjects with any occurrence of potential allergic
reaction. All details of allergic medical history and of allergic
family medical history are listed on an individual basis.
Anti-Insulin Antibodies
[0776] A summary table is provided with the number of subjects for
the anti-insulin antibodies results during the study and from the
post study investigations. Individual subject listing is
provided.
Analysis of Pharmacokinetic Data
Pharmacokinetic Parameters
[0777] The list of PK parameters is shown above. In addition,
T.sub.50%-AUC.sub.0-36 for insulin is derived in the context of the
statistical analysis.
Statistical Analysis
[0778] Pharmacokinetic parameters of insulin glargine are listed
and summarized using at least arithmetic and geometric means,
standard deviation (SD), standard error of the mean (SEM),
coefficient of variation (CV %), minimum, median and maximum for
each treatment.
[0779] All pharmacokinetic analyses encompass data of the
corresponding pharmacokinetic populations as defined above. No
adjustment of the alpha-level is made for multiple analyses.
[0780] Statistical analyses compare test treatments (T.sub.1 to
T.sub.3) versus reference treatment (R).
Analysis of Treatment Ratios
[0781] The analysis is performed for AUC.sub.0-36 for insulin
glargine. Prior to all analysis described below, AUC.sub.0-36
values are log-transformed (natural log).
[0782] Log-transformed parameters are analyzed with a linear mixed
effects model with fixed terms for sequence, period and
treatment
log(parameter)=sequence+period+treatment+error,
and with an unstructured R matrix of treatment (i, i) variances and
covariances for subject within sequence blocks, using SAS PROC
MIXED.
[0783] Estimate and 90% confidence interval (CI) for the ratio of
treatments geometric means (T.sub.1/R, T.sub.2/R, T.sub.3/R) are
obtained by computing estimate and 90% CI for the difference
between treatment means within the linear mixed effects model
framework, and then converting to ratio of geometric means by the
antilog transformation. Bioequivalence is concluded if the 90% CI
for the ratio is entirely within the 0.80 to 1.25 equivalence
reference interval.
[0784] Listings of individual treatment ratios (T.sub.1/R,
T.sub.2/R, T.sub.3/R) are provided with the corresponding
descriptive statistics.
T.sub.50%-AUC.sub.0-36 for Insulin
[0785] The distribution of T.sub.50%-AUC.sub.0-36 values for
insulin is represented by histogram plots for each treatment. In
addition, a histogram of differences in T.sub.50%-AUC.sub.0-36
between treatments (T.sub.1-R, T.sub.2-R, T.sub.3-R) is
provided.
[0786] T.sub.50%-AUC.sub.0-36 (h) is analyzed
non-parametrically.
Dose Exposure Relationship for Insulin Glargine U300
Descriptive Analyses of Dose Exposure Relationship
[0787] Dose exposure relationship for insulin glargine U300 is
described graphically by [0788] plots per subject of exposure over
total dose per subject [0789] plots per subject of exposure over
dose per kg bodyweight [0790] plots per subject of dose normalized
exposure over dose per kg bodyweight (dose normalization on 0.6
U/kg)
[0791] If deemed necessary for interpretation of results,
additional descriptive analyses are added.
Statistical Analysis of Dose Exposure Relationship
[0792] For AUC of insulin glargine calculated for the test
treatments T.sub.1-T.sub.3, dose exposure relationship is assessed
using the empirical power model (PK-parameter=a*dose.sup.b), along
with an "estimation" interpretation, according to the
recommendations in Gough et al. (Gough K, Hutchison M, Keene O et
al. Assessment of dose proportionality: report from the
pharmaceutical industry. Drug Information Journal 1995;
29:1039-1048).
[0793] The empirical power model provides a readily and
interpretable measure of the degree of non-proportionality, which
can be used both to confirm proportionality and to assess the
pharmacokinetic and clinical significance of any departures. The
analysis of dose proportionality studies, however, requires
estimation rather than significance testing in order that the
pharmacokinetic and clinical significance of any
non-proportionality can be assessed.
[0794] The power model is fit on the log-transformed scale using a
random coefficients power model for dose (in U/kg body weight):
log(parameter)=(log(alpha)+alpha[i])+(beta+beta[i])*log(dose)
where log(alpha) and beta are the population intercept and slope,
respectively, and alpha[i] and beta[i] are the random deviations
from alpha and beta, respectively, for the i-th subject.
[0795] Estimates for beta with 90% confidence intervals are
obtained via estimated generalized least squares in the
SAS.RTM./PROC MIXED procedure, with restricted maximum likelihood
(REML) estimates of covariance parameters. Estimates and 90%
confidence intervals for beta are further used to obtain estimates
and 90% confidence intervals for the PK parameter increase
associated with an r-fold increase in dose (r=1.5 and r=2.25 [i.e.
high dose/low dose]), by exponentiating r to the powers of the beta
estimate and confidence limits
[0796] If there is evidence of model lack-of-fit, the mixed effect
model (as used for analysis of treatment ratios) is used for the
analysis. Estimates with 90% CIs for the parameter increases
associated with pairwise dose increases are obtained by first
computing estimates with CIs for pairwise differences between doses
in the mixed effects model framework, and then converting to ratios
using the antilog transformation.
PK/PD Analysis
[0797] If appropriate, graphical displays (scatter plots) are
generated to explore PK/PD relationship.
Example 14
Study Results
Subject Disposition
[0798] A total of 24 subjects with Type 1 diabetes mellitus were
enrolled, randomized and received at least one dose of study
medication. Of the 24 randomized subjects, 2 subject withdrew from
the study on own request. Twenty-two (22) subjects completed the
study according to the protocol and were included in the
pharmacodynamic (PD) and pharmacokinetic (PK) analyses. All 24
treated subjects were included in the safety evaluation.
[0799] There were no major protocol deviations.
Demographics Characteristics
[0800] The following data (Table 12) were collected: sex, age at
screening, height, weight, and race. Body mass indexes (BMI) per
subject were calculated from body weight and height data:
BMI=body weight [kg](height [m]).sup.-2.
TABLE-US-00015 TABLE 12 Demographics BMI Weight Age Race (n) Sex
(kg/m.sup.2) (kg) (years) [%] N 5 F, 19 M 25.55 79.38 42.6
Caucasian / N 1.99 (SD) 9.67 (SD) 10.0 (SD) white 24 24 min min min
19:max [100] 20.5:max 57.3:max 60 28.3 94.3
Clamp Performance
[0801] At the four treatment periods for each subject, R (Lantus
U100), T1 (0.4 U/kg HOE901-U 300), T2 (0.6 U/kg HOE901-U 300) and
T3 (0.9 U/kg HOE901-U 300), the individuals' baseline blood glucose
concentrations prior to insulin medication were similar, defining
the clamp level at 100 mg/dL. The duration of the observation
period of the clamps after dosing was 36 hours and the same in all
treatment periods.
Primary Endpoints
[0802] Equivalence in bio-availability (exposure) and bio-efficacy
(activity) for R and T was not established.
Primary Variables
[0803] The area under the serum insulin glargine concentration time
curve from 0 to 36 hours (INS-AUC.sub.(0-36h)) was not equivalent
for R and T1 and T2 and about equivalent with T3. The exposure was
estimated to be less by about 37% with T1, less by about 43% with
T2 and similar with T3, compared to R.
[0804] The area under the GIR versus time curve from 0 to 36 hours
(GIR-AUC.sub.(0-36h)) was not equivalent for R and T1 and T2 and
about equivalent with T3. The exogenous glucose consumption
required to preserve blood glucose control was estimated to be less
by about 88% with T1, 67% with T2 while about similar with T3.
Secondary Variables
[0805] The time to 50% of INS-AUC.sub.(0-36h) (h) with R was about
14 h and thus shorter as compared to about 16 h, 16 h and 19 h with
T1, T2 and T3, respectively.
[0806] The time to 50% of GIR-AUC.sub.(0-36h) (h) with R was about
12 h and thus shorter as compared to about 17 h, 18 h and 20 h with
T1, T2 and T3, respectively.
Safety
[0807] No serious adverse events (AEs) or withdrawals due to AEs
were reported. Two subjects on R, 2 on T1 and 4 on T3 reported a
total 8 TEAEs, all of which were of mild to moderate intensity, and
resolved without sequalae. The most frequently reported event was
headache. Of note, headache is a common observation for clamp
studies and is related to the infusion of hyper-osmolaric glucose
solutions. However, a link to the investigational products cannot
be excluded. No injection site reactions were reported with T1, T2
and T3 while 2 subjects on R developed hardly perceptible erythema
at the injection site.
CONCLUSIONS
[0808] Same doses of R and T U 300 are not equivalent in
bio-availability (exposure) and bio-efficacy (activity) after
single dose administration. Exposure and activity after T1 (0.4
U/kg) and T2 (0.6 U/kg) were less as compared to exposure and
activity after administration of R (0.4 U/kg). R and T3 were
virtually equivalent as to exposure and exogenous glucose
consumption.
[0809] T1, T2 and T3 did, however, show yet flatter PK (exposure)
and PD (activity) profiles with even less fluctuation around the
averages than R, i.e., a profile as it would be desired for basal
insulin supply. This is particularly evident when comparing R and
T3 which provide nominal equivalent total exposure and total
glucose consumption though of different profiles.
[0810] These surprising and unexpected differences in exposure and
activity between R (Lantus U100) and T (HOE901-U300) formulations
in subjects with type 1 diabetes mellitus are effectively shown in
the figures below.
[0811] Over and above, administration of T (HOE901-U300) was
without safety and tolerability issues.
Example 15
[0812] Study rationale for comparing the glucodynamic activity and
exposure of two different subcutaneous doses of (HOE901-U300) to
Lantus U100 in patients with type 1 diabetes mellitus.
[0813] Results from the study in healthy subjects and in subjects
with Type 1 diabetes mellitus (see foregoing examples) showed
exposure and effectiveness not to be equivalent between Lantus.RTM.
U100 and insulin glargine U300. Subjects received the same dose of
insulin glargine (0.4 U/kg) for U100 and U300, but delivery of the
same unit-amount from U300 produced less exposure at less exogenous
glucose consumption to preserve blood glucose control than delivery
from U100. Though Lantus U100 shows exposure and pharmacodynamic
profiles without pronounced fluctuation around the averages,
HOE901-U300 did, however, show even less fluctuation in exposure
and pharmacodynamic profiles, as it would be desired for basal
insulin supply, with a yet even longer duration of action.
[0814] In order to assess the pharmacokinetic and pharmacodynamic
profile under steady state conditions, a new study described in the
following examples therefore compares two different subcutaneous
doses of insulin glargine U300 versus a standard dose of
Lantus.RTM. U100 as comparator with a final euglycemic clamp
setting in patients with type 1 diabetes mellitus. This study aims
to estimate an U300 dose that is equieffective to 0.4 U/kg
Lantus.RTM. U100 as assessed by parameters of blood glucose control
and blood glucose disposal provided by the clamp technique.
[0815] Insulin glargine exposure is assessed from
concentration-time profiles after repeated subcutaneous
administration at steady state, and activity as glucose utilization
per unit insulin at steady state.
[0816] The study comprises two cross-over treatments (R and T1, and
R and T2) in 2 parallel groups, with 2 treatment periods (TP1, TP2)
and 2 sequences, each. There are one screening visit (D-21 to D-3),
treatment visits (D1 to D10 in TP1 and TP2 with evening dosing),
with in-house periods (D1 to D4 morning and D8 morning to D10
evening for clamp assessments) and one end-of-study visit (between
D7 to D10 after last dosing) with final assessment of safety
parameters.
[0817] The Lantus.RTM. U100 dose of 0.4 U/kg selected for the study
is well characterized to provide euglycaemic blood glucose control
in type 1 diabetes patients and has been readily investigated in
other clamp studies with type 1 diabetes patients.
[0818] Two different doses are tested for insulin glargine U300,
0.4 and 0.6 U/kg. This dose range allows interpolating an
approximate dose equieffective to 0.4 U/kg Lantus.RTM. U100. The
dose of 0.4 U/kg of insulin glargine U300 has already been tested
in healthy volunteers and subjects with type diabetes mellitus (see
foregoing examples) and was found to be less active than 0.4 U/kg
Lantus.RTM. U100 within 30 and 36 hours, respectively, the
predefined ends of the observation periods. Blood glucose control
with 0.4 U/kg insulin glargine U300 required less total glucose
disposition than that of reference medication (0.4 U/kg Lantus.RTM.
U100). A correspondingly higher dose of insulin glargine U300, e.g.
0.6 U/kg insulin glargine U300, is expected to result in even
tighter blood glucose control at less total glucose disposition.
Moreover, the proportional dose escalation allows exploring
exposure and effect profiles for dose-proportionality.
[0819] A study in patients with type 1 diabetes avoids confounding
impact of endogenous insulin and better permits assessment of
exposure and duration of action.
[0820] This study has a cross over design; based on the outcome of
previous studies not more than two HOE901-U300 doses will be
compared to Lantus.RTM. U100. Assessment of glucodynamic activity
of long acting insulin products requires a euglycemic clamp setting
beyond 24 hours, the predefined injection interval, owed to the
extended duration of action.
[0821] The active pharmaceutical ingredient, insulin glargine, is
the same in both formulations, U100 and U300. The doses used in
this study are within the range of regular use. Although an overall
risk of hypoglycemia is not completely excluded, it is controlled
by the euglycemic clamp technique.
Pharmacodynamics
[0822] The pharmacodynamic activity of insulin glargine is
evaluated by the euglycemic clamp technique in type 1 diabetes
patients, which is the established standard procedure to evaluate
the effect of exogenous administered insulin products on blood
glucose disposal.
[0823] Parameters specific for assessment of glucose disposition in
a euglycemic clamp setting are the body weight standardized glucose
infusion rate (GIR), total glucose disposed within 24 and 36 hours,
respectively, GIR-AUC.sub.0-24 and GIR-AUC.sub.0-36, and times to a
given percentage of GIR-AUC.sub.0-24 and GIR-AUC.sub.0-36 such as
time to 50% of GIR-AUC.sub.0-36.
[0824] Ancillary parameters are the maximum smoothed body weight
standardized GIR, GIR.sub.max, and Time to GIR.sub.max,
GIR-T.sub.max.
[0825] Duration of action of insulin glargine is derived from the
time between dosing and pre-specified deviations above the
euglycemic (clamp) level.
[0826] Glucose monitoring is performed for 36 hours due to the long
duration of action of insulin glargine after subcutaneous
administration
Pharmacokinetics
[0827] Due to the sustained release nature of insulin glargine
there is a lack of pronounced peaks in the concentration profile.
Therefore, the time to 50% of INS-AUC (e.g. T.sub.50%
INS-AUC.sub.0-36) is calculated as a measure for the time location
of the insulin glargine exposure profile, and INS-C.sub.max and
INS-T.sub.max will serve as additional measures.
Primary Study Objectives
[0828] The primary objective of the study is to assess the blood
glucose control and the required exogenous glucose consumption of
two different insulin glargine U300 doses versus 0.4 U/kg
Lantus.RTM. U100 in steady state.
Secondary Study Objectives
[0829] The secondary objectives of the study are to assess in
steady state, the exposure ratios of two different insulin glargine
U300 doses versus 0.4 U/kg Lantus.RTM. U100, to compare the
duration of action of two different insulin glargine U300 doses
versus 0.4 U/kg Lantus.RTM. U100, to explore the dose response and
dose exposure relationship of insulin glargine U300, and to asses
the safety and tolerability of insulin glargine U300 in subjects
with type 1 diabetes.
Example 16
Change of Dissolution Properties of Acidic Formulations of
Long-Acting Insulins at Higher Concentrations
[0830] The influence of the higher concentrations of insulin
glargine formulations with regard to dissolution properties are
investigated by using an in-vitro test system. To do so,
precipitation studies are performed using a phosphate buffer with a
pH of 7.4, simulating the in-vivo conditions.
[0831] The supernatant of the precipitated insulin is investigated
using HPLC technique to determine the insulin glargine content.
[0832] Detailed Description of the Studies:
Preparation of the Precipitation Buffer Solution:
[0833] 19.32 mg sodium dihydrogen phosphate monohydrate (M: 137.98
g/mol) are dissolved per mL water. 0.1 M sodium hydroxide or 0.1 M
hydrochloric acid is used for adjustment of the pH to 7.4.
Performance of the Precipitation Studies:
[0834] Solutions of insulin glargine drug product having
concentrations of up to 1000 U/mL and comprising the same total
amount of insulin glargine and the buffer are placed in plastic
tubes and are slightly shaken. After precipitation of the insulin
glargine the dispersions are centrifuged at slow rotations for a
pre-defined time period. A defined volume of the dissolution medium
is taken out and replaced with fresh buffer medium.
Determination of the Insulin Content:
[0835] The content of insulin glargine in the samples from the
supernatant is quantified against the respective insulin reference
standard by reverse-phase-HPLC using a two mobile phase system,
containing a sodium dihydrogenphosphate buffer in water, sodium
chloride (NaCl) and different amounts of acetonitrile.
[0836] As stationary phase an octadodecyl-column is used, detection
wavelength is 215 nm.
[0837] The release profile of insulin glargine from the higher
concentrated solutions (e.g. U500 and U1000) is flatter and
prolonged compared to Lantus U100.
Example 17
Microscopic Investigation of Precipitates
[0838] The precipitates of insulin glargine formulations having
concentrations of 100 U/mL, 300 U/mL, 500 U/mL 700 U/mL and 1000
U/mL have been investigated by microscopy. Said formulations (with
an identical amount of 60 U of insulin glargine) have been
precipitated in 200 .mu.L of a phosphate buffer, pH 7.4 and were
investigated by transmitted light optical microscope (Olympus Model
BX61) with the magnitudes 100.times., the pictures are shown in the
following also presenting the maximum diameters. These
investigations revealed differences in the precipitations
characteristics, leading to remarkable bigger particles with
increasing concentrations. The results are shown in FIGS.
8A-8E.
Example 18
Blood Glucose Lowering Effect of Insulin Glargine in Dogs
[0839] The blood glucose lowering effect of insulin glargine was
evaluated in healthy, normoglycemic Beagle dogs. The dogs received
single subcutaneous injections of 0.3 IU/kg. Venous blood glucose
was determined before the first injection and subsequently up to 24
h.
[0840] Animals were taken from cohort of .about.30 healthy,
normoglycemic male Beagle dogs, originally obtained from Harlan.
The dogs were maintained in kennel groups under standardized
conditions. The day before study start the dogs were randomly
distributed to study cages. They were fasted 18 hours prior to
start and throughout the experiment with free access to tap water.
Body weight of the dogs in the present study was between 13 and 27
kg. After each experiment the dogs were allowed to recover for at
least two weeks.
[0841] The animals were randomized to groups of n=6. At time point
zero the animals were treated with single doses of the test
compound. Insulin glargine was administered as a single
subcutaneous injection a dose of 0.3 IU/kg.
[0842] Blood sampling was performed consecutively via puncture of
the forearm vein (Vena cephalica) before drug administration (0 h)
and thereafter up to 24 hours. Blood glucose was determined
enzymatically (Gluco-quant.RTM. Glucose/HK kit on Roche/Hitachi
912).
[0843] The effect on blood glucose following subcutaneous injection
of differently concentrated preparations of insulin glargine, 100
and 300 units/mL, was tested in healthy, normoglycemic
Beagle Dogs
[0844] With increasing insulin glargine concentration the mean time
of action increased from 6.8 h (U100) to 7.69 h (U300),
respectively.
[0845] By increasing the glargine concentration from 100 to 300
U/mL the blood glucose decreasing time-action profile was changed
towards a flatter and prolonged activity in the dog
[0846] The current data in dogs is consistent with data in humans
showing that higher drug concentrations of insulin glargine are
positively correlated with profile and longer duration of
action.
LIST OF ABBREVIATIONS
.degree. C. Degrees Celsius
ABE Average Bioequivalence
AE Adverse Event
ALT Alanine Aminotransferase
[0847] aPPT activated Partial Thromboplastin Time
ARF Acute Renal Failure
AST Aspartate Aminotransferase
.beta.-HCG Beta-Human Choriongonadotropine
[0848] bpm beats per minute cm centimeter
CPK Creatinine Phosphokinase
CRF Case Report Form
DRF Discrepancy Resolution Form
ECG Electrocardiogram
[0849] EOS End-of-study (visit)
GCP Good Clinical Practice
[0850] GGT Gamma-glutamyl transferase
Hb Hemoglobin
[0851] HbA1c Glycocylated hemoglobin HBs Hepatitis B surface
Hct Hematocrit
HCV Hepatitis C Virus
HIV Human Immunodeficiency Virus
HR Heart Rate
INN International Nonproprietary Name
[0852] INR International Normalized Ratio (prothrombin time)
IP Investigational Product
IRB/IEC Institutional Review Board/Independent Ethics Committee
Kg Kilogram
[0853] LOQ Limit of quantification
PT Prothrombin Time
[0854] QTc QT interval automatically corrected by the ECG machine
QTcB QT interval corrected by Bazett formula QTcF QT interval
corrected by Fridericia formula QtcN QT interval corrected by a
population approach QtcNi QT interval corrected by individual
population approach RBC Red Blood Cell count
SBP Systolic Blood Pressure
[0855] SCR Screening (visit)
UDS Urine Drug Screen
[0856] ULN Upper Limit of Normal range WBC White Blood Cell
count
* * * * *