JP2010213707A - Albumin-fused ciliary neurotrophic factor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for treating obesity in mammals, and also a method for potentially minimizing adverse effects (e.g. nausea, headache) associated to the treatment of the mammals by using a suitably high concentration CNTF (ciliary neurotrophic factor). <P>SOLUTION: This fused protein contains albumin, a fragment, variant or derivative thereof, and at least one biologically active peptide or protein activating ciliary neurotrophic factor (CNTF) receptor, a fragment, variant or derivative thereof. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

Field of Invention

  The present invention relates to albumin, or a fragment or variant or derivative thereof, and at least one biologically active peptide or protein that activates ciliary neurotrophic factor (CNTF) receptor, or a fragment or variant thereof or The present invention relates to a fusion protein comprising a derivative.

Background of the Invention

  The regulation of daily energy homeostasis is under central control of a small number of individual nuclei [1] mainly in the basal hypothalamus (ventral medial, dorsal medial, paraventricular, and lateral hypothalamic areas). And other central nervous structures (cerebral cortex, marginal zone, brain stem, pituitary gland, autonomic ganglion cells, vagus dorsal complex) and related peripheral nerve structures (sympathetic ganglion cells) [2 ].

  In addition to the regulation of central and peripheral nerves, the peripheral organs involved in the balance of energy homeostasis are the gastrointestinal tract (stomach, intestine), pancreas, adipose tissue, muscle tissue, adrenal gland and thyroid.

  The process of regulation is complex, and peripheral organs such as the gastrointestinal tract release hormones (eg, CCK (cholecystokinin)) that decrease appetite-enhancing hormones in the hypothalamus after food intake. Furthermore, leptin released by adipose tissue after food intake has a negative regulatory effect on, for example, NPY (neuropeptide Y), one of the main appetite-enhancing hormones acting on the center. On the other hand, centrally released hormones may also have peripheral effects that increase heat production as well (eg, β3 adrenergic agonists, uncoupling protein (UCP)). If you are interested, see the actual review [1-6], which covers the full range.

  Axocaine (AXOKINE ™) (REGENERON, Terrytown, NY, USA) is a variant of CNTF. Axocaine ™ is a truncated form of CNTF from which the last 15 amino acids at the C-terminus have been removed. In order to improve the stability of the molecule, the 63rd glutamine is replaced by arginine and the 17th free cysteine is replaced by alanine [7].

  During clinical trials in patients suffering from motor neuron disease, it was discovered by chance that CNTF lost weight [8]. Further studies have revealed that the mechanism of weight loss-inducing action caused by CNTF is similar to leptin, and that CNTF is also effective in diet-induced obesity [7]. In animal experiments using Axocaine ™, it was confirmed that the ability to induce weight loss by this CNTF mutant is similar to the mechanism of CNTF.

  CNTF has a negative regulatory effect on the synthesis of NPY, agouti-related peptide (AGRP) and γ-aminobutyric acid (GABA), all known to stimulate feeding.

CNTF has been shown to pass through the blood brain barrier (BBB) as it is [10]. Recently, CNTF has been shown to be transported via a saturable transport system with a Ki influx of 4.60 (± 0.78) × 10 ⁻⁴ mL / g [ 11].

  The BBB is a highly regulated barrier that prevents the entry of molecules from the blood into brain tissue [13]. It is made up of brain capillary endothelial cells.

  Lambert et al. [7] show that Axocaine ™ works in leptin-deficient (ob / ob) and wild-type (diet-induced obesity, DIO) mice. The most effective dose was 300 μg / kg body weight for Axocaine ™, but the effect was also found at 100 μg / kg body weight. The achieved weight loss is mainly due to the loss of adipose tissue and the loss due to lean body mass is avoided.

  In addition, mice treated with Axocain ™ had no rebound effect, but mice that received the same diet as those consumed by animals treated with Axocaine ™ without treatment with Axocaine ™ ) Immediately recovered the original weight.

  Phase I data has been published by Guler et al. In the International Journal of Obesity [14]. Axocaine ™ was well tolerated, no subjects dropped out, and the majority of all adverse events (AEs) were “mild”. Dose limiting toxicity was vomiting and nausea at 16 μg / kg body weight in Part A. Injection site reactions were most frequently reported in drugs treated subjects, followed by decreased appetite, nausea, headache, and diarrhea. Some subjects had herpes-like lesions in the mouth.

One subject had transient Bell paralysis 10 days after the end of treatment with Axokine ™ at 1 μg / kg body weight / day (facial nerve paralysis, which is the VII cranial nerve with facial facial muscle paralysis). Suffered. At higher doses, increased C-reactive protein and erythrocyte sedimentation rate (ESR) and decreased serum Fe ⁺ were seen. The dose-dependent type tended to increase heart rate and increase body temperature.

  A multicenter, randomized, double-blind, placebo-controlled, dose-controlled phase II study [15] for 170 severe or morbidly obese patients was evaluated and the optimal amount of Axocaine ™ (1. Patients who received (0 μg / kg) lost an average of 10 pounds more [16] body weight than placebo recipients (p <0.001).

  Weight loss was maintained for 4 months after the last dose of Axocaine ™ to patients in the 8-week treatment group [17, 18]. No serious adverse events were reported. The most frequently reported adverse event was dose-dependent, mild injection site reaction (site redness) that occurred in all patients, including the placebo group. Administration of Axocaine ™ was associated with cough and nausea, most frequently after administration of 2.0 μg / kg body weight of drug. No increase in herpes simplex virus infection was found in Axocaine ™ recipients compared to placebo. A comparable proportion of Axocaine ™, and (58-74%), and placebo (61%), the recipient completed the entire 12-week study.

  In a Phase III placebo-controlled trial, 1467 Axocaine ™ treated patients and 501 placebo treated patients demonstrated the following:

  Axocaine ™ treatment achieved a statistical advantage for both endpoints of the study compared to placebo.

  The percentage of at least 5% reduction in initial body weight compared to placebo-treated patients was greater in Axocaine ™ -treated patients (25.1% vs 17.6%, p <0.001).

  Participants who received Axocaine ™ had a greater average weight loss than those who received placebo (6.2 pounds versus 2.6 pounds, p <0.001).

  Axocaine (TM) treatment statistically increased to 2 out of 3 secondary endpoints, such as the proportion of patients who lost at least 10% of their initial body weight (11.3% vs. 4.2%, p <0.001). Significant results were achieved.

  Axocaine ™ treatment was generally well tolerated. Adverse events were generally characterized as mild to moderate and did not show a trend for serious or severe adverse events. The most notable adverse effects compared to placebo were injection site reactions, nausea and cough, which were mainly characterized as mild.

  The weight loss associated with Axocaine ™ was limited by antibody production beginning approximately 3 months after Axocaine ™ treatment. However, more than 30% of all 1467 patients treated with Axocaine ™ did not produce antibodies by the end of the year.

  In one aspect of the invention, the invention provides albumin, in particular human serum albumin, or a fragment or variant or derivative thereof having albumin activity and at least one that activates ciliary neurotrophic factor (CNTF) receptor. A fusion protein comprising a biologically active peptide or protein, or a fragment or variant or derivative thereof.

  In different embodiments, CNTF or albumin may be a fragment or derivative, or both in the case of Axocaine ™, or a variant. The albumin fusion protein may be a therapeutic agent.

  In another aspect, the present invention is a method of extending the half-life of CNTF in a mammal. This method involves coupling CNTF with albumin to form albumin-fused CNTF and administering albumin-fused CNTF to the mammal. In general, the half-life of albumin-fused CNTF is extended at least 2 to at least 50 times the half-life of CNTF lacking linked albumin.

  By using a CNTF transport system or a non-specific transport system that crosses the blood brain barrier (BBB) such as, for example, transcellular transport, the concentration of albumin fusion Axokine ™ in the brain is expected to increase. Over time, the plasma concentration of albumin-fused Axocaine ™ increases in the BBB compared to unfused Axocaine ™, resulting in an influx of albumin-fused Axocaine ™ by more transcellular transport.

  Furthermore, the present invention involves a method of treating obesity in a mammal. The method comprises linking CNTF with albumin to form albumin-fused CNTF and administering albumin-fused CNTF to the mammal. The invention also encompasses methods that potentially minimize side effects (eg, nausea, headaches) associated with treating mammals with moderately high concentrations of CNTF. The method includes linking the CNTF with albumin to form an albumin fused CNTF and administering the albumin fused CNTF to the mammal.

Detailed description of the invention

Definition Ciliary neurotrophic factor (CNTF) means any molecule that is an analog, homologue, fragment, or derivative of a naturally occurring CNTF that has a single biological activity of the naturally occurring CNTF. . A preferred CNTF is Axocaine (AXOKINE ™). Another CNTF variant (Ser166Asp / Gln167His) is described in European Patent No. WO 98/22128, which has the following amino acid sequence from number 159 to number 178: Leu Lys Val Leu Gln Glu Leu Asp His Trp Thr Val Arg Ser Ile His Asp Leu Arg Phe (159-178; SEQ ID NO: 4)

  Axocaine (trademark) is a variant of CNTF. Axocaine ™ is a truncated form of CNTF from which the last 15 amino acids at the C-terminus have been removed. In order to increase the stability of the molecule, the 63rd glutamine is replaced by arginine and the free cysteine is replaced by alanine at the 17th position [7].

  N-terminal-Axocaine ™ is a fusion of the C-terminus of Axocaine ™ and the N-terminus of human serum albumin as described in Example 1.

  C-terminal-Axocaine ™ is a fusion of the N-terminus of Axocaine ™ and the C-terminus of human serum albumin as described in Example 1.

  The cleavable Axocaine ™ described in Example 1 is a non-fused Axocaine ™ used as a cleavable or N- and C-terminal fusion control between the CNTF moiety and albumin. C-terminal fusion of Axocaine ™ with human serum albumin having the enterokinase cleavage site used to generate.

The terms albumin human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms “albumin” and “serum albumin” are broader and include human serum albumin (and fragments and variants thereof) and albumin from other species (and fragments and variants thereof).

  As used herein, “albumin” collectively refers to an albumin protein or amino acid sequence or albumin fragment or variant having one or more functional activities (eg, biologically active) of albumin. In particular, “albumin” refers to human albumin or a fragment thereof (see EP2012239, EP322094, WO97 / 24445, WO95 / 23857), in particular WO01 / 79480, which is incorporated herein by reference in its entirety. As shown in FIG. 15 (SEQ ID NO: 18), mature albumin of human albumin, or albumin derived from other plants or fragments thereof, or analogs or variants of these molecules or fragments thereof.

  In the present application, this arrangement of FIG. 15 of WO01 / 79480 is referred to as a “WO′480 arrangement”.

  The human serum albumin protein used in the albumin fusion protein of the present invention has the following series of point mutations with respect to the WO'480 sequence: Leu No. 407 to Ala, Leu No. 408 to Val, Val No. 409 to Ala And Arg from 410 to Ala; or Arg from 410 to Ala; Lys from 413 to Gln; and Lys from 414 to Gln (for example, by reference) One or both of international publication W095 / 23857). In another embodiment, an albumin fusion protein of the invention comprising one or both of the above series of point mutations has improved stability / resistance to yeast Yap3p proteolytic cleavage and is expressed in a yeast host cell. Increases production of recombinant albumin fusion protein.

  As used herein, a portion of albumin sufficient to extend or extend the in vivo half-life, therapeutic activity, or shelf-life of CNTF is the infusion of CNTF in the unfused state. long enough to stabilize, extend or extend the in vivo half-life, therapeutic activity, or shelf life of the CNTF portion of an albumin fusion protein, as compared to the in vivo half-life, therapeutic activity, or shelf life The albumin part of the structure. The albumin portion of the albumin fusion protein can include a full length HA sequence as described above, or can include one or more fragments thereof that can stabilize or prolong therapeutic activity. Such a fragment is 10 or more amino acids in length, or contains about 15, 20, 25, 30, 50, or more adjacent amino acids from the HA sequence, or one of the specific domains of HA. Part or all.

  The albumin portion of the albumin fusion protein of the present invention may be a normal HA variant. The CNTF portion of the albumin fusion protein of the invention may also be a variant of a therapeutic protein as described herein. “Variants” include insertions, deletions, and conservative or non-conservative substitutions, where such changes may result in albumin conferring therapeutic activity of the therapeutic protein, or in the active site or domain. It does not substantially alter the useful ligand binding and non-immunogenicity of one or more swellings.

  In particular, the albumin fusion protein of the present invention comprises a naturally occurring polymorphic variant of human albumin and a fragment of human albumin, such as the fragment disclosed in EP 320994 (ie, HA (Pn) where n is 369 to 419). May include. Albumin may be derived from any vertebrate, especially mammals such as humans, cows, sheep, or pigs. Non-mammalian-derived albumin includes, but is not limited to, hens and pupae. The albumin portion of the albumin fusion protein may be from an animal different from the CNTF portion.

  Generally speaking, an HA fragment or variant will be at least 100 amino acids long and optionally at least 150 amino acids long. The HA variant may consist of or contain at least one whole domain of HA. For example, domain 1 (amino acids 1 to 194 of the WO′480 sequence), 2 (amino acids 195 to 387 of the WO′480 sequence), 3 (amino acids 388 to 585 of the WO′480 sequence), 1 + 2 (1 to 2 of the WO′480 sequence) 387) 2 + 3 (195-585 of the WO'480 sequence) or 1 + 3 (amino acids 1-194 of the WO'480 sequence + amino acids 388-585 of the WO'480 sequence). Each domain itself consists of two homologous subdomains, namely 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, Lys106-Glu119, Glu292-Va1315 and Glu492-Ala511 It includes a flexible sub-domain interlinker region comprising residues.

  The albumin portion of the albumin fusion protein of the invention may comprise at least one subdomain or domain of HA or a conservative modification thereof. If the fusion is based on a subdomain, some or all of the adjacent linkers may optionally be used for linking with the CNTF moiety.

Albumin fusion proteins The present invention relates generally to albumin fusion proteins and methods for treating, preventing or ameliorating diseases and disorders. As used herein, “albumin fusion protein” refers to the fusion of at least one molecule of albumin (or a fragment or variant thereof) and at least one molecule of CNTF (or a fragment or variant thereof). Refers to the protein that is formed. The albumin fusion protein of the present invention comprises at least one fragment or variant of CNTF and at least one fragment or variant of human serum albumin, which are linked to each other, such as by fusion of genes to each other (ie, An albumin fusion protein is produced by translation of a nucleic acid in which a polynucleotide encoding all or part of CNTF is linked in frame with a polynucleotide encoding all or part of albumin). Once CNTF and albumin protein are part of an albumin fusion protein, they may be referred to as “parts”, “regions” or “parts” of the albumin fusion protein.

  In one embodiment, the present invention provides an albumin fusion protein comprising or consisting of CNTF and serum albumin protein. In another aspect, the present invention provides an albumin fusion protein comprising or consisting of biologically active and / or therapeutically active fragments of CNTF and serum albumin protein. In another aspect, the present invention provides an albumin fusion protein comprising or consisting of biologically active and / or therapeutically active variants of CNTF and serum albumin protein. In a further aspect, the serum albumin protein component of the albumin fusion protein is a mature portion of serum albumin.

  In a further aspect, the present invention provides an albumin fusion protein comprising or consisting of CNTF and biologically active and / or therapeutically active fragments of serum albumin. In a further aspect, the present invention provides an albumin fusion protein comprising or consisting of CNTF and biologically active and / or therapeutically active variants of serum albumin. In some embodiments, the CNTF portion of the albumin fusion protein is the mature portion of the therapeutic protein.

  In a further aspect, the invention provides biologically active and / or therapeutically active fragments or variants of CNTF and biologically active and / or therapeutically active fragments or variants of serum albumin. An albumin fusion protein comprising or consisting of is provided. In some embodiments, the invention provides an albumin fusion protein comprising or consisting of the mature portion of CNTF and the mature portion of serum albumin.

  In one embodiment, the albumin fusion protein comprises HA as the N-terminal portion and CNTF as the C-terminal portion. Alternatively, an albumin fusion protein comprising HA as the C-terminal portion and CNTF as the N-terminal portion may be used.

  In other embodiments, the albumin fusion protein has CNTF fused to both the N-terminus and C-terminus of albumin. In one embodiment, the plurality of CNTF proteins fused at the N and C termini are the same CNTF protein. In another embodiment, the plurality of CNTF proteins fused at the N and C termini are different CNTF proteins. In another embodiment, the multiple CNTF proteins fused at the N and C terminus are different CNTF proteins that may be used for the treatment or prevention of the same disease, disorder or condition. In another aspect, multiple CNTF proteins fused at the N and C terminus may be used in the treatment or prevention of diseases or disorders commonly known in the art to occur simultaneously in multiple patients. CNTF protein.

  In addition to albumin fusion proteins in which the albumin moiety is fused to the N-terminus and / or C-terminus of the CNTF portion, the albumin fusion protein of the present invention may be produced by inserting the desired CNTF or peptide into the internal region of HA. . For example, in the protein sequence of an HA molecule, there are a number of loops or turns between the end of the HA helix and the start of the α-helix, stabilized by disulfide bonds. These loops extend out of the body of the molecule, as determined from the crystal structure of HA (PDB identification numbers 1AO6, 1BJ5, 1BKE, 1BM0, 1E7E-1E7l and 1UOR). Such a loop essentially produces an albumin molecule with a specific biological activity and therefore requires a therapeutically effective peptide, particularly one that requires a secondary structure that should be functional or the insertion or interior of a therapeutic protein. Useful for fusion.

  Loops in human albumin structures into which peptides or polypeptides can be inserted to produce albumin fusion proteins of the invention include Val54-Asn61, Thr76-Asp89, Ala92-Glu100, Gln170-Ala176, His247-Glu252, Glu266- Glu277, Glu280-His288, Ala362-Glu368, Lys439-Pro447, Val462-Lys475, Thr478-Pro486, and Lys560-Thr566. In other embodiments, the peptide or polypeptide is inserted into the Val54-Asn61, Gln170-Ala176, and / or Lys560-Thr566 loops of mature human albumin (WO'480 sequence).

  The inserted peptide may be derived from phage display or a synthetic peptide library screened for a specific biological activity, or derived from the active portion of the molecule containing the desired function. It may be. In addition, a random peptide library may be created within a specific loop, created by inserting a randomized peptide into a specific loop of the HA molecule to represent all possible amino acid combinations. May be.

Such a library can be generated on HA or a domain fragment of HA by one of the following methods.
(A) Randomized amino acid mutations within one or more peptide loops of HA or HA domain fragments. Both can mutate more or all residues in the loop in this way;
(B) substitution of one or more loops of the HA or HA domain fragment of a randomized peptide of length Xn, where X is an amino acid and n is the number of residues, or one or more thereof Insertion into the loop (ie internal fusion);
(C) N-, C- or N- and C-terminal peptide / protein fusions in addition to (a) and / or (b).

  HA or HA domain fragments may be made multifunctional by grafting peptides from different screens of different loops against different targets to the same HA or HA domain fragment.

  The peptide inserted into the loop of human serum albumin is CNTF peptide or a peptide fragment or peptide variant thereof. More specifically, the invention relates to at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, inserted into a loop of human serum albumin. Albumin fusion proteins comprising peptide fragments or peptide variants that are 25, at least 30, at least 35, or at least 40 amino acids long are included. The invention also provides at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, fused to the N-terminus of human serum albumin. Albumin fusion proteins comprising peptide fragments or peptide variants of at least 35, or at least 40 amino acids are included. The invention also provides at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, fused to the C-terminus of human serum albumin. Albumin fusion proteins comprising peptide fragments or peptide variants of at least 35, or at least 40 amino acids are included.

  In general, the albumin fusion protein of the present invention may have one HA-derived region and one CNTF protein-derived region. However, the albumin fusion protein of the present invention may be prepared using a plurality of regions of each protein. Similarly, the albumin fusion protein of the present invention may be prepared using one or more CNTFs. For example, CNTF may be fused with both the N and C termini of HA. In such an arrangement, the CNTF portions can be the same or different CNTF molecules. The structure of the bifunctional albumin fusion protein is X-HA-Y or Y-HA-X or XY-HA or HA-XY or HA-Y-X-HA or HA-XX-HA Or HA-Y-Y-HA or HA-X-HA-Y or X-HA-Y-HA or combinations thereof and / or can be represented as X and / or Y insertions at any position within the HA sequence .

  Bifunctional or multifunctional albumin fusion proteins may be produced in various proportions depending on function, half-life and the like.

  Bifunctional or multifunctional albumin fusion proteins may also be made to target the CNTF portion of the fusion to the target organ or cell type via the protein or peptide at the opposite end of the HA.

  As an alternative to known therapeutic molecule fusions, the N-, C- or N- and C-terminus of HA, or generally 6, 8, 12, 20 or 25 or Xn (where X is an amino acid (aa And n is equal to the number of residues.) Screening of a library constructed as a fusion of randomized amino acids with domain fragments of HA will result in peptides so that all possible amino acid combinations are represented. It can be obtained. The particular advantage of this approach is that the peptide can be selected in situ on the HA molecule, such as in the case of peptides derived by any other method where the properties of the peptide are subsequently bound to HA. Rather, it can be chosen as to what is possibly modified.

  In addition, the albumin fusion proteins of the present invention may include a linker peptide between the fusion moieties to provide greater physical separation between the moieties, and thus, for example, access to the CNTF moiety for binding to its cognate receptor. It is possible to maximize performance. The linker peptide may be composed of amino acids so that it is flexible or more robust.

Therefore, as described above, the albumin fusion protein of the present invention has the following formulas R2-R1; R1-R2; R2-R1-R2; R2-L-R1-L-R2; R1-L-R2-R2-L Or R1-L-R2-L-R1 wherein R1 is at least one therapeutic protein, peptide or polypeptide sequence (including fragments or variants thereof) and is not necessarily the same therapeutic protein Need not be, L may be a linker and R2 may be a serum albumin sequence (including fragments or variants thereof). Examples of linkers, _{(GGGGS) N} (SEQ ID NO: 8) or _{(GGGS) N} (SEQ ID NO: 9) or _{(GGS) N,} wherein, N is an integer of 1 or more, G in the formula represents a glycine , S represents serine. When R1 is two or more therapeutic protein, peptide or polypeptide sequences, these sequences may be connected by a linker if desired.

  In a further aspect, an albumin fusion protein of the invention comprising a CNTF protein has a shelf life or in vivo half-life or a shelf life or in vivo half-life or therapeutic activity of the same CNTF that is not fused to albumin. The therapeutic activity is prolonged. The shelf life generally refers to the period during which the CNTF protein therapeutic activity is stable in solution or other storage formulation form without undue loss of therapeutic activity. Many CNTF proteins are extremely unstable in their unfused state. As described below, the general shelf life of these CNTF proteins was significantly extended by incorporation into the albumin fusion proteins of the present invention.

  The albumin fusion proteins of the present invention with “prolonged” or “extended” shelf life exhibit greater therapeutic activity compared to standards subjected to the same storage and handling conditions. The standard can be a non-fused full length CNTF protein. If the CNTF portion of the albumin fusion protein is an analog, variant, otherwise modified form, or does not contain the complete sequence of the protein, an extension of therapeutic activity, or an analog, variant, change thereof Comparable to the non-fused equivalent of the truncated peptide or incomplete sequence. As an example, the albumin fusion protein of the present invention, when subjected to the same storage and handling conditions as the standard substance, is about 100% or more of the therapeutic activity or about 105 of the therapeutic activity of the standard substance when compared at a given time point. %, 110%, 120%, 130%, 150%, or 200% or more. However, the therapeutic activity depends on the stability of CNTF protein and may be 100% or less.

  The shelf life may also be evaluated with respect to the therapeutic activity remaining after storage by normalizing the therapeutic activity at the beginning of storage. An albumin fusion protein of the present invention with an extended or extended shelf life exhibited by extended or extended therapeutic activity may retain about 50% or more of the therapeutic activity and is subjected to the same conditions. May retain about 60%, 70%, 80%, or 90% or more of the therapeutic activity of equivalent unfused CNTF.

をエクソン１に、そしてプライマー：

をエクソン２に用い、それぞれ標準条件を用いて２つのエクソンを増幅してヒトゲノムＤＮＡからクローン化した。両断片を標準条件下で連結した後、プライマー：

を用いてＰＣＲによって再増幅し、ベクターｐＣＲ４(Invitrogen)へクローニングした。Lambertら(PNAS 98: 4652-4657; 2001)に開示の通りアクソカイン（商標）を生成するため、位置指定突然変異誘発を用いてＣ１７Ａ（ＴＧＴ->ＧＣＴ）およびＱ６３Ｒ（ＣＡＧ->ＡＧＡ）突然変異を導入した。ＤＮＡシーケンシングも、サイレントＴ->Ｃ置換Ｖ８５Ｖ（ＧＴＴ->ＧＴＣ）の存在を明らかにした。 Example 1
Manufacture of albumin fusion axocaine (AXOKINE ™) CNTF, primer:

To exon 1 and primer:

Was used for exon 2, and two exons were amplified using standard conditions, respectively, and cloned from human genomic DNA. After ligating both fragments under standard conditions, primers:

Was amplified again by PCR and cloned into the vector pCR4 (Invitrogen). C17A (TGT-> GCT) and Q63R (CAG-> AGA) mutations using site-directed mutagenesis to generate Axocaine ™ as disclosed in Lambert et al. (PNAS 98: 4652-4657; 2001) Was introduced. DNA sequencing also revealed the presence of silent T-> C substitution V85V (GTT-> GTC).

およびＭＨ３５：

を用いる突然変異誘発ＰＣＲによってヒトアルブミンをコードするｃＤＮＡと連結させ、１４アミノ酸ＧＳ−（−Ｇｌｙ−Ｇｌｙ−Ｓｅｒ−Ｇｌｙ−Ｇｌｙ−Ｓｅｒ−Ｇｌｙ−Ｇｌｙ−Ｓｅｒ−Ｇｌｙ−Ｇｌｙ−Ｓｅｒ−Ｇｌｙ−Ｇｌｙ−）ペプチドスペーサーを導入した。成熟ｒＨＡ−ＧＳ−アクソカイン（商標）融合体のアミノ酸配列を図８に示す。 To create a C-terminal rHA-GS-Axokine ™ fusion, an Axokine ™ cDNA was added to a single standard oligonucleotide primer MH32:

And MH35:

And ligated with cDNA encoding human albumin by mutagenesis PCR using a 14 amino acid GS-(-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly -) A peptide spacer was introduced. The amino acid sequence of the mature rHA-GS-Axokine ™ fusion is shown in FIG.

およびＣＦ８３：

を用いる突然変異誘発ＰＣＲによって、アクソカイン（商標）ｃＤＮＡを、ヒトアルブミンをコードするｃＤＮＡと連結させ、２２アミノ酸３×ＦＬＡＧ（−Ａｓｐ−Ｔｙｒ−Ｌｙｓ−Ａｓｐ−Ｈｉｓ−Ａｓｐ−Ｇｌｙ−Ａｓｐ−Ｔｙｒ−Ｌｙｓ−Ａｓｐ−Ｈｉｓ−Ａｓｐ−Ｉｌｅ−Ａｓｐ−Ｔｙｒ−Ｌｙｓ−Ａｓｐ−Ａｓｐ−Ａｓｐ−Ａｓｐ−Ｌｙｓ−）ペプチドスペーサー(Sigma-Aldrich Company Ltd.)をアルブミンとアクソカイン（商標）との配列間へ導入した。成熟Ｃ末端ｒＨＡ３×ＦＬＡＧ−アクソカイン（商標）融合体のアミノ酸配列を図９に示す。ＷＯ９０／０１０６３に開示のＨＳＡ／ＭＦ□−１融合体分泌リーダー配列を準備して確実に融合タンパク質を分泌するようにする。 To create a C-terminal rHA-3 × FLAG-Axokine ™ (cleavable Axokine ™) fusion, a single-stranded oligonucleotide primer MH32:

And CF83:

The Axocaine ™ cDNA was ligated with cDNA encoding human albumin by mutagenesis PCR using a 22 amino acid 3 × FLAG (-Asp-Tyr-Lys-Asp-His-Asp-Gly-Asp-Tyr- Introduction of Lys-Asp-His-Asp-Ile-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-) peptide spacer (Sigma-Aldrich Company Ltd.) between sequences of albumin and Axocaine ™ did. The amino acid sequence of the mature C-terminal rHA3 × FLAG-Axokine ™ fusion is shown in FIG. The HSA / MF □ -1 fusion secretion leader sequence disclosed in WO90 / 01063 is prepared to ensure secretion of the fusion protein.

およびＭＨ３６：

を用いる突然変異誘発ＰＣＲによってアクソカイン（商標）ｃＤＮＡを、ヒトアルブミンをコードするｃＤＮＡと連結させ、各１４のアミノ酸ＧＳ（−Ｇｌｙ−Ｇｌｙ−Ｓｅｒ−Ｇｌｙ−Ｇｌｙ−Ｓｅｒ−Ｇｌｙ−Ｇｌｙ−Ｓｅｒ−Ｇｌｙ−Ｇｌｙ−Ｓｅｒ−Ｇｌｙ−Ｇｌｙ−）ペプチドスペーサーをアクソカイン（商標）とアルブミンとの配列間に導入した。成熟アクソカイン−ＧＳ−ｒＨＡ融合体のアミノ酸配列を図１０に示す。 To create an N-terminal Axocaine ™ -GS-rHA fusion, single stranded oligonucleotide primer MH33:

And MH36:

The Axocaine ™ cDNA was ligated with cDNA encoding human albumin by mutagenesis PCR using the following 14 amino acids: GS (-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly A -Gly-Ser-Gly-Gly-) peptide spacer was introduced between the sequence of Axocaine ™ and albumin. The amino acid sequence of the mature axocaine-GS-rHA fusion is shown in FIG.

  Maps of the rHA-GS-Axokine ™ sequence, the rHA-3xFLAG-Axokine ™ sequence and the Axokine ™ -GS-rHA sequence are shown in Figures 11, 12 and 13, respectively.

Suitable transcription promoter and transcription terminator sequences already disclosed in WO00 / 44772 and described in Sleep, D., et al. (1991) Bio / Technology 9,183-187, respectively, from yeast PRB1 promoter and yeast ADH1 terminator I was drowned. A suitable vector sequence was provided by the “degraded” plasmid pSAC35, generally disclosed in EP-A-286424 and described in Sleep, D., et al. (1991) Bio / Technology 9, 183-187. .
The rHA fusion was expressed and the shake flask culture expression level was determined.

Example 2
The purified C-terminal axocaine ™ contained material that was cut at a high level. It was purified using standard rHA SP-FF conditions (see US Pat. No. 6,034,221) but in a negative mode, whereby the fusion was in permeate. The permeate was adjusted to pH 8 and 2.5 mS · cm ⁻¹ and loaded onto standard rHA DE-FF equilibrated in 15 mM potassium tetraborate. For SP-FF, DEFF was operated in negative mode. The conductivity of the DE-FF permeate increased to 15 mS · cm ⁻¹ and the material was then purified using standard rHA DBA chromatography eluting with excess 50 mM octanoate. The eluate was then concentrated and diafiltered with 5 mM phosphoric acid pH 8.3.

The N-terminal Axocaine ™ contained some material that was cut. It was purified using standard rHA SP-FF conditions, but in negative mode, whereby the fusion was in the permeate. The permeate was adjusted to pH 8 and 2.5 mS · cm ⁻¹ and loaded onto standard rHA DE-FF equilibrated in 15 mM potassium tetraborate. In this case, the fusion ratio was fixed and eluted with a standard eluate containing 200 mM NaCl. The eluate conductivity was reduced to 15 mS · cm ⁻¹ and the material was then purified using standard rHA DBA chromatography by elution with an excess of 50 mM octanoate. The eluate was then concentrated and diafiltered with 5 mM phosphoric acid pH 8.3.

The cleavable Axocaine ™ contained material that was cut at a high level. It was purified using standard rHA SP-FF conditions, but in negative mode, whereby the fusion was in the permeate. The permeate was adjusted to pH 8 and 2.5 mS · cm ⁻¹ and loaded onto standard rHA DE-FF equilibrated in 15 mM potassium tetraborate. For SP-FF, this was operated in negative mode. The permeate conductivity increased to 15 mS · cm ⁻¹ and the material was purified using standard rHA DBA chromatography by elution with excess 50 mM octanoate. This material was then concentrated and diafiltered into the cleavage buffer. Cleavage was performed overnight at room temperature and enterokinase was removed using an Ekapture gel. The material was then concentrated and diafiltered with 5 mM phosphoric acid pH 8.3. Wurde hier nicht erneut gereinigt?

Example 3
Evaluation of half-life and bioavailability of N-terminal and C-terminal albumin fused axokine ™ against pharmacokinetic non-fused axokine ™, and further addition of N-terminal and C-terminal albumin fused axokine ™ to non-fused axokine ™ Evaluation of pharmacokinetic parameters.

Administration protocol:
Test article 1: Non-fusion axocaine (trademark)
Application amount: 0.33mL / kg
Single dose / route: 10 μg / kg intravenous or subcutaneous frequency: once (t = 0)
Test article 2: N-terminal albumin-fused axocaine (trademark)
Application amount: 0.33mL / kg
Single dose / route: 40 μg / kg intravenous or subcutaneous frequency: once (t = 0)
Test article 3: C-terminal albumin-fusion axocaine (trademark)
Application amount: 0.33mL / kg
Single dose / route: 40 μg / kg intravenous or subcutaneous frequency: once (t = 0)

試験動物
種／系統： ウサギ
性別／齢： 雄１２匹、雌１２匹；３〜４ヶ月
総数： ２４
供給者： Fa. Bauer (Neuenstein-Lohe, Germany) Test plan

Test animals Species / strain: Rabbit gender / age: 12 males, 12 females; 3-4 months total: 24
Supplier: Fa. Bauer (Neuenstein-Lohe, Germany)

On day 0 of animal model , 2 male and 2 female rabbits per group were treated with cleavable Axocain ™ (1 μg / kg), C-terminal albumin fusion Axocaine ™ (40 μg / kg), or N Terminal albumin-fused axocaine ™ (40 μg / kg) was administered by a single intravenous or subcutaneous injection. After intravenous administration of each test substance, baseline, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours (1 day), 48 hours ( 2 days), 72 hours (3 days), 7 days, 9 days, 11 days and 14 days, and after subcutaneous injection, baseline, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours Blood samples were taken to measure the respective antigen levels at (1 day), 48 hours (2 days), 72 hours (3 days), 5 days, 7 days, 9 days, 11 days and 14 days. Plasma levels of Axocaine ™ and albumin-fused Axocaine ™ were measured by ELISA.

Pharmacokinetic (PK) variables:
Elimination half-life, area under the plasma concentration curve up to 14 days (AUC _0-14 ), maximum concentration (C _max ). Area under estimated concentration curve to infinity (AUC _0-∞ ), maximum concentration time (t _max ), average residence time, absorption and distribution half-life (if applicable), and distribution clearance.

Analytical Method ELISA measurement of unfused Axocaine ™ plasma concentration was performed using monoclonal mouse anti-hu CNTF antibody (R & D Systems, clone no. 21809.111) and biotinylated polyclonal goat anti-hu CNTF antibody (R & D Systems, catalog) No. BAF257) in combination. Human CNTF was used as a standard according to the instructions of the ELISA kit.

  An ELISA measurement of albumin-fused Axocaine ™ plasma concentration was used in combination with a monoclonal anti-hu albumin antibody (Aventis Behring GmbH, Laboratory) and a biotinylated polyclonal goat anti-hu CNTF antibody (R & D Systems, catalog number BAF257). went. Each albumin-fused Axocaine ™ helped generate a standard curve.

  Using a commercially available human CNTF ELISA (R & D Systems), it was impossible to detect albumin-fused axokine ™. Probably because albumin sterically interferes with the binding of anti-CNTF antibodies.

  As a solution, an anti-albumin monoclonal antibody was used as a capture antibody and an internal anti-albumin assay was established, which bound to the plate. As the next step, a commercially available CNTF antibody obtained from R & D Systems was used as an antibody for detecting albumin-fused axokine (trademark).

Individual Plasma Level Analysis Plasma concentration-time profiles of C-terminal and N-terminal albumin fused axocaine ™ and non-fused axocaine ™ were analyzed from animal to animal using non-linear regression. This data was fitted to an exponential model by the method of least squares. An open two-compartment model was used for the profile following intravenous administration. The profile after subcutaneous administration used an open 1-compartment model with primary input and lag time. A weight coefficient of 1 / (estimated concentration) ² was applied to the intravenous administration model.

AUC fulfills (AUC _0-14 ) by a) using a linear trapezoidal formula up to the last measurement (AUC _0-14 ) and b) estimating the period between 14 days and infinity. Calculated.

Summary and comparative analysis Individual PK results were descriptively summarized by treatment and application route (minimum, median, maximum, mean, standard deviation).

Two-way analysis of variance was performed for elimination half-life, AUC and C _max (all log transformed). The fixed factors were gender and treatment group. Appropriate contrast between treatment groups was evaluated. Non-uniform distribution was also taken into account.

In this analysis, it was assumed that In (half-life), In (AUC) and In (C _max ) each follow a normal distribution.

The elimination half-life was compared between substances, and bioavailability in terms of AUC and C _max was compared between administration groups of albumin fusion Axokine ™ at alpha level 0.1 using a two-sided 90% confidence interval.

Results The mean and standard deviation of Axocain ™ concentrations at each time point are shown in FIG. 1 for the intravenously treated non-fused Axocaine ™ group and in FIG. 2 for the intravenously treated albumin-fused Axocaine ™ group and subcutaneously treated. FIG. 3 shows the albumin-fused axokine (trademark) group. Concentration measurements could not be made for the subcutaneously treated non-fused axocaine ™ group.

  In animals treated intravenously with unfused Axokine ™, levels were below 1 pg / mL 4 hours after injection. In the albumin-fused Axocaine ™ group, this level exceeded 1 ng / mL for 7 days. In animals treated subcutaneously with the albumin-fused Axocaine ™ product, this level peaked after about 1 day and exceeded 1 ng / mL for 7 days. Table 2 shows the pharmacokinetic results for the intravenous treatment group and Table 3 for the subcutaneous treatment group. The unfused axokine ™ results have been converted to the same units of the albumin fused axokine ™ group, but cannot be compared to these due to differences in the assay, except for half-life and average residence time.

  Table 4 shows the results of analysis of variance for elimination half-life. The difference between non-fused and albumin-fused axocaine ™ after intravenous injection was very significant. Animal gender had no significant effect on half-life.

Table 5 shows the results of analysis of variance for absolute bioavailability. For both albumin fusion products, the difference between the two routes of administration was not statistically significant with respect to elimination half-life. The differences with respect to AUC and _Cmax were very significant.

  The value of the area under the concentration curve and the maximum plasma level of unfused Axocaine ™ cannot be compared directly with the values of N- and C-terminal albumin fusion Axocaine ™. On the other hand, comparison of half-life is reasonable.

  Both albumin-fused Axokine ™ formulations showed significant prolongation of plasma excretion after intravenous administration compared to non-fused Axokine ™. The C-terminal albumin fused axocaine ™ showed a mean elimination half-life that was 72 times longer than the non-fused axocaine ™. N-terminal albumin fused Axocaine ™ showed a 48-fold longer mean elimination half-life than non-fused Axocaine ™.

  For AUC, the absolute bioavailability after subcutaneous injection was 76% for C-terminal albumin fusion axocaine ™ and 23% for N-terminal albumin fusion axocaine ™. Since plasma levels of unfused Axocaine ™ were below the detection limit after subcutaneous application, comparison with intravenous application was not possible.

Example 4
Pharmacodynamics The purpose of this experiment was to demonstrate the effectiveness of N- and C-terminal albumin-fused axocaine ™ in reducing weight in leptin-deficient or diet-induced obese mice compared to placebo or unfused axocaine ™. It was to evaluate.

Pharmacodynamic animal study study design, Part I
This study consisted of a total of 70 female C57BL / 6 Jlep ^ob (ob / ob), and 41 male and 41 female C57BL / 6J mice (2 Designed as an experimental setting (leptin deficiency-induced obesity versus diet-induced obesity).

Test animals C57BL / 6 Jlep ^ob (ob / ob) mice were fed a standard diet for about 3 months. During this time, C57BL / 6 Jlep ^ob (ob / ob) mice gained weight strongly due to uncontrolled food intake associated with leptin deficiency. In wild-type C57BL / 6 mice, obesity was induced by eating a high-calorie diet with 45% fat. Body weight was recorded once a week during this obesity induction phase prior to therapeutic treatment. Treatment with the test substance was started when the average body weight increased to at least 130% of baseline. Test substances (non-fused axocaine ™, albumin fusion axocaine ™, placebo) were administered daily by subcutaneous injection for 7 days. Body weight was measured daily during the treatment phase. The average weight loss compared to baseline and placebo was calculated to assess the relative effect of the test substance.

Test drug and dose Test article 1: Placebo (pH 8.3, 5 mM phosphate buffer)
Endotoxin content: 0.007 EU / mL
Storage concentration: Not applicable Application amount: 250 μl ^a
1 dose / route: not fit / subcutaneous: daily injection for 7 days

Test item 2: Non-fusion Axocaine (trademark)
Endotoxin content: 14.9 EU / m2L
Storage concentration: 0.1 mg / mL
Application amount: 250μl ^a
1 dose / route: according to Tables 1 and 2 / subcutaneous: 7 days daily injection

Test item 3: N-terminal albumin fusion axokine (trademark)
Endotoxin content: 1.8 EU / mL
Storage concentration: 5 mg / mL
Application amount: 250μl ^a
1 dose / route: according to Tables 1 and 2 / subcutaneous: 7 days daily injection

Test article 4: C-terminal albumin fusion axokine (trademark)
Endotoxin content: 64 EU / mL and 32 EU / mL
Storage concentration: 0.2 mg / mL
Application amount: 250μl ^a
1 dose / route: according to Tables 1 and 2 / subcutaneous: 7 days daily injection
^{a On} day 1 of treatment (day 83) all mice received 250 μl of test substance and then the dosage was adapted to changes in body weight by adjusting the dose. Mice in group 13 (1200 μg / kg C-terminal axocaine ™) received about 390 μg on day 83.

The following dose reductions were required for both ob / ob as well as wild type mice.
Non-fused axocaine (trademark) manufactured by Delta: 300 μg / kg from 1 to 2 days to 200 μg / kg from 3 to 7 days
N, C-terminal albumin fusion axocaine ™: 280 μg / kg from 1-2 days to 200 μg / kg from 3-7 days
N, C-terminal albumin fusion axocaine ™: 1200 μg / kg from 1-2 days to 800 μg / kg from 3-7 days
Randomization was performed separately for C57BL / 6Jlep ^ob (ob / ob) and C57BL / 6J mice according to the randomization list. After the mice were randomized and placed in cages, the cages were randomized and treated.
Efficacy variable: body weight (measured daily from day 0 to day 7)

Analytical method The body weight of the awakened animal was measured and recorded.

Statistical method
Primary efficacy variable : weight difference between 7 and 0 and up to 102 days. Dose-response relationship to non-fused axokine ™, N-terminal albumin fused axokine ™, and C-terminal albumin fused axokine ™ classified into C57BL / 6Jlep ^ob (ob / ob) and C57BL / 6J Were analyzed within one analysis method of the dispersion model.

  Sequential comparison of various doses, including placebo, using a contrast (eg, Helmart contrast or reverse Helmart contrast) to determine the minimum effective dose. Comparison of equimolar dose pairs using a two-tailed t-test for differences and a two-sided 95% confidence interval.

  An overall evaluation of the N-terminal albumin-fused axocaine ™ and the C-terminal albumin-fused axocaine ™ with respect to the non-fused axocaine ™ was performed by a parallel line test at logarithmically transformed doses. The derived efficacy was supplemented with a 95% confidence interval.

Results <br/> Statistical analysis of primary endpoints
Endpoint : Weight change from 82 days to 91, 92, 93, 94, 95, 96, 102 days (g)
Statistics : F test within ANOVA for oeded hypotheses families. Starting on day 92, if the corresponding F test is significant, one hypothesis is rejected and the previous hypothesis is also rejected.
Reference : Bauer P: Multiple tests in clinical trials. Statistics in Medicine, 10: 871 890, 1991

Weight loss of ob / ob mice FIGS. 4, 5, 6 and 7 compare equimolar doses of non-fused axocaine ™ and albumin-fused axocaine ™ in leptin-deficient mice.

  In summary, pharmacodynamic data show that albumin-fused axokine ™ is more statistically significant than unfused axokine ™ for dose groups 11, 12, and 13 in leptin-deficient mice. Show. In wild-type mice, albumin-fused axokine ™ is more statistically significant in group 12 than non-fused axokine ™.

Pharmacodynamic animal study study design, part II
The study was originally a total of 82 female B6. Absence in two experimental settings (leptin deficiency-induced obesity versus diet-induced obesity), including V-Lep ^ob (ob / ob) mice and 41 male and 41 female C57BL / 6J mice Designed as a randomized partially blinded parallel 11 arm study. Due to the limited availability of unfused Axocaine ™, only selected leptin-deficient treatment groups were included in the treatment phase of the study (Table 8).

Schedule B6. V-Lep ^ob mice were fed a standard diet up to 80 days and gained weight. 7 consecutive days (81, 82, 83, 84, 85, 86, 87 days) or 1, 4, 7 days (81, 82) with either non-fused axokine ™ or C-albumin fused axokine ™ 84, 87 days) treatment only started on day 81.

Body weight was evaluated up to 21 days (108 days) after cessation of treatment. Body weight changes and related analysis were related to body weight at 81 days.
The corresponding time points are summarized in the following table.

Administration of test article Test article 1: placebo (5 mM phosphate buffer, pH 8.3)
Manufacturer: Aventis Behring (Laboratory Dr. H. Metzner)
Batch number: −
Endotoxin content: not carried out Storage concentration: not suitable Application amount: 5 μl / g
1 dose / route: not fit / subcutaneous: daily injection for 7 days

Test article 2: Non-fused axocaine (trademark) (enterokinase cleavage type)
C-terminal albumin fusion axocaine (registered trademark))
Manufacturer: Delta Biotechnology Ltd., Laboratory Dr. D. Sleep
Batch number: 1675 # 40
Endotoxin content: 18 EU / mL
Storage concentration: about 0.1 mg / mL (compared with CNTF as a standard,
Assumptions based on SDS PAGE with Coomassie staining, Appendix B)
Application amount: 5 μl / g
One dose / route: According to Table 1 / subcutaneous frequency: Single injection on days 1, 4, 7 or daily injection for 7 days

Test article 3: C-terminal albumin fusion axokine (trademark)
Manufacturer: Delta Biotechnology Ltd., Laboratory Dr. D. Sleep /
Aventis Behring GmbH, Laboratory Dr. H. Metzner
Batch number: 091002
Endotoxin content: 16 EU / mL
Storage concentration: about 0.1 mg / mL (compared with HSA as standard,
Assumptions based on SDS PAGE with Coomassie staining, Appendix B)
Application amount: 5 μl / ga
One dose / route: According to Table 1 / subcutaneous frequency: Single injection on days 1, 4, 7 or daily injection for 7 days

Test article 4: C-terminal albumin fusion axocaine stored for 14 days at room temperature
(Trademark)
Manufacturer: Delta Biotechnology Ltd., Laboratory Dr. D. Sleep /
Aventis Behring GmbH, Laboratory Dr. H. Metzner
Batch number: 091002
Endotoxin content: 16 EU / mL
Storage concentration: about 0.4 mg / mL (compared with HSA as standard,
Assumptions based on SDS PAGE with Coomassie staining, Appendix B)
Application amount: 5 μl / g ^a
One dose / route: According to Table 1 / subcutaneous frequency: Single injection on days 1, 4, 7 or daily injection for 7 days
^a All mice received 5 μl of test substance. However, 10 μl / g was administered to mice treated with C-terminal Axocaine ™ 3600 μg / kg.

Animal model B6. V-Lep ^ob mice were fed a standard diet for 12 weeks. During this time, mice gained weight strongly due to uncontrolled food intake associated with leptin deficiency. During this obesity-inducing phase preceding therapeutic treatment, body weights were recorded once a week except for the days 49-66 when animals were not weighed. Test substances (Axocaine ™, C-terminal albumin fusion Axocaine ™, placebo) were administered daily by subcutaneous injection for 7 days or 3 times, once a day on 1, 4, 7 treatment days. . Body weight was measured daily during the treatment phase. Thereafter, the body weight was recorded every other day for 14 days (ie, 3 times per week), and another 21 days after the treatment (28 days after the start of treatment = 108 days of the survey). The average weight loss compared to baseline and placebo was calculated to assess the relative effect of the test substance.

Randomization Randomization was performed according to a randomized list. After the mice were randomized and placed in cages, the cages were randomized and treated.

Efficacy variable Main variable: change in body weight (g) from treatment day 1 (survey day 81) to treatment day 7 (survey days 88, 87, 86, 85, 84, 83, and 82).
Secondary variable: body weight 28 days after the start of treatment (survey date 108 days). Changes in body weight (g) from the 81st survey day to 89, 91, 94, 96, 98, 101, 108.

Analytical method The body weight of the awakened animal was measured and recorded.

F-test within ANOVA of statistical method order hypothesis group. Starting on the 88th, and then proceeding downward, if the corresponding F-test is significant (p ≦ 0.05), one hypothesis is rejected and the previous hypothesis is also rejected (p ≦ 0. 05). The same procedure was used upwards from 89 to 108 days. This procedure controlled multiple levels of 0.05 in a series of comparisons consisting of seven hypotheses related to the day.

  An analysis consisting of 4 blocks was performed. Tables 10 and 11 tabulated the trial decisions of the trials compared to placebo, ie, to check the validity of the model, the effective treatment group (groups 2-11) was compared to placebo (group 1). An equimolar dose analysis is shown in Tables 12 and 13, while treatment schedules are compared in Tables 14 and 15. Finally, efficacy assessment using parallel line test for log dose at 88 days body weight change as an alternative to response criteria is summarized in Table 16. There have been no tests (ie linearity, parallelism) on the suitability of the verification approach.

Results Effect on body weight Study treatment was administered from 81 to 87 days.
Comparison with placebo All groups that received the test substance showed significant differences relative to placebo between days 82 and 101 (Tables 10 and 11).

Comparison of equimolar doses

Treatment schedule comparison

Efficacy evaluation

  It was very surprising that during the administration of the fusion protein subcutaneously, an increase in the plasma half-life of albumin-fused axocaine (72 times longer than unfused axocaine) was found as investigated in rabbits. First, subcutaneous administration is known to reduce absorption, but this is not the case here. Secondly, it is also generally known that the plasma half-life of human plasma proteins can be dramatically reduced in animals, indicating that this prolongation is even more pronounced in humans. This was confirmed by the inventors' pharmacodynamic findings in mice that it is possible to administer fusion proteins every three days, which has an effect comparable to daily application of unfused axocaine. As a result, the inventors speculate that it may be possible to administer the fusion protein to a human once a week or at longer intervals. Since the production rate of antibodies against CNTF may be reduced, further effects and safety may increase.

Six of the clinical findings animals withdrew from the study prematurely and all withdrew after completing the following treatments:

  All animals in groups 8, 10, and 11 starting on day 84 (3 daily doses of 1200 μg / kg C-AFP or 3600 μg / kg C-AFP) were sluggish, disturbed, and the skin was red throughout the body. And systemic symptoms decreased. Up to 10 out of 12 animals received 1200 μg and all animals treated with 3600 μg suffered from hemorrhagic diarrhea over the next 2 days, resulting in severe dehydration with reduced water intake.

Therefore, one animal from groups 10 and 11 on 89th and 3 animals in 8 groups on 91th were euthanized just before death. In addition, one animal from 10 groups died on the 96th.
At necropsy, severe obesity, dehydration, and liver and kidney steatosis were found in all animals tested with enlarged intestines.

Conclusion Prior to the start of treatment (day 81), a total of 70 animals were available, 10 in the placebo group (1 group) and 6 in each of the 10 effective treatment groups. A total of 6 animals were euthanized or died during the study, all died after treatment was completed. That is, one animal from groups 10 and 11 died on 89th, 3 animals from 8 groups on 91 days, and finally another animal from group 10 on 96 days. These cases as well as the clinical symptoms found are limited to the highest dose group and are therefore considered to be treatment related.

  The weight of the placebo-treated animals was approximately constant between 81 and 88 days (88 days average weight change: -0.4%), but gained by 108 days in the course of further testing ( An average change of 108 days: 7.2%) was observed.

  Treatment with an effective substance (Groups 2-11) resulted in a significant dose-dependent weight loss compared to placebo (Table 10). Within 21 days after completion of treatment, animals treated with the active ingredient showed significantly higher weight loss than placebo animals (Table 11).

  When compared at equimolar doses, albumin-fused axokine ™ was significantly better than non-fused axokine ™ for weight loss, regardless of which treatment schedule was applied (Table 12, Figure 14). . This effect continued in a dose-dependent manner after the end of treatment (Table 13), and groups 7 and 11 continued to 108 days.

  Daily injections over 7 days resulted in a more pronounced effect than injections at 1, 4 and 7 days in both the treatment period and the 21-day follow-up period (Tables 7 and 8). This was the same for comparisons within the unfused Axocaine ™ and within the C-albumin fusion Axocaine ™.

  Efficacy assessment was limited to 88 days of weight change. Albumin-fused Axokine ™ was 1.9 and 2.3 times more potent than the non-fused Axokine ™ and the 7-day treatment schedule and 1, 4, 7-day treatment schedule, respectively (Table 16). The 1-7 day treatment was more potent than the 1, 4, 7 day treatment. A potency of 1.85 times for non-fused axocaine ™ and 9.13 times potency for albumin-fused axocaine ™ was calculated.

The 1, 4 and 7 day albumin fusion Axocaine ™ injections were almost as effective as 7 consecutive daily injections with non-fusion Axocaine ™.

Pharmacokinetics of unfused axocaine (AXOKINE ™) in rabbits (intravenous injection). Pharmacokinetics of C-terminal and N-terminal fusion axocaine (AXOKINE ™) in rabbits (intravenous injection). Pharmacokinetics of C-terminal and N-terminal fusion axocaine (AXOKINE ™) in rabbits (subcutaneous injection). Weight loss curve of leptin-deficient mice treated with unfused axocaine (AXOKINE ™). Weight loss curve of leptin-deficient mice treated with C-terminal fusion axocaine (AXOKINE ™). Weight loss curve of wild type mice treated with unfused axocaine (AXOKINE ™). Weight loss curve of wild type mice treated with C-terminal fusion axocaine (AXOKINE ™). Amino acid sequence of mature C-terminal axocaine (AXOKINE ™) (SEQ ID NO: 1). Amino acid sequence of mature C-terminal rHA-3xFLAG- (cleavable) axocaine (AXOKINE ™) (SEQ ID NO: 2). Amino acid sequence of mature N-terminal axocaine (AXOKINE ™) (SEQ ID NO: 3). Map of C-terminal fusion axocaine (AXOKINE ™). Map of C-terminal rHA-3xFLAG- (cleavable) axokine (AXOKINE ™). Map of N-terminal fusion axocaine (AXOKINE ™). Weight loss curves of leptin-deficient mice treated with unfused axocaine (AXOKINE ™) and C-terminal fused axocaine ™ every 3 days. Weight loss curves of leptin-deficient mice treated daily with unfused axocaine (AXOKINE ™) and C-terminal fused axocaine ™.

Claims (32)

Albumin, or a fragment or variant or derivative thereof;
A fusion protein comprising at least one biologically active peptide or protein that activates ciliary neurotrophic factor (CNTF) receptor, or a fragment or variant or derivative thereof.   The fusion protein of claim 1, wherein the at least one peptide or protein that activates the ciliary neurotrophic factor (CNTF) receptor is CNTF or a fragment or variant or derivative thereof.   The fusion protein according to claim 2, wherein CNTF is AXOKINE.   The fusion protein according to any one of claims 1 to 3, wherein the in-vivo half-life of the fusion protein is longer than the in-vivo half-life of the non-fused biologically active peptide or protein.   The fusion protein according to any one of claims 1 to 3, wherein the shelf life of the fusion protein is longer than that of the non-fused biologically active peptide or protein.   The fusion protein according to any one of claims 1 to 5, which is expressed in yeast.   The fusion protein according to any one of claims 1 to 5, which is expressed in a mammalian cell.   The fusion protein according to any one of claims 1 to 5, wherein the mammalian cell is a human cell.   A pharmaceutical composition comprising an effective amount of the fusion protein according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier or excipient.   Use of the fusion protein according to any one of claims 1 to 8 for the manufacture of a medicament for the treatment of obesity and related diseases.   Use according to claim 10, wherein the disease associated with obesity is diabetes, hyperglycemia or hyperinsulinemia. A method for increasing the half-life of a biologically active peptide or protein, or a fragment or variant or derivative thereof, that activates ciliary neurotrophic factor (CNTF) receptor in a mammal, comprising:
The biologically active peptide or protein and albumin are linked to form an albumin-fused biologically active peptide or protein, and the albumin-fused biologically active peptide or protein To the mammal, whereby the half-life of the albumin fusion biologically active peptide or protein is at least twice the half-life of the biologically active peptide or protein lacking linked albumin A method comprising extending.   13. A method according to claim 12, wherein the biologically active peptide or protein is CNTF or a fragment or variant or derivative thereof.   14. The half-life of an albumin-fused biologically active peptide or protein is increased by at least 5 times the half-life of a biologically active peptide or protein lacking linked albumin. The method according to one item.   14. The half-life of an albumin-fused biologically active peptide or protein is extended at least 10 times the half-life of a biologically active peptide or protein lacking linked albumin. The method according to one item.   14. The half-life of an albumin-fused biologically active peptide or protein is extended at least 50 times the half-life of a biologically active peptide or protein lacking linked albumin. The method according to one item. A method of increasing the concentration of biologically active peptides or proteins that cross the blood brain barrier,
Linking the biologically active peptide or protein and albumin to form an albumin fusion biologically active peptide or protein, and transferring the albumin fusion biologically active peptide or protein to the mammal Administration, thereby increasing the concentration of the albumin-fused biologically active peptide or protein that crosses the blood brain barrier above the concentration of biologically active peptide or protein lacking linked albumin Comprising a method.   18. The method of claim 17, wherein the biologically active peptide or protein activates ciliary neurotrophic factor (CNTF) receptor, or a fragment or variant or derivative thereof.   18. A method according to claim 17, wherein the biologically active peptide or protein is CNTF or a fragment or variant or derivative thereof. A method of minimizing side effects associated with treating a mammal with a biologically active peptide or protein that activates ciliary neurotrophic factor (CNTF) receptor, or a fragment or variant or derivative thereof, comprising:
The biologically active peptide or protein and albumin are linked to form an albumin-fused biologically active peptide or protein, and the albumin-fused biologically active peptide or protein Administering to the mammal.   21. The method of claim 20, wherein the biologically active peptide or protein activates ciliary neurotrophic factor (CNTF) receptor, or a fragment or variant or derivative thereof.   21. The method of claim 20, wherein the biologically active peptide or protein is CNTF or a fragment or variant or derivative thereof.   23. A method according to any one of claims 20 to 22, wherein the side effect is nausea and / or headache.   A nucleic acid molecule comprising a polynucleotide sequence encoding the fusion protein according to any one of claims 1-8.   A vector comprising the nucleic acid molecule of claim 24.   A host cell comprising the nucleic acid molecule of claim 24.   A method for activating a CNTF receptor in a cell, comprising the step of contacting the cell with an effective concentration of the fusion protein according to any one of claims 1-8.   28. The method of claim 27, wherein the cell is a mammalian cell.   30. The method of claim 28, wherein the cell is a human cell.   A method for activating a CNTF receptor in a cell, wherein the nucleic acid molecule according to claim 24 is introduced into a cell, and the fusion protein according to any one of claims 1 to 8 is therapeutically effective. A method comprising providing a cell with an effective concentration of the fusion protein according to any one of claims 1 to 8 by allowing the cell to produce a sufficient amount.   32. The method of claim 30, wherein the cell is a mammalian cell.   32. The method of claim 31, wherein the cell is a human cell.

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