NZ710882B2 - Novel insulin analog and use thereof - Google Patents
Novel insulin analog and use thereof Download PDFInfo
- Publication number
- NZ710882B2 NZ710882B2 NZ710882A NZ71088214A NZ710882B2 NZ 710882 B2 NZ710882 B2 NZ 710882B2 NZ 710882 A NZ710882 A NZ 710882A NZ 71088214 A NZ71088214 A NZ 71088214A NZ 710882 B2 NZ710882 B2 NZ 710882B2
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- New Zealand
- Prior art keywords
- insulin
- insulin analog
- immunoglobulin
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Classifications
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
- A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Abstract
The present invention relates to: an insulin analog, wherein the insulin titer is reduced compared with a natural form and the binding force to an insulin receptor is reduced in order to increase the half-life of insulin in the blood; a conjugate, wherein the insulin analog and a carrier are connected; an extended release preparation containing the conjugate; and a method for preparing the conjugate. ed; an extended release preparation containing the conjugate; and a method for preparing the conjugate.
Description
[DESCRIPTION]
[Invention Title]
Novel insulin analog and use thereof
[Technical Field]
The present invention relates to an insulin analog that
has a d insulin titer and a reduced insulin or
binding affinity compared to the native form for the purpose
of increasing the blood half-life of insulin, a conjugate
prepared by linking the insulin analog and a carrier, a longacting
formulation including the conjugate, and a method for
preparing the conjugate.
[Background Art]
In vivo proteins are known to be eliminated via various
routes, such as degradation by proteolytic enzymes in blood,
ion through the , or clearance by receptors. Thus,
many efforts have been made to improve therapeutic efficacy by
avoiding the protein clearance mechanisms and increasing halflife
of physiologically active proteins.
On the other hand, insulin is a hormone secreted by the
as of the human body, which tes blood glucose
levels, and plays a role in maintaining normal blood glucose
levels while carrying surplus glucose in the blood to cells to
provide energy for cells. In diabetic ts, however,
insulin does not function properly due to lack of insulin,
resistance to insulin, and loss of beta-cell function, and
thus glucose in the blood cannot be utilized as an energy
source and the blood glucose level is elevated, leading to
hyperglycemia. Eventually, urinary excretion occurs,
contributing to development of various complications.
Therefore, insulin therapy is essential for patients with
abnormal insulin secretion (Type I) or insulin resistance
(Type II), and blood glucose levels can be normally regulated
by insulin administration. However, like other n and
peptide hormones, insulin has a very short in vivo half-life,
and thus has a disadvantage of repeated administration. Such
frequent administration causes severe pain and fort for
the patients. For this reason, in order to improve quality of
life by increasing in vivo half-life of the protein and
reducing the administration frequency, many s on protein
formulation and chemical conjugation (fatty acid conjugate,
polyethylene r ate) have been conducted.
Commercially ble long-acting insulin includes insulin
glargine manufactured by Sanofi Aventis s, lasting for
about 20-22 hours), and insulin detemir (levemir, lasting for
about 18-22 hours) and tresiba (degludec, lasting for about 40
hours) manufactured by Novo Nordisk. These long-acting insulin
formulations produce no peak in the blood insulin
concentration, and thus they are suitable as basal insulin.
However, e these formulations do not have sufficiently
long half-life, the disadvantage of one or two injections per
day still remains. Accordingly, there is a limitation in
achieving the ed goal that administration frequency is
remarkably reduced to improve convenience of diabetic patients
in need of long-term administration.
The previous research reported a specific in vivo insulin
clearance process; 50% or more of insulin is removed in the
kidney and the rest is d via a receptor mediated
clearance (RMC) process in target sites such as , fat,
liver, etc.
In this , many studies, including J Pharmacol Exp
Ther (1998) 286: 959, Diabetes Care (1990) 13: 923, Diabetes
(1990) 39: 1033, have reported that in vitro activity is
reduced to avoid RMC of insulin, thereby increasing the blood
level. However, these insulin analogs having reduced receptor
g ty cannot avoid renal clearance which is a main
clearance mechanism, although RMC is reduced. Accordingly,
there has been a limit in remarkably increasing the blood
half-life.
Under this background, the present inventors have made
many efforts to se the blood half-life of insulin. As a
result, they found that a novel insulin analog having no
native insulin sequence but a non-native n sequence
shows a reduced in-vitro titer and a reduced insulin receptor
binding affinity, and therefore, its renal clearance can be
reduced. They also found that the blood half-life of insulin
can be further sed by linking the insulin analog to an
immunoglobulin Fc fragment as a representative carrier
effective for half-life improvement, thereby completing the
present invention.
[Disclosure]
[Technical Problem]
An object of the t invention is to provide an
insulin analog that is prepared to have a reduced in-vitro
titer for the purpose of prolonging in vivo ife of
insulin, and a conjugate prepared by linking a carrier thereto.
Specifically, one object of the t invention is to
provide an insulin analog having a reduced insulin titer,
compared to the native form.
Another object of the present invention is to provide an
insulin analog conjugate that is prepared by g the
insulin analog to the carrier.
Still r object of the present invention is to
provide a long-acting insulin formulation including the
insulin analog conjugate.
Still another object of the t invention is to
provide a method for preparing the n analog conjugate.
Still another object of the present invention is to
provide a method for increasing in vivo half-life using the
insulin analog or the insulin analog conjugate prepared by
linking the insulin analog to the carrier.
Still another object of the present invention is to
provide a method for treating insulin-related diseases,
including the step of administering the insulin analog or the
insulin analog conjugate to a subject in need thereof.
[Technical Solution]
In one aspect to achieve the above s, the present
invention es an insulin analog having a reduced insulin
titer, compared to the native form, in which an amino acid of
B chain or A chain is modified.
In one specific embodiment, the present invention
provides an insulin analog having a reduced insulin or
binding affinity.
In another specific ment, the present invention
provides an insulin analog having a d insulin titer
compared to the native form, wherein an amino acid in insulin
is modified by substituting the 14th amino acid of the A chain
with glutamic acid or asparagine.
In another specific embodiment, the present invention
provides a tive insulin analog, in which one amino acid
selected from the group consisting of 8th amino acid, 23th amino
acid, 24th amino acid, and 25th amino acid of B chain and 1th
amino acid, 2th amino acid, and 19th amino acid of A chain is
substituted with alanine, or in which 14th amino acid of A
chain is substituted with glutamic acid or asparagine in the
n analog according to the present invention.
In still another specific embodiment, the t
invention provides an insulin analog, in which the insulin
analog according to the present invention is selected from the
group consisting of SEQ ID NOs. 20, 22, 24, 26, 28, 30, 32, 34
and 36.
In another , the present invention provides an
insulin analog conjugate that is prepared by linking the above
described insulin analog to a carrier capable of prolonging
half-life.
In one specific embodiment, the present invention
provides an insulin analog conjugate, in which the insulin
analog conjugate is prepared by g (i) the above
described insulin analog and (ii) an immunoglobulin Fc region
via (iii) a e linker or a non-peptidyl linker selected
from the group consisting of polyethylene glycol,
polypropylene glycol, copolymers of ethylene -propylene
glycol, polyoxyethylated polyols, polyvinyl alcohols,
polysaccharides, n, polyvinyl ethyl ether, biodegradable
polymers, lipid polymers, chitins, hyaluronic acid, and
combination thereof.
In still another aspect, the present invention provides a
long-acting insulin formulation including the above described
insulin analog conjugate, in which in vivo duration and
stability are increased.
In one specific embodiment, the present invention
provides a long-acting formulation that is used for the
ent of diabetes.
In another ment, the present invention provides a
method for preparing the above described insulin analog
conjugate.
In still another specific embodiment, the present
invention provides a method for increasing in vivo half-life
using the insulin analog or the insulin analog conjugate that
is prepared by linking the insulin analog and the carrier.
In still another specific embodiment, the present
invention provides a method for treating insulin-related
diseases, including the step of administering the insulin
analog or the insulin analog conjugate to a subject in need
thereof.
[Advantageous s]
A non-native insulin analog of the present ion has
a d insulin titer and a reduced insulin or binding
affinity, compared to the native form, and thus avoids in vivo
clearance mechanisms. Therefore, the insulin analog has
increased blood half-life in vivo, and an n analogimmunoglobulin
Fc conjugate prepared by using the same shows
ably increased blood half-life, thereby improving
convenience of patients in need of insulin administration.
[Description of Drawings]
shows the result of analyzing purity of an insulin
analog by protein electrophoresis, which is the result of the
representative n analog, Analog No. 7 (Lane 1: size
marker, Lane 2: native insulin, Lane 3: insulin analog (No. 7);
shows the result of analyzing purity of an insulin
analog by high pressure tography, which is the result of
the representative insulin analog, Analog No. 7 ((A) RP-HPLC,
(B) SE-HPLC);
shows the result of peptide mapping of an insulin
, which is the result of the representative insulin
analog, Analog No. 7 ((A) native insulin, (B) insulin analog
(No. 7));
shows the result of analyzing purity of an insulin
analog-immunoglobulin Fc conjugate by protein electrophoresis,
which is the result of the representative insulin analog,
Analog No. 7 (Lane 1: size marker, Lane 2: insulin analog (No.
7)-immunoglobulin Fc conjugate);
shows the result of analyzing purity of an insulin
analog-immunoglobulin Fc conjugate by high pressure
tography, which is the result of the representative
insulin analog, Analog No. 7 ((A) C, (B) SE-HPLC, (C)
IE-HPLC); and
shows the result of analyzing pharmacokinetics of
native insulin-immunoglobulin Fc ate and insulin analogimmunoglobulin
Fc conjugate in normal rats, which is the
result of the representative insulin , Analog No. 7 (○:
native insulin-immunoglobulin Fc conjugate (21.7 nmol/kg), ●:
native insulin-immunoglobulin Fc conjugate (65.1 nmol/kg), □:
insulin analog-immunoglobulin Fc conjugate (21.7 nmol/kg), ■:
insulin analog-immunoglobulin Fc conjugate (65.1 nmol/kg). (A)
native insulin-immunoglobulin Fc conjugate and insulin analog
(No. 7)-immunoglobulin Fc conjugate, (B) native insulinimmunoglobulin
Fc conjugate and insulin analog (No. 8)-
globulin Fc ate, (C) native insulin-immunoglobulin
Fc conjugate and insulin analog (No. 9)-immunoglobulin Fc
conjugate).
[Best Mode]
The t invention s to an insulin analog having
a reduced in-vitro titer. This insulin analog is characterized
in that it has the non-native insulin sequence and therefore,
has a reduced insulin receptor binding affinity, compared to
the native insulin, and consequently, receptor-mediated
nce is remarkably reduced by increased dissociation
constant, resulting in an increase in blood half-life.
As used herein, the term “insulin analog” includes
various s having reduced insulin titer, compared to the
native form.
The insulin analog may be an insulin analog having
reduced insulin titer, compared to the native form, in which
an amino acid of B chain or A chain of insulin is modified.
The amino acid sequences of the native insulin are as follows.
-A chain:
Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-
Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO. 37)
-B chain:
Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-
Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-
Thr (SEQ ID NO. 38)
The insulin analog used in Examples of the present
invention is an insulin analog prepared by a genetic
ination technique. However, the present ion is not
limited thereto, but es all insulins having reduced invitro
titer. Preferably, the insulin analog may include
inverted insulins, insulin variants, insulin fragments or the
like, and the preparation method may include a solid phase
method as well as a genetic ination technique, but is
not limited thereto.
The insulin analog is a peptide retaining a function of
controlling blood e in the body, which is identical to
that of insulin, and this e includes insulin agonists,
derivatives, fragments, variants thereof or the like.
The insulin agonist of the present invention refers to a
substance which is bound to the in vivo receptor of insulin to
exhibit the same biological activities as insulin, regardless
of the structure of insulin.
The insulin analog of the t invention denotes a
peptide which shows a sequence homology of at least 80% in an
amino acid sequence as compared to A chain or B chain of the
native insulin, has some groups of amino acid residues altered
in the form of chemical substitution (e.g., alpha-methylation,
alpha-hydroxylation), removal (e.g., deamination) or
modification (e.g., N-methylation), and has a function of
controlling blood glucose in the body. With t to the
objects of the present invention, the insulin analog is an
insulin analog having a reduced insulin receptor binding
affinity, compared to the native form, and n analogs
having a reduced insulin titer compared to the native form are
included t limitation.
As long as the insulin analog is able to exhibit low
receptor-mediated internalization or receptor-mediated
clearance, its type and size are not particularly limited. An
insulin analog, of which major in vivo clearance mechanism is
the receptor-mediated internalization or receptor-mediated
clearance, is le for the objects of the present
invention.
The insulin fragment of the present invention denotes the
type of insulin in which one or more amino acids are added or
deleted, and the added amino acids may be non-native amino
acids (e.g., D-type amino acid). Such n fragments retain
the on of controlling blood glucose in the body.
The insulin variant of the present invention denotes a
peptide which differs from insulin in one or more amino acid
sequences, and retains the function of controlling blood
glucose in the body.
The respective methods for preparation of insulin
agonists, derivatives, fragments and variants of the present
invention can be used independently or in ation. For
example, peptides of which one or more amino acid ces
differ from those of n and which have deamination at the
amino-terminal amino acid residue and also have the function
of controlling blood glucose in the body are included in the
present invention.
Specifically, the insulin analog may be an insulin analog
in which one or more amino acids selected from the group
consisting of 8th amino acid, 23th amino acid, 24th amino acid,
and 25th amino acid of B chain and 1th amino acid, 2th amino
acid, 14th amino acid and 19th amino acid of A chain are
substituted with other amino acid, and preferably, in which
one or more amino acids selected from the group consisting of
8th amino acid, 23th amino acid, 24th amino acid, and 25th amino
acid of B chain and 1th amino acid, 2th amino acid, and 19th
amino acid of A chain are substituted with alanine or in which
14 th amino acid of A chain is tuted with glutamic acid or
asparagine. In addition, the insulin analog may be selected
from the group consisting of SEQ ID NOs. 20, 22, 24, 26, 28,
, 32, 34 and 36, but may include any insulin analog having a
d insulin receptor binding affinity without limitation.
ing to one embodiment of the present invention, the
insulin analogs of SEQ ID NOs. 20, 22, 24, 26, 28, 30, 32, 34
and 36, in particular, the representative insulin analogs, 7,
8, and 9 (SEQ ID NOs. 32, 34, and 36) were found to have
reduced n receptor binding affinity in vitro, compared
to the native form (Table 4).
In another aspect, the present invention provides an
insulin analog conjugate that is prepared by linking the
insulin analog and a carrier.
As used herein, the term er” denotes a substance
capable of increasing in vivo half-life of the linked insulin
analog. The insulin analog according to the t invention
is characterized in that it has a remarkably reduced insulin
receptor binding affinity, compared to the native form, and
avoids receptor-mediated clearance or renal clearance.
Therefore, if a carrier known to se in vivo ife
when linked to the known various physiologically active
polypeptides is linked with the insulin analog, it is apparent
that in vivo half-life can be improved and the resulting
conjugate can be used as a cting formulation.
For example, because half-life improvement is the first
ty, the carrier to be linked with the novel insulin
having a reduced titer is not limited to the immunoglobulin Fc
region. The carrier includes a biocompatible material that is
able to g in vivo half-life by linking it with any one
biocompatible material, capable of reducing renal clearance,
selected from the group consisting of various polymers (e.g.,
polyethylene glycol and fatty acid, albumin and fragments
thereof, ular amino acid sequence, etc.), albumin and
fragments thereof, albumin-binding materials, and polymers of
repeating units of particular amino acid ce, antibody,
antibody fragments, FcRn-binding materials, in vivo connective
tissue or derivatives thereof, nucleotide, fibronectin,
transferrin, saccharide, and polymers, but is not limited
thereto. In addition, the method for linking the biocompatible
material e of prolonging in vivo half-life to the
insulin analog having a reduced titer includes genetic
recombination, in vitro conjugation or the like. Examples of
the biocompatible al may e an FcRn-binding
material, fatty acid, polyethylene glycol, an amino acid
fragment, or albumin. The FcRn-binding material may be an
immunoglobulin Fc .
The n analog and the biocompatible material as the
carrier may be linked to each other via a peptide or a nonpeptidyl
polymer as a linker.
The insulin conjugate may be an insulin analog conjugate
that is prepared by linking (i) the insulin analog and (ii)
the immunoglobulin Fc region via (iii) a peptide linker or a
non-peptidyl linker selected from the group consisting of
polyethylene glycol, polypropylene glycol, copolymers of
ethylene glycol-propylene glycol, polyoxyethylated polyol,
polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl
ether, biodegradable polymer, lipid polymers, chitins,
onic acid and combination thereof.
In one ic embodiment of the insulin analog
conjugate of the present invention, a non-peptidyl polymer as
a linker is linked to the amino terminus of B chain of the
n analog. In another ic embodiment of the
conjugate of the present invention, a non-peptidyl polymer as
a linker is linked to the residue of B chain of the insulin
analog. The modification in A chain of insulin leads to a
reduction in the activity and safety. In these embodiments,
therefore, the non-peptidyl polymer as a linker is linked to B
chain of insulin, thereby maintaining insulin activity and
improving safety.
As used herein, the term “activity” means the ability of
insulin to bind to the insulin receptor, and means that
insulin binds to its receptor to exhibit its action. Such
binding of the non-peptidyl polymer to the amino terminus of B
chain of n of the present invention can be achieved by
pH control, and the preferred pH range is 4.5 to 7.5.
As used herein, the term “N-terminus” can be used
interchangeably with “N-terminal region”.
In one specific Example, the present inventors prepared
an insulin analog-PEG-immunoglobulin Fc conjugate by g
PEG to the inus of an immunoglobulin Fc region, and
selectively coupling the N-terminus of B chain of n
o. The serum half-life of this insulin analog-PEG-
immunoglobulin Fc conjugate was increased, compared to nonconjugate
, and it showed a hypoglycemic effect in disease
animal models. Therefore, it is apparent that a new longacting
insulin ation maintaining in vivo activity can be
prepared.
The immunoglobulin Fc region is safe for use as a drug
carrier because it is a biodegradable polypeptide that is in
vivo metabolized. Also, the immunoglobulin Fc region has a
relatively low molecular weight, as compared to the whole
immunoglobulin les, and thus, it is advantageous in
terms of preparation, purification and yield of the conjugate.
The immunoglobulin Fc region does not n a Fab fragment,
which is highly non-homogenous due to different amino acid
sequences according to the antibody subclasses, and thus it
can be expected that the globulin Fc region may greatly
increase the homogeneity of substances and be less antigenic
in blood.
As used herein, the term “immunoglobulin Fc region”
refers to a protein that contains the chain constant
region 2 (CH2) and the heavy-chain constant region 3 (CH3) of
an globulin, ing the variable regions of the heavy
and light chains, the heavy-chain constant region 1 (CH1) and
the light-chain constant region 1 (CL1) of the immunoglobulin.
It may further include a hinge region at the heavy-chain
constant region. Also, the immunoglobulin Fc region of the
present invention may contain a part or all of the Fc region
including the heavy-chain constant region 1 (CH1) and/or the
light-chain constant region 1 (CL1), except for the variable
regions of the heavy and light chains of the immunoglobulin,
as long as it has an effect substantially similar to or better
than that of the native form. Also, it may be a fragment
having a deletion in a relatively long portion of the amino
acid sequence of CH2 and/or CH3.
That is, the immunoglobulin Fc region of the present
ion may include 1) a CH1 domain, a CH2 domain, a CH3
domain and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3)
a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3
domain, 5) a combination of one or more domains and an
immunoglobulin hinge region (or a portion of the hinge region),
and 6) a dimer of each domain of the chain nt
regions and the light-chain constant region.
Further, the immunoglobulin Fc region of the present
invention includes a sequence derivative (mutant) thereof as
well as a native amino acid sequence. An amino acid sequence
derivative has a sequence that is different from the native
amino acid sequence due to a deletion, an insertion, a servative
or vative substitution or combinations
thereof of one or more amino acid residues. For example, in an
IgG Fc, amino acid residues known to be important in binding,
at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331,
may be used as a suitable target for modification.
In addition, other various derivatives are possible,
ing derivatives having a deletion of a region capable of
forming a disulfide bond, a deletion of several amino acid
residues at the N-terminus of a native Fc form, or an addition
of methionine residue to the N-terminus of a native Fc form.
Furthermore, to remove effector functions, a deletion may
occur in a ment-binding site, such as a C1q-binding site
and an ADCC (antibody dependent cell mediated cytotoxicity)
site. Techniques of preparing such sequence derivatives of the
immunoglobulin Fc region are disclosed in WO 97/34631 and WO
96/32478.
Amino acid exchanges in proteins and peptides, which do
not generally alter the activity of les, are known in
the art (H.Neurath, R.L.Hill, The Proteins, Academic Press,
New York, 1979). The most commonly occurring exchanges are
Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn,
Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,
Leu/Val, Ala/Glu, Asp/Gly, in both directions.
The Fc , if desired, may be ed by
phosphorylation, sulfation, acrylation, glycosylation,
ation, farnesylation, acetylation, amidation or the like.
The aforementioned Fc derivatives are derivatives that
have a biological activity cal to that of the Fc region
of the present invention or improved structural stability
against heat, pH, or the like.
In addition, these Fc regions may be obtained from native
forms isolated from humans and other animals including cows,
goats, swine, mice, rabbits, hamsters, rats and guinea pigs,
or may be recombinants or derivatives thereof, obtained from
transformed animal cells or microorganisms. Here, they may be
obtained from a native globulin by isolating whole
immunoglobulins from human or animal organisms and treating
them with a proteolytic enzyme. Papain digests the native
immunoglobulin into Fab and Fc regions, and pepsin treatment
results in the production of pF’c and F(ab)2. These fragments
may be subjected to size-exclusion chromatography to isolate
Fc or pF’c.
Preferably, a human-derived Fc region is a recombinant
immunoglobulin Fc region that is obtained from a microorganism.
In addition, the immunoglobulin Fc region may be in the
form of having native sugar chains, increased sugar chains
compared to a native form or decreased sugar chains compared
to the native form, or maybe in a deglycosylated form. The
se, decrease or removal of the immunoglobulin Fc sugar
chains may be achieved by methods common in the art, such as a
al method, an enzymatic method and a genetic ering
method using a microorganism. Here, the removal of sugar
chains from an Fc region results in a sharp decrease in
binding affinity to the complement (c1q) and a se or
loss in antibody-dependent cell-mediated cytotoxicity or
ment-dependent xicity, thereby not inducing
unnecessary immune responses in-vivo. In this regard, an
immunoglobulin Fc region in a deglycosylated or aglycosylated
form may be more suitable to the object of the present
invention as a drug carrier.
The term cosylation”, as used herein, means to
enzymatically remove sugar moieties from an Fc region, and the
term “aglycosylation” means that an Fc region is produced in
an unglycosylated form by a prokaryote, preferably, E. coli.
On the other hand, the immunoglobulin Fc region may be
derived from humans or other animals including cows, goats,
swine, mice, rabbits, hamsters, rats and guinea pigs, and
preferably humans. In addition, the immunoglobulin Fc region
may be an Fc region that is derived from IgG, IgA, IgD, IgE
and IgM, or that is made by combinations thereof or hybrids
thereof. Preferably, it is derived from IgG or IgM, which is
among the most abundant proteins in human blood, and most
preferably, from IgG which is known to enhance the half-lives
of ligand-binding proteins.
On the other hand, the term “combination”, as used herein,
means that polypeptides encoding single-chain immunoglobulin
Fc regions of the same origin are linked to a single-chain
polypeptide of a different origin to form a dimer or multimer.
That is, a dimer or multimer may be formed from two or more
nts selected from the group consisting of IgG Fc, IgA Fc,
IgM Fc, IgD Fc and IgE Fc fragments.
The term “hybrid”, as used herein, means that sequences
encoding two or more immunoglobulin Fc regions of different
origin are present in a single-chain immunoglobulin Fc region.
In the t ion, s types of s are
le. That is, domain hybrids may be composed of one to
four domains selected from the group ting of CH1, CH2,
CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc, and
may include the hinge region.
On the other hand, IgG is divided into IgG1, IgG2, IgG3
and IgG4 subclasses, and the present invention includes
combinations and hybrids f. Preferred are IgG2 and IgG4
subclasses, and most preferred is the Fc region of IgG4 rarely
having effector functions such as CDC (complement dependent
cytotoxicity). That is, as the drug carrier of the present
invention, the most preferable immunoglobulin Fc region is a
human IgG4-derived non-glycosylated Fc . The
humanderived Fc region is more preferable than a non-human
derived Fc region which may act as an antigen in the human
body and cause undesirable immune responses such as the
production of a new antibody against the antigen.
In the specific embodiment of the n analog
ate, both ends of the non-peptidyl polymer may be linked
to the N-terminus of the globulin Fc region and the
amine group of the N-terminus of B chain of the insulin analog
or the ε-amino group of the internal lysine residue or the
thiol group of B chain, respectively.
The Fc region-linker-insulin analog of the present
invention is made at various molar ratios. That is, the number
of the Fc fragment and/or linker linked to a single insulin
analog is not limited.
In addition, the linkage of the Fc region, a certain
linker, and the n analog of the present invention may
include all types of covalent bonds and all types of noncovalent
bonds such as en bonds, ionic ctions, van
der Waals forces and hydrophobic interactions when the Fc
region and the insulin analog are expressed as a fusion
protein by genetic recombination. However, with respect to the
physiological activity of the insulin analog, the linkage is
ably made by covalent bonds, but is not limited thereto.
On the other hand, the Fc region of the present invention,
a certain linker and the insulin analog may be linked to each
other at an N-terminus or C-terminus, and preferably at a free
group, and especially, a covalent bond may be formed at an
amino terminal end, an amino acid residue of lysine, an amino
acid residue of histidine, or a free ne residue.
In addition, the linkage of the Fc region of the present
invention, a certain linker, and the insulin analog may be
made in a certain direction. That is, the linker may be linked
to the N-terminus, the C-terminus or a free group of the
globulin Fc , and may also be linked to the N-
terminus, the C-terminus or a free group of the insulin analog.
The non-peptidyl linker may be linked to the N-terminal
amine group of the immunoglobulin fragment, and is not limited
to any of the lysine residue or cysteine residue of the
immunoglobulin fragment sequence.
Further, in the specific embodiment of the insulin analog
conjugate, the end of the non-peptidyl polymer may be linked
to the internal amino acid residue or free ve group
capable of binding to the reactive group at the end of the
non-peptidyl polymer, in addition to the N-terminus of the
immunoglobulin Fc , but is not limited thereto.
In the present invention, the non-peptidyl polymer means
a biocompatible r including two or more ing units
linked to each other, in which the repeating units are linked
by any covalent bond excluding the peptide bond. Such nonpeptidyl
polymer may have two ends or three ends.
The non-peptidyl polymer which can be used in the present
invention may be selected from the group consisting of
polyethylene glycol, polypropylene glycol, mers of
ne glycol and propylene glycol, polyoxyethylated polyols,
polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl
ether, biodegradable polymers such as PLA lactic acid))
and PLGA (polylactic-glycolic acid), lipid polymers, chitins,
hyaluronic acid, and combinations thereof, and preferably,
polyethylene glycol. The derivatives thereof well known in the
art and being easily prepared within the skill of the art are
also included in the scope of the present invention.
The peptide linker which is used in the fusion protein
obtained by a conventional inframe fusion method has drawbacks
in that it is easily in-vivo cleaved by a lytic enzyme,
and thus a sufficient effect of increasing the blood half-life
of the active drug by a carrier cannot be obtained as expected.
In the present invention, r, the conjugate can be
prepared using the non-peptidyl linker as well as the peptide
linker. In the non-peptidyl linker, the polymer having
resistance to the lytic enzyme can be used to maintain
the blood half-life of the peptide being similar to that of
the carrier. Therefore, any non-peptidyl polymer can be used
without limitation, as long as it is a polymer having the
aforementioned function, that is, a polymer having resistance
to the in-vivo proteolytic enzyme. The non-peptidyl polymer
has a molecular weight g from 1 to 100 kDa, and
preferably, ranging from 1 to 20 kDa.
The non-peptidyl polymer of the present invention, linked
to the immunoglobulin Fc , may be one polymer or a
combination of different types of polymers.
The non-peptidyl polymer used in the present invention
has a reactive group capable of binding to the globulin
Fc region and the protein drug.
The ptidyl polymer has a reactive group at both
ends, which is preferably selected from the group consisting
of a reactive aldehyde group, a propionaldehyde group, a
butyraldehyde group, a maleimide group and a succinimide
derivative. The succinimide derivative may be succinimidyl
propionate, hydroxy succinimidyl, succinimidyl carboxymethyl,
or succinimidyl carbonate. In particular, when the nonpeptidyl
polymer has a reactive aldehyde group at both ends
thereof, it is effective in linking at both ends with a
physiologically active polypeptide and an globulin with
minimal non-specific reactions. A final product generated by
reductive alkylation by an aldehyde bond is much more stable
than that linked by an amide bond. The aldehyde reactive group
ively binds to an N-terminus at a low pH, and binds to a
lysine residue to form a covalent bond at a high pH, such as
pH 9.0.
The ve groups at both ends of the non-peptidyl
polymer may be the same as or different from each other. For
example, the non-peptide polymer may possess a maleimide group
at one end, and an aldehyde group, a propionaldehyde group or
a butyraldehyde group at the other end. When a polyethylene
glycol having a reactive hydroxy group at both ends thereof is
used as the ptidyl polymer, the y group may be
activated to various reactive groups by known al
reactions, or a polyethylene glycol having a commercially
available modified reactive group may be used so as to prepare
the single chain insulin analog conjugate of the present
invention.
The insulin analog conjugate of the present invention
ins in vivo activities of the conventional insulin such
as energy metabolism and sugar metabolism, and also increases
blood half-life of the insulin analog and markedly increases
duration of in-vivo efficacy of the peptide, and therefore,
the conjugate is useful in the treatment of diabetes.
In one Example of the present invention, it was confirmed
that the insulin analog having a reduced insulin receptor
g affinity exhibits much higher in vivo half-life than
the native insulin conjugate, when linked to the carrier
capable of prolonging in vivo half-life (.
In another aspect, the present invention provides a longacting
insulin ation including the n analog
ate. The long-acting n ation may be a longacting
insulin formulation having increased in vivo duration
and stability. The long-acting formulation may be a
pharmaceutical composition for the treatment of diabetes.
The pharmaceutical composition including the conjugate of
the present invention may include pharmaceutically acceptable
carriers. For oral administration, the ceutically
acceptable carrier may include a binder, a lubricant, a
disintegrator, an excipient, a solubilizer, a dispersing agent,
a stabilizer, a suspending agent, a coloring agent, a perfume
or the like. For injectable preparations, the pharmaceutically
acceptable carrier may include a buffering agent, a preserving
agent, an analgesic, a solubilizer, an isotonic agent, and a
stabilizer. For preparations for topical administration, the
pharmaceutically acceptable carrier may include a base, an
excipient, a lubricant, a preserving agent or the like. The
pharmaceutical composition of the present invention may be
formulated into a variety of dosage forms in combination with
the aforementioned pharmaceutically acceptable carriers. For
example, for oral administration, the pharmaceutical
ition may be formulated into tablets, s, capsules,
elixirs, suspensions, syrups or wafers. For injectable
preparations, the pharmaceutical ition may be formulated
into single-dose ampule or multidose container. The
pharmaceutical composition may be also formulated into
solutions, suspensions, tablets, pills, capsules and ned
release preparations.
On the other hand, examples of carriers, excipients and
diluents suitable for formulation include e, se,
e, sorbitol, mannitol, xylitol, erythritol, maltitol,
starch, acacia, alginate, gelatin, calcium phosphate, calcium
silicate, ose, cellulose, microcrystalline
cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate,
propylhydroxybenzoate, talc, magnesium stearate, mineral oils
or the like.
In addition, the ceutical formulations may further
include fillers, anti-coagulating agents, lubricants,
humectants, perfumes, antiseptics or the like.
In still another aspect, the present invention provides a
method for treating n-related es, including
administering the insulin analog or the insulin analog
conjugate to a subject in need f.
The conjugate according to the present invention is
useful in the treatment of diabetes, and therefore, this
disease can be treated by administering the pharmaceutical
composition including the same.
The term “administration”, as used herein, means
introduction of a predetermined substance into a patient by a
certain suitable method. The conjugate of the present
invention may be administered via any of the common routes, as
long as it is able to reach a desired tissue. Intraperitoneal,
intravenous, intramuscular, subcutaneous, intradermal, oral,
topical, intranasal, intrapulmonary and intrarectal
administration can be performed, but the present invention is
not limited o. However, since peptides are digested upon
oral stration, active ingredients of a composition for
oral administration should be coated or formulated for
protection against degradation in the h. Preferably, the
present composition may be administered in an injectable form.
In addition, the pharmaceutical composition may be
administered using a certain apparatus capable of transporting
the active ingredients into a target cell.
Further, the pharmaceutical composition of the present
invention may be determined by several related factors
including the types of diseases to be treated, stration
routes, the patient’s age, gender, weight and severity of the
illness, as well as by the types of the drug as an active
component. Since the pharmaceutical composition of the t
invention has ent in vivo duration and titer, it has an
advantage of greatly reducing administration frequency of the
pharmaceutical formulation of the present invention.
In still another aspect, the present invention es a
method for preparing the insulin analog conjugate, ing
preparing the insulin analog; preparing the carrier; and
linking the insulin analog and the carrier.
In still another aspect, the present invention provides a
method for increasing in vivo half-life using the insulin
analog or the insulin analog ate which is prepared by
linking the insulin analog and the r.
[Mode for Invention]
Hereinafter, the present invention will be described in
more detail with reference to es. However, these
Examples are for rative es only, and the invention
is not intended to be limited by these Examples.
Example 1: Preparation of single chain insulin analogexpressing
vector
In order to e insulin analogs, each of them having
a modified amino acid in A chain or B chain, using the native
insulin-expressing vector as a template, forward and reverse
oligonucleotides were synthesized (Table 2), and then PCR was
carried out to amplify each analog gene.
In the following Table 1, amino acid sequences modified
in A chain or B chain and analog names are given. That is,
Analog 1 represents that 1st glycine of A chain is substituted
with alanine, and Analog 4 represents that 8th glycine of B
chain is substituted with alanine.
[Table 1]
Primers for n analog amplification are given in the
following Table 2.
[Table 2]
PCR for insulin analog amplification was carried out
under conditions of 95°C for 30 seconds, 55°C for 30 seconds,
68°C for 6 minutes for 18 cycles. The insulin analog fragments
obtained under the conditions were ed into pET22b vector
to be expressed as intracellular inclusion bodies, and the
resulting expression vectors were designated as pET22b-insulin
analogs 1 to 9. The expression vectors ned nucleic acids
encoding amino acid sequences of insulin analogs 1 to 9 under
the control of T7 promoter, and insulin analog proteins were
expressed as ion bodies in host cells.
DNA sequences and protein sequences of insulin analogs 1
to 9 are given in the following Table 3.
[Table 3]
l Ammog ce SEQIDNO.
l Analog 1 DNA TlC (BTT AAC CAA CAC TUB TGT (SGC TCA CAC C76 (3T6 (3AA GCT 19
l CTC TAC CTA GT6 TGC (EGG GAA CGA (JGC TTC TTC TAC ACA CCC
l AAG ACC C(SC CGG GAG («A GAS (SAC CTG CAO (ETC: (366 {AG
i (3T6 (3A6 (:16 GGC GGG (SGC CC”? (SGT GCA GGC AGE CTG CA6
CCC WC) GO: (TO GAG (366 TU: CTG CAG AAG (GT GUS AW GTG
l l3AA CAA TGC lG'l' ACC AGE AK TL’aC TCC CTC TAC CA6 CTG GAG
i LAAC TAC TGC AAC
l Pwe Val Asn Gln His Leu CyS (filly Set His Leu Val Glu Ala Leu Tyl Lem
l Pnflem
Val Cy; (Sly GIL: Alt) (Sly le Plus Tyr ’l'hr Pm Lye Tln' Arr.) Avg (Elm Ala
l (Blu Asp Leu Calm Val Gly Gin Val Glu Leu (Sly Gly Gly Pro (Ely Ala Gly
i Ser Loeu (Eln Pro Leu Ala Leu Cwlu Gly ‘39! ten Gln Lys Arg Ala lle Val
g E(31m Gin Cys Cys Tlxr Se: lle Lys Ber LE‘U Tyr Gln Lem Calm Asn Tyr Cys
l ASH
lAnalqvg 2 'DNA TTC GTTVAAC C A}; (A? TTG €in (SEE—C TC A CA—(f (17:61:31 (3ij GET 21
l CTC TAC {TA (3T6 W3C C166 GAA CCSA 136C TFC TTC TAC ACA CCC
l :AAG ACC CBC 036 GAG GCA GAG GAC (TC: (LAG GT6 EGG CAG
i (3T6 (3A6 CTG GGC (366 C161: (ZCT (BGT (“CA (36C AGC (T6 ("AG
E CCC WC: (301‘. C TO GAG C1663 TICC C713 CA6 AAC: CGT (36C (ECG
GTG 6AA {AA TGC TGT ACE AGC ATC TGC TCC Cle TAC CAG CTG
l GAG AAC TAC TQC AN]
l ‘
l I
Wotan Pm; Val Asn (nln H15 Lea! Ly$ Lxly Ber HlS Len Val blu Ala Leu Tyr Leu_ , _ _
. , .
l rVal Cys (Sly GILL Arg (Sly Phe Phe Ty: th Pro. Lys Tl'n Arg Avg GIL] Ala
'6li Asp leu Gln Val iSly Gln Val Glu Let: (Sly Gly (Sly Pro (Sly Ala: Gly
l Sar Leu Culn Pro Leu Ala Leu Glu (Sly Ser Leu Calm Lys Arg Gly Ala Val
l Glu Gln Cys Cys Thr 39: lle Cys Ser' Leu Tyr Gln Leu‘ (33m ASH Tyr Cys
l l
Analog 3 .DNA THC GTT AAC (AA CAC TTL’B NET 66:: TCA CAC CTG GT6 (3AA GCT IV 9:
l CTC TAC CTA (ETC: TEE: (EGG 6AA CGA GGC FTC TTC TAL’ ACA CCC
AAG ACC CGC COG GAG GCA GAG GAC CTG (AG (3R3 C160 (AG
l (ETC: GAG CTG (36C (366 (36C CCT GGT (ECA Get AGC (TIC: CA6
(ICC TTG GUI CTG GAG (366 TU: (.‘TG CAG AAG CTL’ST GGC ATT GTG
l (3AA (AA TGC TGT ACC AGC ATC TGC TCC (TC TAC CA6 CTG GAG
l AAC (ECG TGC AAC
g Pnflem Pm; Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ty! Leu
l Val Cys Gly (Slu Arg Gly Phe Pile Tyl Tlu Pm Lys th A113 Arg (3le Ala
Glu Asp Lou Gin Val Ely Gin Val GIL: Leu (Sly (Sly (Sly Pro Gly Al.) Gly
l 391' Leu (Sln Pm Lem Ala lgeu Glu Gly Sel Len Gin Lys Arg (Sly Ile Val
l ,Glu Gln Cys Cys Tlir' $9! Ile Eys SBI Lem Ty; Gln Lem lilu Asn Ala ifys
Analog 4 DNA TTC GTT AAC {AA CAC TTG TGT GUS TCA CAC (TC: (,3th 6AA GCT ZS
CTC TAC CTA GTO TESC 6(3er GAA CGA (SGC T—TC TTC TAC ACA CCC
AAG ACC CCIC CGG GAG GCA GAG GAC CTG CAL-1 GTG 666 (AG
GT6 (3A6 CTG GGC (366 GGC CCT GGT (ECA GGC AGC. CTG; (:AG
CCC TTG GCC CTG (SAG 6.th TCL’ CH} CAG AAG CGT GGC ATT GTG
(3AA CAA TGC TGT ACC AGC ATC TGC ICC (TC TA(‘ (AG L'ITG GAG
AAC TAC TGC AAC
Protein Pile Val Am (31m His Leu Cys Ala Ser His Leu Val Glu Ala Lem Tyr Leu [V o.
Val Cys (Sly Glu Arg (sly Phe Phe Tyr Tlu' Pro Lys Thr Arg Arg Glu Ala
Glu Asp Leu Gln Val (Sly (Zulu Val Glu Leu Gly Gly (Sly Pro (Sly Ala (Bly
Ser Lem Gln Pro Leu Ala Leu Glu (Ely Ser Leu (Elm Lys Arg (31y lle Val
GIL} (Sln Cys Cys Thr Ser lle Cyg Ser' Lem Tyr Caln Leu GIL: ASH Tyr Cys
Analog 5 DNA 4'TTC GTT AAL’ CAA {AC TTG TGT GGC TCA CAL" CTG GT6 GAA GCT 27
(TC TAC CTA OTC: TGC C166 (3AA (:GA L303 TTC TTC TA( ACA CCC
AAQ ACC CGC (K36 GATT: GCA GAG CIAC {Tl} CA6 (ST6 (366 CAG
GT6 GAG CTG (36C (36C: GGC (CT GGT GCA GGC AGC CTG CAT}
CCC TTG (SEC CTG GAG £166 TCC CTG CAL} AAG (GT GGC ATT (ETC:
(3AA CAA TGC TGT ACC AGE ATC TCaC TCC ETC TAC (IAG CTG GAG
AAC TAC TGC AAC
n Phe Val Asn Gin His Len Cys Gly Ser Hi5 Leu Val Glu Ala Leu Tyr Leu
Val Cys Gly (3k: Arg Ala Phe Phe Ty: Thr Pro. Lys Tlrr Ar'g Arg Glu Ala
(Zulu Asp Leu Gin Val (Ely C1311 Val Calu Leu Gly Gly Gly Pro My Ala (Sly
Sex Lem Calm Pro Leu Ala Leu Glu (Sly Ser Len Gln Lys Arg Gly Ile Val
Glu Gln Cys Cys Thr $er lle Cys Ber Leu Tyr Calm Leu Glu Asn Tyr Cys
Analog l3 DNA T'T'L' GTT AAC {AA (AC TTG TGT (SGT: TCA CAC CTG (3H3 6AA GET
CTC TA': CTA (3T6 TGC (366 (3AA 013A GCaC GCC: TTC TAT: ACA CCC
AAG ACT: (6C CGG GAG GCA GAG GAC (TC) CA6 GTG (366 CAG
(3T6 (3A6 CTG GGC GGG (36C CCT (SGT GCA GCuC AGC CTG CA6
(CC TTG (fiCC CTG (SAC: GGG TCC CTG CA6 AAG CGT (36C ATT GT6
(3AA CAA TGC TGT ACT; AGC ATC TGC TCC (TTC TAC CAG LC TG GAG
AAC TAC TGC AAC
Protein Phe Val Asn Gln His Leu Cys Gly 891 His Leu Val Glu Ala Leu Tyr Leu
Val Cys [Sly lilu Arg (Sly Ala Phi? Tyr Thr Pro Lys Thr Arq Arg Glu Ala
Glu Aap Leu Gln Val Gly Gln Val Glu Len (Sly (31y (fily Pro Gly Ala Gly
SEW Leu Gln Pro Leu Ala Leu Glu 6!)! Ser Leu Gln Lys Arg (Ely lle Val
(STU Gln Cys Cys Thr Se: lle Cys SEW Len Tyr kiln Leu Glu Asn Tyr Cys
Example 2: Expression of inant insulin analog
fusion e
Expressions of recombinant insulin analogs were carried
out under the control of T7 promoter. E.coli BL21-DE3 (E. coli
B F-dcm ompT B-mB-) gal λDE3); Novagen) was transformed
with each of the recombinant n analog-expressing vectors.
Transformation was performed in accordance with the
recommended protocol (Novagen). Single colonies transformed
with each recombinant expression vector were collected and
inoculated in 2X Luria Broth (LB) ning ampicillin (50
µg/ml) and cultured at 37°C for 15 hours. The recombinant
strain culture broth and 2X LB medium containing 30% glycerol
were mixed at a ratio of 1:1 (v/v). Each 1 ml was dispensed to
a cryotube and stored at -140°C, which was used as a cell
stock for production of the recombinant fusion protein.
To express the recombinant insulin analogs, 1 vial of
each cell stock was thawed and ated in 500 ml of 2X
Luria broth, and cultured with g at 37°C for 14~16 hours.
The cultivation was terminated, when OD600 reached 5.0 or
higher. The culture broth was used as a seed culture broth.
This seed culture broth was inoculated to a 50 L fermentor
(MSJ-U2, B.E.MARUBISHI, Japan) containing 17 L of fermentation
medium, and initial bath fermentation was started. The culture
conditions were maintained at a temperature of 37°C, an air
flow rate of 20 L/min (1 vvm), an agitation speed of 500 rpm,
and at pH 6.70 by using a 30% ammonia solution. Fermentation
was carried out in fed-batch mode by adding a feeding on,
when nutrients were depleted in the culture broth. Growth of
the strain was monitored by OD value. IPTG was introduced in a
final concentration of 500 µM, when OD value was above 100.
After introduction, the cultivation was further carried out
for about 23~25 hours. After terminating the cultivation, the
recombinant strains were harvested by centrifugation and
stored at -80°C until use.
Example 3: Recovery and ing of recombinant insulin
analog
In order to change the recombinant insulin analogs
expressed in Example 2 into soluble forms, cells were
disrupted, followed by refolding. 100 g (wet weight) of the
cell pellet was re-suspended in 1 L lysis buffer (50 mM Tris-
HCl (pH 9.0), 1 mM EDTA (pH 8.0), 0.2 M NaCl and 0.5% Triton
. The cells were disrupted using a microfluidizer
processor M-110EH (AC Technology Corp. Model M1475C) at an
operating pressure of 15,000 psi. The cell lysate thus
disrupted was centrifuged at 7,000 rpm and 4°C for 20 minutes.
The supernatant was discarded and the pellet was re-suspended
in 3 L washing buffer (0.5% Triton X-100 and 50 mM Tris-HCl
(pH 8.0), 0.2 M NaCl, 1 mM EDTA). After centrifugation at
7,000 rpm and 4°C for 20 minutes, the cell pellet was resuspended
in led water, ed by centrifugation in
the same . The pellet thus obtained was re-suspended in
400 ml of buffer (1 M Glycine, 3.78 g ne-HCl, pH 10.6)
and stirred at room temperature for 1 hour. To recover the
recombinant insulin analog thus re-suspended, 400 mL of 8M
urea was added and stirred at 40°C for 1 hour. For refolding
of the solubilized recombinant insulin analogs, centrifugation
was carried out at 7,000 rpm and 4°C for 30 minutes, and the
supernatant was obtained. 2 L of led water was added
thereto using a peristaltic pump at a flow rate of 1000 ml/hr
while stirring at 4°C for 16 hours.
e 4: Cation binding chromatography purification
The sample refolded was loaded onto a Source S (GE
healthcare) column equilibrated with 20 mM sodium citrate (pH
2.0) buffer containing 45% ethanol, and then the insulin
analog proteins were eluted in 10 column volumes with a linear
gradient from 0% to 100% 20 mM sodium citrate (pH 2.0) buffer
containing 0.5 M potassium chloride and 45% ethanol.
Example 5: Trypsin and Carboxypeptidase B treatment
Salts were removed from the eluted samples using a
desalting , and the buffer was exchanged with a buffer
(10 mM Tris-HCl, pH 8.0). With respect to the obtained sample
protein, n corresponding to 1000 molar ratio and
carboxypeptidase B corresponding to 2000 molar ratio were
added, and then stirred at 16°C for 16 hours. To terminate the
reaction, 1 M sodium citrate (pH 2.0) was used to reduce pH to
3.5.
Example 6: Cation g chromatography purification
The sample thus reacted was loaded onto a Source S (GE
healthcare) column equilibrated with 20 mM sodium citrate (pH
2.0) buffer containing 45% ethanol, and then the insulin
analog proteins were eluted in 10 column volumes with a linear
gradient from 0% to 100% 20 mM sodium citrate (pH 2.0) buffer
containing 0.5 M potassium chloride and 45% ethanol.
Example 7: Anion binding chromatography purification
Salts were removed from the eluted sample using a
ing column, and the buffer was exchanged with a buffer
(10 mM Tris-HCl, pH 7.5). In order to isolate a pure insulin
analog from the sample obtained in Example 6, the sample was
loaded onto an anion ge column (Source Q: GE healthcare)
equilibrated with 10 mM Tris (pH 7.5) buffer, and the insulin
analog protein was eluted in 10 column volumes with a linear
gradient from 0% to 100% 10 mM Tris (pH 7.5) buffer containing
0.5 M sodium chloride.
Purity of the n analog thus purified was analyzed
by protein electrophoresis (SDS-PAGE, and high
pressure chromatography (HPLC) (, and modifications of
amino acids were identified by peptide mapping ( and
molecular weight analysis of each peak.
As a result, each insulin analog was found to have the
desired modification in its amino acid sequence.
Example 8: Preparation of insulin analog (No. 7)-
immunoglobulin Fc ate
To pegylate the N-terminus of the beta chain of the
insulin analog using 3.4K ALD2 PEG (NOF, Japan), the n
analog and PEG were reacted at a molar ratio of 1:4 with an
insulin analog concentration of 5 mg/ml at 4°C for about 2
hours. At this time, the reaction was performed in 50 mM
sodium citrate at pH 6.0 and 45% isopropanol. 3.0 mM sodium
cyanoborohydride was added as a ng agent and was allowed
to react. The reaction solution was purified with SP-HP (GE
Healthcare, USA) column using a buffer containing sodium
citrate (pH 3.0) and 45% ethanol, and KCl concentration
gradient.
To e an insulin analog-immunoglobulin Fc fragment
conjugate, the purified mono-PEGylated n analog and the
immunoglobulin Fc fragment were reacted at a molar ratio of
1:1 to 1:2 and at 25°C for 13 hrs, with a total protein
concentration of about 20 mg/ml. At this time, the reaction
buffer conditions were 100 mM HEPES at pH 8.2, and 20 mM
sodium cyanoborohydride as a reducing agent was added thereto.
Therefore, PEG was bound to the N-terminus of the Fc fragment.
After the reaction was ated, the reaction solution
was loaded onto the Q HP (GE Healthcare, USA) column with
Tris-HCl (pH 7.5) buffer and NaCl concentration gradient to
separate and purify unreacted immunoglobulin Fc fragment and
EGylated insulin analog.
fter, Source 15ISO (GE Healthcare, USA) was used as
a secondary column to remove the remaining immunoglobulin Fc
fragment and the ate, in which two or more insulin
analogs were linked to the immunoglobulin Fc fragment, thereby
obtaining the insulin analog-immunoglobulin Fc fragment
conjugate. At this time, elution was carried out using a
concentration gradient of ammonium sulfate containing Tris-HCl
(pH 7.5), and the insulin analog-immunoglobulin Fc conjugate
thus eluted was analyzed by protein electrophoresis (SDS-PAGE,
and high pressure tography (HPLC) (. As
a result, the conjugate was found to have almost 99% purity.
Example 9: Comparison of insulin receptor binding
ty between native insulin, n analog, native
insulin-immunoglobulin Fc conjugate, and insulin analogimmunoglobulin
Fc conjugate
In order to measure the insulin receptor binding affinity
of the insulin analog-immunoglobulin Fc conjugate, Surface
plasmon nce (SPR, BIACORE 3000, GE care) was used
for analysis. Insulin receptors were lized on a CM5 chip
by amine coupling, and 5 dilutions or more of native insulin,
insulin analog, native insulin-immunoglobulin Fc conjugate,
and insulin analog-immunoglobulin Fc ate were applied
o, independently. Then, the insulin receptor binding
affinity of each substance was examined. The binding affinity
of each substance was calculated using BIAevaluation software.
At this time, the model used was 1:1 Langmuir g with
baseline drift.
As a result, compared to human insulin, insulin analog
(No. 6) showed receptor binding affinity of 14.8%, insulin
analog (No. 7) showed receptor binding affinity of 9.9%,
n analog (No. 8) showed receptor binding ty of
57.1%, insulin analog (No. 9) showed receptor binding affinity
of 78.8%, native insulin-immunoglobulin Fc conjugate showed
receptor binding affinity of 3.7-5.9% depending on
experimental runs, insulin analog (No. 6)-immunoglobulin Fc
conjugate showed receptor binding affinity of 0.9% or less,
insulin analog (No. 7)-immunoglobulin Fc conjugate showed
receptor binding affinity of 1.9%, insulin analog (No. 8)-
globulin Fc conjugate showed receptor binding affinity
of 1.8%, and insulin analog (No. 9)-immunoglobulin Fc
conjugate showed receptor binding affinity of 3.3% (Table 4).
As such, it was observed that the insulin analogs of the
present invention had reduced insulin receptor binding
ty, compared to the native insulin, and the insulin
analog-immunoglobulin Fc conjugates also had remarkably
reduced insulin receptor binding affinity.
[Table 4]
e 10: Comparison of in-vitro efficacy between
native insulin-immunoglobulin Fc conjugate and insulin analogimmunoglobulin
Fc conjugate
In order to evaluate in vitro cy of the insulin
analog-immunoglobulin Fc ate, mouse-derived
differentiated 3T3-L1 adipocytes were used to test glucose
uptake or lipid synthesis. 3T3-L1 cells were sub-cultured in
% NBCS (newborn calf -containing DMEM co’s
Modified Eagle’s Medium, Gibco, Cat.No, 12430) twice or three
times a week, and maintained. 3T3-L1 cells were suspended in a
differentiation medium (10% FBS-containing DMEM), and then
inoculated at a density of 5 x 104 per well in a 48-well dish,
and cultured for 48 hours. For yte differentiation, 1
µg/mL human insulin (Sigma, Cat. No. I9278), 0.5 mM IBMX (3-
isobutylmethylxanthine, Sigma, Cat. No.I5879), and 1 µM
Dexamethasone (Sigma, Cat. No. D4902) were mixed with the
differentiation medium, and 250 µl of the mixture was added to
each well, after the previous medium was removed. After 48
hours, the medium was exchanged with the entiation
medium supplemented with only 1 µg/mL of human insulin.
Thereafter, while the medium was exchanged with the
differentiation medium supplemented with 1 µg/mL of human
insulin every 48 hours, induction of adipocyte differentiation
was examined for 7-9 days. To test glucose uptake, the
differentiated cells were washed with serum-free DMEM medium
once, and then 250 µl was added to induce serum depletion for
4 hours. Serum-free DMEM medium was used to carry out 10-fold
serial dilutions for Human insulin from 2 µM to 0.01 µM, and
for native insulin-immunoglobulin Fc conjugate and insulin
analog-immunoglobulin Fc conjugates from 20 µM to 0.02 µM.
Each 250 µl of the s thus prepared were added to cells,
and cultured in a 5% CO2 incubator at 37°C for 24 hours. In
order to measure the residual amount of glucose in the medium
after incubation, 200 µl of the medium was taken and diluted
-fold with D-PBS, followed by GOPOD (GOPOD Assay Kit,
Megazyme, Cat. No. K-GLUC) assay. Based on the absorbance of
glucose standard solution, the concentration of glucose
ing in the medium was converted, and EC50 values for
glucose uptake of native insulin-immunoglobulin Fc conjugate
and insulin analog-immunoglobulin Fc conjugates were
calculated, respectively.
As a result, compared to human insulin, native insulinimmunoglobulin
Fc conjugate showed glucose uptake of 11.6%,
insulin analog (No. unoglobulin Fc conjugate showed
glucose uptake of 0.43%, insulin analog (No. 7)-immunoglobulin
Fc conjugate showed glucose uptake of 1.84%, insulin analog
(No. 8)-immunoglobulin Fc conjugate showed glucose uptake of
16.0%, insulin analog (No. 9)-immunoglobulin Fc conjugate
showed glucose uptake of 15.1% (Table 5). As such, it was
observed that the insulin analog (No. unoglobulin Fc
ate and insulin analog (No. 7)-immunoglobulin Fc
conjugate of the present invention had remarkably reduced in
vitro titer, compared to native insulin-immunoglobulin Fc
conjugate, and insulin analog (No. 8)-immunoglobulin Fc
ate and insulin analog (No. 9)-immunoglobulin Fc
conjugate had in vitro titer similar to that of the native
insulin-immunoglobulin Fc conjugate.
[Table 5]
Example 11: cokinetics of insulin analogimmunoglobulin
Fc conjugate
In order to examine pharmacokinetics of the insulin
analog-immunoglobulin Fc conjugates, their blood concentration
over time was ed in normal rats (SD rat, male, 6-week
old) adapted for 5 days to the laboratory. 21.7 nmol/kg of
native insulin-immunoglobulin Fc conjugate and 65.1 nmol/kg of
insulin analog-immunoglobulin Fc conjugate were subcutaneously
injected, respectively. The blood was collected at 0, 1, 4, 8,
24, 48, 72, 96, 120, 144, 168, 192, and 216 hours. At each
time point, blood concentrations of native insulinimmunoglobulin
Fc conjugate and insulin analog-immunoglobulin
Fc conjugate were measured by enzyme linked sorbent
assay (ELISA), and Insulin ELISA (ALPCO, USA) was used as a
kit. However, as a ion antibody, mouse anti-human IgG4
HRP conjugate (Alpha Diagnostic Intl, Inc, USA) was used.
The results of examining pharmacokinetics of the native
insulin-immunoglobulin Fc conjugate and the insulin analogimmunoglobulin
Fc conjugate showed that their blood
trations increased in proportion to their administration
concentrations, and the n analog-immunoglobulin Fc
conjugates having low insulin receptor binding affinity showed
highly increased half-life, compared to the native insulin-Fc
conjugate (.
These results suggest that when the insulin analogs of
the present invention ed to have reduced n
receptor binding affinity are linked to immunoglobulin Fc
region to prepare conjugates, the conjugates can be provided
as stable insulin formulations due to remarkably increased in
vivo blood half-life, and thus effectively used as therapeutic
agents for diabetes. rmore, since the insulin analogs
according to the present invention themselves also have
reduced insulin receptor binding affinity and reduced titer,
the n analogs also exhibit the same effect although they
are linked to other various carriers.
Based on the above ption, it will be apparent to
those d in the art that various cations and
changes may be made without departing from the scope and
spirit of the invention. Therefore, it should be understood
that the above embodiment is not limitative, but illustrative
in all aspects. The scope of the invention is defined by the
appended claims rather than by the description preceding them,
and therefore all changes and modifications that fall within
metes and bounds of the claims, or equivalents of such metes
and bounds are therefore intended to be embraced by the claims.
Claims (22)
1. An insulin analog having a reduced insulin titer ed to the native form, wherein an amino acid in insulin is ed by substituting the 14th amino acid of the A chain with ic acid or asparagine, and wherein the amino acid sequences of the B chain and the A chain of insulin consist of SEQ ID NOs: 38 and 37, respectively.
2. The insulin analog according to claim 1, wherein the insulin analog is SEQ ID NO. 34 or 36.
3. An insulin analog conjugate, in which (i) the insulin analog according to claim 1 or claim 2 is linked to (ii) FcRn-binding material.
4. The insulin analog conjugate ing to claim 3, n the insulin analog and the FcRn-binding material are linked to each other via a peptide or a nonpeptidyl polymer as a linker.
5. The insulin analog conjugate according to claim 3, wherein the FcRn-binding material is an immunoglobulin Fc region.
6. The insulin analog conjugate according to claim 3, wherein (i) the insulin analog ing to claim 1 or claim 2 is linked to (ii) an immunoglobulin Fc region via (iii) a peptide linker or a non-peptidyl linker selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol-propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitins, hyaluronic acid and combination thereof.
7. The insulin analog conjugate according to claim 6, wherein the non-peptidyl linker is linked to the N-terminus of B chain of the insulin analog.
8. The n analog conjugate according to claim 6, wherein one end of the non-peptidyl polymer is linked to the N-terminus of the immunoglobulin Fc region and the other end of the ptidyl polymer is linked to the N-terminal amine group of the n analog or the ε-amino group of the internal lysine residue or the thiol group of B chain, respectively.
9. The insulin analog conjugate according to claim 6, wherein the immunoglobulin Fc region is aglycosylated.
10. The insulin analog conjugate according to claim 6, wherein the immunoglobulin Fc region is composed of 1 domain to 4 s ed from the group consisting of CH1, CH2, CH3 and CH4 domains.
11. The insulin analog conjugate according to claim 6, wherein the immunoglobulin Fc region is an Fc region derived from IgG, IgA, IgD, IgE or IgM.
12. The insulin analog conjugate according to claim 11, wherein each domain of the immunoglobulin Fc region is a hybrid of domains having different origins and being derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE and IgM.
13. The insulin analog conjugate according to claim 6, n the immunoglobulin Fc region further includes a hinge region.
14. The n analog conjugate according to claim 11, wherein the globulin Fc region is a dimer or a multimer consisting of single-chain immunoglobulins composed of domains of the same origin.
15. The insulin analog conjugate according to claim 11, wherein the immunoglobulin Fc region is an IgG4 Fc region.
16. The insulin analog conjugate according to claim 15, wherein the immunoglobulin Fc region is a human IgG4-derived aglycosylated Fc region.
17. The insulin analog conjugate according to claim 6, wherein a reactive group of the non-peptidyl linker is selected from the group consisting of an de group, a propionaldehyde group, a butyraldehyde group, a maleimide group and a succinimide derivative.
18. The insulin analog conjugate according to claim 17, wherein the succinimide derivative is imidyl propionate, succinimidyl carboxymethyl, hydroxy succinimidyl, or succinimidyl carbonate.
19. The insulin analog conjugate according to claim 6, wherein the non-peptidyl linker has reactive de groups at both ends thereof.
20. A long-acting insulin ation having improved in vivo on and stability, comprising the insulin analog conjugate of claim 3.
21. The long-acting insulin ation according to claim 20, wherein the formulation is a therapeutic agent for diabetes.
22. A method for preparing the insulin analog ate of claim 3, comprising: linking the insulin analog according to claim 1 or claim 2 to FcRn-binding material. [ [ [ (A) Glu-C cleavage fragment of native insulin sequence (B) Glu-C ge fragment of insulin analog (No. 7) sequence [ [ [
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