Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-18 are pending.
Applicant’s election a method for prevention or treatment of a neurodegenerative disease that read on (A) SEQ ID NO: 42 as the species of peptide and (B) Parkinson as the species of neurodegenerative disease in the reply filed on December 4, 2025 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claim 15 is withdrawn from further consideration by the examiner, 37 C.F.R. 1.142(b) as being drawn to non-elected inventions.
Claims 1-14 and 16-18, drawn to a method for prevention or treatment of a neurodegenerative disease that read on (A) SEQ ID NO: 42 as the species of peptide and (B) Parkinson as the species of neurodegenerative disease, are being acted upon in this Office Action.
Priority
Receipt is acknowledged of papers submitted under 35 U.S.C. 119(a)-(d), which papers have been placed of record in the file.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on November 1, 2024 and March 24, 2023 have been considered by the examiner and an initialed copy of the IDS is included with this Office Action.
Drawings
The drawings filed on March 24, 2023 are acceptable.
Specification
The amendment to the specification filed on March 24, 2023 has been entered.
The lengthy specification has not been checked to the extent necessary to determine the presence of all possible minor errors. Applicant's cooperation is requested in correcting any errors of which applicant may become aware in the specification.
Claim Objection
Claims 1-14 and 16-18 are objected to because of the following informality: the [ ] in claims 1-14 and 16-18 should be deleted.
Claims 1 and 2 are objected to because of the following informality: “containing” should have been “comprising”.
Claims 4-6 are objected to because of the following informality: “contains” should have been “comprises”.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 2 and 8-13 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 2, which depends on claim 1, recites “the method according to claim 1, wherein the peptide is in the form of a long-acting conjugate and the long-acting conjugate is represent by Formula 1 below:[Formula 1] X-L-F wherein X represents a peptide containing an amino sequence of any one of SEQ ID NOS: 1 to 102; L represents a linker containing ethylene glycol repeat units; F represents an immunoglobulin Fc region; and "-" symbols represent covalent linkages between X and L and between L and F, respectively. However, the conjugate in claim 2 is not further limiting the subject matter, e.g., peptide containing SEQ ID NO 42 of claim 1 as the parent claim 1 is drawn to a method for prevention or treatment of a neurodegenerative disease by administering the subject a pharmaceutical acceptable vehicle and a therapeutically effective of a peptide containing an amino acid sequence of SEQ ID NO: 42.
Claim 8, which depends on claims 2 and 1, recites “the method according to claim 2, wherein L is polyethylene glycol, which do not further limit the peptide containing amino acid sequence of SEQ ID NO 42 in claim 1.
Claim 9, which depends on claims 2 and 1, recites “the method according to claim 2, wherein the formula weight of an ethylene glycol repeat unit moiety in L is in a range of 1 to 100 kDa, which do not further limit the peptide containing amino acid sequence of SEQ ID NO 42 in claim 1.
Claim 10, which depends on claims 2 and 1, recites “the method according to claim 2, wherein the immunoglobulin Fc region is aglycosylated, which do not further limit the peptide containing amino acid sequence of SEQ ID NO 42 in claim 1.
Claim 11, which depends on claims 2 and 1, recites “the method according to claim 2, wherein F is an IgG Fc region, which do not further limit the peptide containing amino acid sequence of SEQ ID NO 42 in claim 1.
Claim 12, which depends on claims 2 and 1, recites “the method according to claim 2, wherein the immunoglobulin Fc region is a dimer consisting of two polypeptide chains, and one end of L is linked only to one of the two polypeptide chains, which do not further limit the peptide containing amino acid sequence of SEQ ID NO 42 in claim 1.
Claim 13, which depends on claims 2 and 1, recites “the method according to claim 2, wherein in the conjugate, one end of L is linked to F via a covalent linkage formed by reaction with an amine or thiol group of F, and the other end of L is linked to X via a covalent linkage formed by reaction with an amine or thiol group of X, which do not further limit the peptide containing amino acid sequence of SEQ ID NO 42 in claim 1.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim rejections under - 35 U.S.C. 112
The following is a quotation of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), first paragraph:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-14 and 16-18 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
The MPEP lists factors that can be used to determine if sufficient evidence of possession has been furnished in the disclosure of the application. These include "level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention. Disclosure of any combination of such identifying characteristics that distinguish the claimed invention from other materials and would lead one of skill in the art to the conclusion that the applicant was in possession of the claimed species is sufficient" (MPEP 2163).
A claimed genus may be satisfied through sufficient description of a representative number of species or disclosure of relevant, identifying characteristics such as functional characteristics coupled with a known or disclosed correlation between function and structure (MPEP 2163(3)a(II)). The number of species that describe the genus must be adequate to describe the entire genus; if there is substantial variability, a large number of species must be described.
The claims encompass a method for prevention or treatment of any neurodegenerative disease (claims 1-2) such as Parkinson’s disease (claims 14 and 16) in a subject comprising administering to the subject a pharmaceutical composition comprising a pharmaceutical acceptable vehicle and a therapeutically effective amount of any peptide containing an amino acid sequence of SEQ ID NO: 42 (elected species, claim 1, 3-6, 17-18) or a long-acting conjugate represented by Formula I X-L-F wherein X represents a peptide containing SEQ ID NO: 42, L represents a linker containing ethylene glycol repeat units, and F represents any immunoglobulin Fc region (claim 2, 8-13).
Regarding neurodegenerative disease, the specification discloses:
[0245] As used herein, the term “neurodegenerative disease” refers to a disease that causes several symptoms resulting from degenerative changes appearing in neurons of the central nervous system and is a collective term for diseases that cause several symptoms resulting from degenerative changes appearing in neurons. Specifically, the neurodegenerative disease may include neuropathy that causes impairments in cognitive function, learning, or memory, impairments in hand/foot sensation, or malfunctions of organs including the stomach. Specifically, the neurodegenerative disease is known to be caused by the gradual loss of functions of a specific brain cell population in the brain and spinal cord to result in the death of brain neurons, which are most important for information transmission in the brain nervous system, problems with the formation or function of synapses that transmit information between brain neurons, or an abnormal increase or decrease in electrical activity of brain nerves.
[0246] Among the neurodegenerative diseases, degenerative brain diseases may be classified considering the main symptoms and the brain areas affected, and the degenerative brain diseases may include Parkinson’s disease (PD), Alzheimer’s disease, Huntington’s disease (HD) also known as Huntington’s chorea, amyotrophic lateral sclerosis (ALS) also known as Lou Gehrig’s disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), Lewy body dementia, Parkinson’s disease dementia, epilepsy, stroke, Huntington’s chorea, cerebral hypoxia, peripheral neuropathy, memory impairment, memory loss, forgetfulness, Pick’s disease, Creutzfeldt-Jakob disease, and nerve damage caused by complications of diabetes.
[0247] In an embodiment of the present invention, the neurodegenerative disease may be Parkinson’s disease or stroke.
[0248] In another embodiment, the neurodegenerative disease is Parkinson’s disease. Parkinson’s disease may be associated with oxidative stress, inflammatory response, apoptosis, loss of neurons, especially loss of dopaminergic neurons, e.g., loss of the substantia nigra, resulting in dopamine deficiency.
[0249] In another embodiment of the present invention, the neurodegenerative disease may be Alzheimer’s disease. “Alzheimer’s disease” is one of the most common degenerative brain diseases that cause dementia, and is a collective term for diseases that cause various symptoms resulting from degenerative changes appearing in neurons of the central nervous system. Specifically, Alzheimer’s disease may include neuropathy that causes impairments in cognitive function, learning, or memory, impairments in hand/foot sensation, or malfunctions of organs including the stomach. Alzheimer’s disease is accompanied by cognitive function as well as neurobehavioral symptoms, such as personality changes, restlessness, depression, delusions, hallucination, increased aggression, and sleep disorders, during the progression; and neurological disorders, such as muscle rigidity and gait abnormalities, or physical complications, such as incontinence, infections, and bedsores, in the later stage. When the brain tissue of an Alzheimer’s disease patient is examined under a microscope, characteristic lesions, such as a neuritic plaque and a neurofibrillary tangle, are observed, and when observed with the naked eye, overall brain atrophy is seen due to loss of neurons. In the early stages of the disease, these brain pathological findings are mainly limited to the hippocampus, which is the main brain region responsible for memory, and the entorhinal cortex, but gradually spreads to the entire brain via the parietal lobe and frontal lobe. In accordance with the progression of the brain pathology invasion area, memory decline mainly appears in the early stage, and as it progresses, clinical symptoms vary and become more severe while showing a gradual course.
[0250] Specifically, the neurodegenerative disease is known to be caused by the gradual loss of functions of a specific brain cell population in the brain and spinal cord to result in the death of brain neurons, which are most important for information transmission in the brain nervous system, problems with the formation or function of synapses that transmit information between brain neurons, or an abnormal increase or decrease in electrical activity of brain nerves. Alzheimer’s disease (AD) may also be referred to as Alzheimer’s disorder. Depending on the age of onset, Alzheimer’s disease may be divided into early-onset (elderly) Alzheimer’s disease if it develops under the age of 65, and late-onset (elderly) Alzheimer’s disease if it develops over the age of 65 years, but is not limited thereto.
[0251] Alzheimer’s disease may be associated with oxidative stress and neuronal loss.
[0252] In another embodiment, the neurodegenerative disease is progressive supranuclear palsy. Progressive supranuclear palsy may be associated with neuronal loss, particularly, loss of dopaminergic neurons.
[0253] In another embodiment, the neurodegenerative disease is multiple system atrophy. Multiple system atrophy may be associated with loss of neurons, particularly, dopaminergic neurons.
[0254] In another embodiment, the neurodegenerative disease is Lewy body dementia. Lewy body dementia may be associated with loss of neurons, particularly, dopaminergic neurons. Lewy body dementia is known to account for about 20% of the most common causes of dementia, after Alzheimer’s disease, among neurodegenerative diseases observed in the elderly. Lewy body dementia may be associated with Parkinson’s disease.
[0255] In another embodiment, the neurodegenerative disease is epilepsy. Epilepsy refers to a brain disease in which symptoms of temporary paralysis of brain functions, such as loss of consciousness, seizures, and behavioral changes, occur chronically and repeatedly, due to the temporary abnormality of brain neurons resulting in excessive excitement. In the cerebrum, neurons connected to each other exchange information through minute electrical signals. Seizures occur when these normal electrical signals are emitted abnormally and incorrectly.
[0256] In another embodiment, the neurodegenerative disease is Parkinson’s disease dementia. Parkinson’s disease may be associated with loss of neurons, particularly, dopaminergic neurons. Especially, Parkinson’s disease dementia is associated with Parkinson’s disease.
[0257] Parkinson’s disease dementia is caused by lack of dopamine due to loss of dopamine-producing cells, extensive nervous system abnormalities due to degeneration of the alpha-synuclein protein, and the like. Dopamine is an important neurotransmitter substance that acts on the basal ganglia of the brain to allow the body to move precisely as desired. Dopamine-producing cells are present in the substantia nigra in the midbrain, and when these cells are lost for a certain reason to be deficient in dopamine, resulting in movement disorders. The accumulation of Lewy bodies, generated by the denaturation of the protein called alpha-synuclein in the brain cortex causes Lewy body dementia, while the first accumulation of Lewy bodies in parts of the brain that are involved in behavior and physical functions causes Parkinson’s disease. That is, these indicate that the accumulation of the abnormal protein in the brain causes the death of brain cells, resulting in disorders in brain functions responsible for behavior and physical functions. Due to this, Parkinson’s disease patients undergo a wide range of abnormalities in the nervous system, such as hallucinations, visual hallucinations, REM sleep disorders, and olfactory disorders.
[0258] In still another embodiment, the neurodegenerative disease is stroke. Stroke may be associated with loss of neurons caused by ischemia, where ischemia may be caused by blockage (e.g., thrombosis or arterial embolism) or hemorrhage.
The specification defines prevention and treatment as follow:
[0242] As used herein, the term “prevention” refers to any action that inhibit or delay the occurrence of a neurodegenerative disease by administration of the above peptide (e.g., the peptide itself or a long-acting conjugate form in which a biocompatible substance is bound to the peptide) or a composition containing the peptide, while the term “treatment” refers to any action that alleviates or advantageously change the symptoms of a neurodegenerative diseases by administration of the above peptide (e.g., the peptide itself or a long-acting conjugate form in which a biocompatible substance is bound to the peptide) or a composition containing the peptide.
Regarding long-acting conjugate of Formula 1, the specification discloses:
[0150] Herein, the term “long-acting conjugate of Formula 1” refers to a form in which a peptide containing an amino acid sequence of any one of SEQ ID NOS: 1 to 102 is linked to an immunoglobulin Fc region via a linker, and the conjugate may exhibit an increase in the duration of efficacy compared with a peptide containing an amino acid sequence of any one of the amino acid sequences of SEQ ID NOS: 1 to 102, to which the immunoglobulin Fc region is not bound.
Regarding Fc region, the specification discloses:
[0193] Meanwhile, F may be an immunoglobulin Fc region and, more specifically, the immunoglobulin Fc region may be derived from IgG, but is not particularly limited thereto.
[0194] In a specific embodiment of the present invention, F (the immunoglobulin Fc region) is a dimer consisting of two polypeptide chains and has a structure in which one end of L is linked to only one polypeptide chain of the two polypeptide chains, but is not limited thereto.
[0195] In the present invention, the term “immunoglobulin Fc region” refers to a region that includes heavy chain constant region 2 (CH2) and/or heavy chain constant region 3 (CH3) portions, excluding heavy chain and light chain variable regions in an immunoglobulin. The immunoglobulin Fc region may be one element constituting a moiety of the conjugate of the present invention. The immunoglobulin Fc region may be used interchangeably with the term “immunoglobulin Fc fragment”.
[0196] Herein, the Fc region includes not only native sequences obtained by papain digestion of immunoglobulins, but also derivatives thereof, for example, variants, such as sequences in which one or more amino acid residues in the native sequence have been altered through deletion, insertion, non-conservative or conservative substitution, or a combination of these and is thus different from that of the native form. The derivatives, modifications, and variants are based on the assumption that these retain the ability to bind to FcRn. In the present invention, F may be a human immunoglobulin region, but is not limited thereto. Herein, the “biocompatible substance” or “carrier” may mean the Fc region.
[0197] F (immunoglobulin Fc region) has a structure in which two polypeptide chains are linked by a disulfide linkage, with the two chains being linked through only a nitrogen atom of one of the chains, but is not limited thereto. The linking through a nitrogen atom may be performed on the epsilon amino group or the N-terminal amino group of lysine through reductive amination.
[0223] Meanwhile, the immunoglobulin Fc region may be originated from humans, or other animals including cows, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, and in a more specific embodiment, the immunoglobulin Fc region is originated from humans.
[0224] In addition, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In a still more specific embodiment, the immunoglobulin Fc region is derived from IgG or IgM, which is most abundant in the human blood, and in a still more specific embodiment, the immunoglobulin Fc region is derived from IgG, which is known to increase the half-lives of ligand-binding proteins. In a still more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the immunoglobulin Fc region is an aglycosylated Fc region derived from human IgG4, but is not limited thereto.
[0225] In a specific embodiment, the immunoglobulin Fc region, which is a fragment of human IgG4 Fc, may be in the form of a homodimer in which two monomers are linked through a disulfide bond (inter-chain form) between cysteines, which are the third amino acids of the monomers, respectively. In particular, each monomer of the homodimer independently have/may have an inter-disulfide bond between cysteines at positions 35 and 95 and an inter-disulfide bond between cysteines at positions 141 and 199, that is, two inter-disulfide bonds (intra-chain form). With respect to the number of amino acids, each monomer may consist of 221 amino acids, and the number of the amino acids forming the homodimer may be a total of 442, but the number of amino acids is not limited thereto. Specifically, in the immunoglobulin Fc fragment, two monomers having the amino acid sequence of SEQ ID NO: 123 (consisting of 221 amino acids) form a homodimer through an inter-disulfide bond between cysteines, which are the 3rd amino acid of each monomer, wherein the monomers of the homodimer independently form an inter-disulfide bond between the cysteines at positions 35 and 95 and an inter-disulfide bond between the cysteines at positions 141 and 199, respectively, but the immunoglobulin Fc fragment is not limited thereto.
Regarding “an amino acid sequence” in claims 1 and 2, the phrase “an amino acid sequence” encompasses a full-length sequence as well as any fragment thereof.
The specification discloses
[0303] Triple agonists showing activities for all of GLP-1, GIP, and glucagon receptors were prepared, and amino acid sequences thereof are shown in Table 1 (SEQ I D NO: 1 to 102).
Example 2: Preparation of Long-Acting Conjugates of Triple Agonists
a conjugate in which a triple agonist, obtained by amidation of the C-terminus of the triple agonist of SEQ ID NO: 42, is bound to the immunoglobulin Fc via PEG was named “conjugate including SEQ ID NO: 42 and immunoglobulin Fc” or “long-acting conjugate of SEQ ID NO: 42”, and these may be used interchangeably herein.
[0322] The therapeutic effect of the triple agonist of the present invention was examined in acute and chronic animal models of Parkinson’s disease, one of the typical neurodegenerative diseases. The long-acting conjugate of the triple agonist of SEQ ID NO: 42 (long-acting conjugate of SEQ ID NO: 42) was selected as a representative example of a long-acting conjugate of a triple agonist and then experimented.
[0323] An acute Parkinson’s disease animal model was constructed by administering 30 mg/kg 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneally to C57BL/6 mice once a day for 7 days to induce the loss of dopaminergic neurons. During the experiment, the mice were housed in groups and had free access to water. Test groups were assigned into Group 1 (vehicle control group), Group 2 (MPTP-vehicle control group), and Group 3 (MPTP-long acting conjugate of SEQ ID NO: 42, 5.03 nmol/kg), with 10 mice per group. The vehicle and the long-acting conjugate of SEQ ID NO: 42 were subcutaneously administered once, 30 minutes after the first day of administration of MPTP. The experiment was terminated on the 7th day. Statistical analysis was performed by one-way ANOVA.
1) Behavioral Test (Rotarod Test)
[0324] After the experiment was completed, the symptoms of ataxia were evaluated using the Rotarod test. The mouse was allowed to move on a rotating cylinder for up to 180 seconds. The rotation speed of the cylinder was set to start at 10 rpm and gradually increase to 25 rpm after 100 seconds. After 100 seconds, the rotation speed of the cylinder was fixed at 25 rpm. The time until the mouse fell off the cylinder rotating in the above manner was measured, and a total of three trials were performed to calculate the average of the fall latency.
[0325] As a result, as can be seen in FIG. 1, the fall latency was reduced due to toxicity of administered MPTP in Group 2, while the fall latency was significantly improved in Group 3 receiving the long-acting conjugate of SEQ ID NO: 42.
2) Dopamine Cell Protective Activity
[0326] After the behavioral test (Rotarod test) was completed, the mice in each group were sacrificed and brain tissue was extracted. The extracted brain tissue was immersed and fixed in a 4% paraformaldehyde solution, freshly prepared, for 24 hours, followed by hydration, and then frozen section slides were made for immunohistological staining. Subsequently, immunostaining was performed using an antibody for tyrosine hydroxylase (TH) involved in dopamine synthesis. For dopamine cell protection activity, the optical density and number of cells for stained TH in the stratum and substantia nigra were determined and quantified using Image J program.
[0327] As a result, as can be seen in FIG. 2, the number of TH-stained cells in the substantia nigra was reduced due to MPTP toxicity in Group 2, while the reduction in the number of TH-stained cells was significantly suppressed in Group 3 receiving the long-acting conjugate of SEQ ID NO: 42.
[0328] A chronic Parkinson’s disease animal model was constructed by administering 25 mg/kg of MPTP and 250 mg/kg probenecid subcutaneously and intraperitoneally, respectively, to C57BL/6 mice twice a week (3.5-day interval) for a total of 5 weeks to induce the death of dopaminergic neurons. Test groups were assigned into Group 1 (vehicle control group), Group 2 (MPTP/Probenecid-vehicle control group), and Group 3 (MPTP/Probenecid-long acting conjugate of SEQ ID NO: 42, 5.03 nmol/kg), with 10 mice per group. The vehicle and the long-acting conjugate of SEQ ID NO: 42 were subcutaneously administered once a week for a total of 6 weeks after the administration of MPTP/Probenecid.
3) Effect of Inhibiting Excessive Immune Action in Parkinson’s Disease
[0329] Microglia, which are responsible for immunity in the brain, regulate immunity in the brain through phagocytosis or cytokine secretion in a situation, such as infection. Microglia in sections of the striatal region of the brain were stained using an antibody for Iba1 protein present in the microglia. The stained area was quantified using the image J program.
[0330] As a result, as can be seen in FIG. 3, the microglia were increased due to the toxicity by the administration of MPTP/Probenecid in Group 2, while the microglia were significantly reduced in Group 3 receiving the long-acting conjugate of SEQ ID NO: 42.
4) Effect of Reducing Parkinson’s Disease Causative Substance
[0331] Alpha-synuclein is a representative protein that causes Parkinson’s disease. The increase and entanglement of alpha-synuclein in the brain causes the death of neurons. Among the proteins in the brain, alpha-synuclein was examined by an enzyme-linked immunoassay (ELISA) method.
[0332] As a result, as can be seen in FIG. 4, the amount of alpha-synuclein was increased due to the toxicity by the administration of MPTP/Probenecid in Group 2, while the amount of alpha-synuclein was significantly reduced in Group 3 receiving the long-acting conjugate of SEQ ID NO: 42.
[0333] It could be therefore confirmed that the long-acting conjugate of SEQ ID NO: 42, a representative triple agonist of the present invention, had an effect of treating Parkinson’s disease as a representative example of neurodegenerative diseases.
Experimental Example 3: Therapeutic Effect of Long-Acting Conjugates of Triplet Agonist for Alzheimer’s Disease
[0334] In order to examine the therapeutic effect of the triple agonist of the present invention on Alzheimer’s disease, a representative neurodegenerative disease, db/db mice were used. The long-acting conjugate of the triple agonist of SEQ ID NO: 42 (long-acting conjugate of SEQ ID NO: 42) was selected as a representative example of a long-acting conjugate of a triple agonist and then experimented.
[0335] The db/db mice are used for a representative animal model of diabetes and known to show representative features of Alzheimer’s disease through may studies.
[0336] Specifically, 6-week-old db/db mice were subcutaneously administered with vehicle or the long-acting conjugate of SEQ ID NO: 42 (1.08 nmol/kg) every 2 days for a total of 12 weeks. As normal controls for the experiment, db/m mice of the same week age and db/db mice of 6 weeks of age were used. During the experiment, the mice were housed in groups and had free access to water. The test groups and control group had 7 animals for each group, and after 12-week administration, the brain tissue was extracted by autopsy and tested. Statistical analysis was performed by one-way ANOVA.
1) Amyloid Beta-Protein Reducing Effect
[0337] Amyloid beta-protein has been known to be a largest cause of Alzheimer’s disease, along with excessive phosphorylation of tau protein. After 12-week drug administration, the amount of amyloid beta 1-42 protein in the cerebral cortex of db/db mice was measured by ELISA.
[0338] As a result, as can be seen in FIG. 5, the amyloid beta 1-42 protein was significantly reduced in the group administered the long-acting conjugate of SEQ ID NO: 42 compared with the control groups administered vehicle.
2) Advanced Glycation End Product Reducing Effect
[0339] Advanced glycation end products (AGEs), which are sugar-modified proteins or lipids, are associated with aging and thus aggravate degenerative diseases, such as diabetes, arteriosclerosis, and Alzheimer’s disease. The advanced glycation end products in the cerebral cortex were quantified by ELISA.
[0340] As a result, as can be seen in FIG. 6, the advanced glycation end products were significantly reduced in the group administered the long-acting conjugate of SEQ ID NO: 42 compared with the control groups receiving vehicle.
3) Anti-Inflammatory Effect
[0341] Upon completion of 12-week administration, the anti-inflammatory effect was evaluated by measuring inflammatory cytokines in the extracted cerebral cortical tissue. Interleukin-1 beta among the inflammatory cytokines was measured by ELISA.
[0342] As a result, as can be seen in FIG. 7, the interleukin-1 beta was significantly reduced in the group administered the long-acting conjugate of SEQ ID NO: 42 compared with the control groups receiving vehicle.
4) Protection Effect Against Oxidative Stress
[0343] 4-Hydroxynonenal (HNE), which is a byproduct produced by lipid peroxidation under oxidative stress, forms a HNE-protein conjugate by conjugation to an in vivo protein. To examine the protection effect against oxidative stress, the amount of HNE-protein conjugate in the cerebral cortex was measured by ELISA.
[0344] As a result, as can be seen in FIG. 8, the HNE-protein conjugate was significantly reduced in the group administered the long-acting conjugate of SEQ ID NO: 42 compared with the control groups receiving vehicle.
However, there is no objective evidence of prevention or treatment of any and all neurodegenerative diseases, including but not limited to neuropathy that causes impairments in cognitive function, learning, or memory, impairments in hand/foot sensation, or malfunctions of organs including the stomach, Alzheimer’s disease, Huntington’s disease (HD) also known as Huntington’s chorea, amyotrophic lateral sclerosis (ALS) also known as Lou Gehrig’s disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), Lewy body dementia, Parkinson’s disease dementia, epilepsy, stroke, Huntington’s chorea, cerebral hypoxia, peripheral neuropathy, memory impairment, memory loss, forgetfulness, Pick’s disease, Creutzfeldt-Jakob disease, and nerve damage caused by complications of diabetes or stroke by administering any peptide such as SEQ ID NO: 42 or long-acting conjugate thereof comprising X-L-F containing peptide such as SEQ ID NO 42 linked to an immunoglobulin Fc region via a polyethylene glycol linker.
Regarding treating any neurodegenerative disease such as the ones recite in claims 14-16, there are no objective evidence of treating any such disease by administering the peptide comprises SEQ ID NO: 42.
Regarding long-acting conjugate comprising the peptide of SEQ ID NO 42 linked to an IgG Fc via a linker comprising polyethylene glycol ranging from 1 to 100 kDa, there are no in vivo working examples of treating neurodegenerative diseases such as Huntington’s disease (HD) also known as Huntington’s chorea, amyotrophic lateral sclerosis (ALS) also known as Lou Gehrig’s disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), Lewy body dementia, Parkinson’s disease dementia, epilepsy, stroke, Huntington’s chorea, cerebral hypoxia, peripheral neuropathy, memory impairment, memory loss, forgetfulness, Pick’s disease, Creutzfeldt-Jakob disease, and nerve damage caused by complications of diabetes or stroke.
Given the broad definition of the neurodegenerative disease and Fc (from humans, or other animals including cows, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, variants, such as sequences in which one or more amino acid residues in the native sequence have been altered through deletion, insertion, non-conservative or conservative substitution, or a combination), the breadth of the position/location of the residues that involved one or more non-conservative or conservative substitution, deletion, addition, and a combination thereof in the Fc, the disclosure does not describe a representative number of species of such long-acting conjugates for the claimed methods.
The specification provides no description of the structure required for the aforementioned modified Fc to maintain their binding specificity for FcRn and/or Fcγ Receptors while maintains effector functions, e.g., ADCC, CDC.
Regarding Fc variant, Moore et al (mAbs 2(2): 181-189, 2010; PTO 892) teaches that the triple mutation Ser267Glu, His268Phe, and serine 324 threonine (Ser324Thr) in the Fc has been found to largely improve CDC at the expense of reduced ADCC and ADCP via increasing the affinity to inhibitory FcγRIIb, see entire document, Table 3, p. 186, right col.
Further, Meng et al (ACS Nano 19: 307-321, 2025; PTO 892) teaches that the blood-brain barrier (BBB) remains a major obstacle for effective for effective delivery of therapeutics to treat central nervous system (CNS) disorders. Limited BBB permeation is one of the most important factors that limit the effectivity of CNS drugs, it is not clear large long-acting conjugate where the PEG linker cannot easily cross the blood-brain barrier (BBB).
One cannot extrapolate the teaching of the specification to the breadth of claims because it is well known that the art of preventing neurodegenerative disease is highly unpredictable, for example, Chou et al (Faculty Reviews 10(81): 1-7, 2021 PTO 892) teaches that despite progress, therapies to prevent or decrease disease progression and restore neuronal function remain a challenge and an ongoing focus in both research and clinical practice. Thus, further investigation into the neurodegenerative pathways and the identification and development of neuroprotective agents are needed to develop promising disease-modifying therapeutic approaches for the treatment of neurodegenerative disease.
In light of this, one of skilled in the art would reasonably conclude that Applicant was not in possession of the genus of long-acting conjugates for prevention or treatment of all neurodegenerative diseases at the time of filing. Thus, the claims lack written description.
Vas-Cath Inc. v. Mahurkar, 19 USPQ2d 1111, makes clear that “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the written description inquiry, whatever is now claimed.” (See page 1117.) The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed.” (See Vas-Cath at page 1116.).
Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method for isolating it. See Fiers v. Revel, 25 USPQ2d 1601, 1606 (CAFC 1993) and Amgen Inc. v. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016.
One cannot describe what one has not conceived. See Fiddles v. Baird, 30 USPQ2d 1481, 1483. In Fiddles v. Baird, claims directed to mammalian FGF’s were found unpatentable due to lack of written description for the broad class. The specification provided only the bovine sequence. Thus, the specification fails to describe these DNA sequences.
For genus claims, an adequate written description of a claimed genus requires more than a generic statement of an invention's boundaries. A patent must set forth either a representative number of species falling within the scope of the genus or structural features common to the members of the genus. Kubin, Exparte, 83 USPQ2d 1410 (Bd. Pat. App. & Int. 2007); Ariad Pharms., Inc. v. Eli Lilly& Co., 598 F.3d 1336, 1350 (Fed. Cir. 2010).
Therefore, only a method of treating Parkinson’s disease, and Alzheimer’s disease comprising administering to a subject in needed thereof an effective amount of a long-acting conjugate represent by form I: X-L-F wherein X is a peptide consisting of the amino acid sequence of SEQ ID NO: 42, wherein L is a linker comprising polyethylene glycol of 10 kDa and F is an Fc comprises the amino acid sequence of SEQ ID NO: 123 and a pharmaceutical acceptable vehicle, but not the full breadth of the claims meets the written description provision of 35 U.S.C. § 112, first paragraph. Applicant is reminded that Vas-Cath makes clear that the written description provision of 35 U.S.C. § 112 is severable from its enablement provision (see page 1115).
Claims 1-14 and 16-18 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method of treating Parkinson’s disease, and Alzheimer’s disease comprising administering to a subject in needed thereof an effective amount of a long-acting conjugate represent by form I X-L-F wherein X is a peptide consisting of the amino acid sequence of SEQ ID NO: 42, wherein L is a linker comprising polyethylene glycol of 10 kDa and F is an Fc comprises the amino acid sequence of SEQ ID NO: 123 and a pharmaceutical acceptable vehicle, does not reasonably provide enablement for a method for “prevention” of all neurodegenerative disease, including but not limited to Parkinson’s disease, by administering any peptide such as SEQ ID NO: 42 or a long-acting conjugate thereof or treating all neurodegenerative disease, including but not limited to Parkinson’s disease, by administering peptide such as SEQ ID NO: 42. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(a)). These factors include, but are not limited to: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. . In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988).
The claims encompass a method for prevention or treatment of any neurodegenerative disease (claims 1-2) such as Parkinson’s disease (claims 14 and 16) in a subject comprising administering to the subject a pharmaceutical composition comprising a pharmaceutical acceptable vehicle and a therapeutically effective amount of any peptide containing an amino acid sequence of SEQ ID NO: 42 (elected species, claim 1, 3-6, 17-18) or a long-acting conjugate represented by Formula I X-L-F wherein X represents a peptide containing SEQ ID NO: 42, L represents a linker containing ethylene glycol repeat units, and F represents any immunoglobulin Fc region (claim 2, 8-13).
Regarding neurodegenerative disease, the specification discloses:
[0245] As used herein, the term “neurodegenerative disease” refers to a disease that causes several symptoms resulting from degenerative changes appearing in neurons of the central nervous system and is a collective term for diseases that cause several symptoms resulting from degenerative changes appearing in neurons. Specifically, the neurodegenerative disease may include neuropathy that causes impairments in cognitive function, learning, or memory, impairments in hand/foot sensation, or malfunctions of organs including the stomach. Specifically, the neurodegenerative disease is known to be caused by the gradual loss of functions of a specific brain cell population in the brain and spinal cord to result in the death of brain neurons, which are most important for information transmission in the brain nervous system, problems with the formation or function of synapses that transmit information between brain neurons, or an abnormal increase or decrease in electrical activity of brain nerves.
[0246] Among the neurodegenerative diseases, degenerative brain diseases may be classified considering the main symptoms and the brain areas affected, and the degenerative brain diseases may include Parkinson’s disease (PD), Alzheimer’s disease, Huntington’s disease (HD) also known as Huntington’s chorea, amyotrophic lateral sclerosis (ALS) also known as Lou Gehrig’s disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), Lewy body dementia, Parkinson’s disease dementia, epilepsy, stroke, Huntington’s chorea, cerebral hypoxia, peripheral neuropathy, memory impairment, memory loss, forgetfulness, Pick’s disease, Creutzfeldt-Jakob disease, and nerve damage caused by complications of diabetes.
[0247] In an embodiment of the present invention, the neurodegenerative disease may be Parkinson’s disease or stroke.
[0248] In another embodiment, the neurodegenerative disease is Parkinson’s disease. Parkinson’s disease may be associated with oxidative stress, inflammatory response, apoptosis, loss of neurons, especially loss of dopaminergic neurons, e.g., loss of the substantia nigra, resulting in dopamine deficiency.
[0249] In another embodiment of the present invention, the neurodegenerative disease may be Alzheimer’s disease. “Alzheimer’s disease” is one of the most common degenerative brain diseases that cause dementia, and is a collective term for diseases that cause various symptoms resulting from degenerative changes appearing in neurons of the central nervous system. Specifically, Alzheimer’s disease may include neuropathy that causes impairments in cognitive function, learning, or memory, impairments in hand/foot sensation, or malfunctions of organs including the stomach. Alzheimer’s disease is accompanied by cognitive function as well as neurobehavioral symptoms, such as personality changes, restlessness, depression, delusions, hallucination, increased aggression, and sleep disorders, during the progression; and neurological disorders, such as muscle rigidity and gait abnormalities, or physical complications, such as incontinence, infections, and bedsores, in the later stage. When the brain tissue of an Alzheimer’s disease patient is examined under a microscope, characteristic lesions, such as a neuritic plaque and a neurofibrillary tangle, are observed, and when observed with the naked eye, overall brain atrophy is seen due to loss of neurons. In the early stages of the disease, these brain pathological findings are mainly limited to the hippocampus, which is the main brain region responsible for memory, and the entorhinal cortex, but gradually spreads to the entire brain via the parietal lobe and frontal lobe. In accordance with the progression of the brain pathology invasion area, memory decline mainly appears in the early stage, and as it progresses, clinical symptoms vary and become more severe while showing a gradual course.
[0250] Specifically, the neurodegenerative disease is known to be caused by the gradual loss of functions of a specific brain cell population in the brain and spinal cord to result in the death of brain neurons, which are most important for information transmission in the brain nervous system, problems with the formation or function of synapses that transmit information between brain neurons, or an abnormal increase or decrease in electrical activity of brain nerves. Alzheimer’s disease (AD) may also be referred to as Alzheimer’s disorder. Depending on the age of onset, Alzheimer’s disease may be divided into early-onset (elderly) Alzheimer’s disease if it develops under the age of 65, and late-onset (elderly) Alzheimer’s disease if it develops over the age of 65 years, but is not limited thereto.
[0251] Alzheimer’s disease may be associated with oxidative stress and neuronal loss.
[0252] In another embodiment, the neurodegenerative disease is progressive supranuclear palsy. Progressive supranuclear palsy may be associated with neuronal loss, particularly, loss of dopaminergic neurons.
[0253] In another embodiment, the neurodegenerative disease is multiple system atrophy. Multiple system atrophy may be associated with loss of neurons, particularly, dopaminergic neurons.
[0254] In another embodiment, the neurodegenerative disease is Lewy body dementia. Lewy body dementia may be associated with loss of neurons, particularly, dopaminergic neurons. Lewy body dementia is known to account for about 20% of the most common causes of dementia, after Alzheimer’s disease, among neurodegenerative diseases observed in the elderly. Lewy body dementia may be associated with Parkinson’s disease.
[0255] In another embodiment, the neurodegenerative disease is epilepsy. Epilepsy refers to a brain disease in which symptoms of temporary paralysis of brain functions, such as loss of consciousness, seizures, and behavioral changes, occur chronically and repeatedly, due to the temporary abnormality of brain neurons resulting in excessive excitement. In the cerebrum, neurons connected to each other exchange information through minute electrical signals. Seizures occur when these normal electrical signals are emitted abnormally and incorrectly.
[0256] In another embodiment, the neurodegenerative disease is Parkinson’s disease dementia. Parkinson’s disease may be associated with loss of neurons, particularly, dopaminergic neurons. Especially, Parkinson’s disease dementia is associated with Parkinson’s disease.
[0257] Parkinson’s disease dementia is caused by lack of dopamine due to loss of dopamine-producing cells, extensive nervous system abnormalities due to degeneration of the alpha-synuclein protein, and the like. Dopamine is an important neurotransmitter substance that acts on the basal ganglia of the brain to allow the body to move precisely as desired. Dopamine-producing cells are present in the substantia nigra in the midbrain, and when these cells are lost for a certain reason to be deficient in dopamine, resulting in movement disorders. The accumulation of Lewy bodies, generated by the denaturation of the protein called alpha-synuclein in the brain cortex causes Lewy body dementia, while the first accumulation of Lewy bodies in parts of the brain that are involved in behavior and physical functions causes Parkinson’s disease. That is, these indicate that the accumulation of the abnormal protein in the brain causes the death of brain cells, resulting in disorders in brain functions responsible for behavior and physical functions. Due to this, Parkinson’s disease patients undergo a wide range of abnormalities in the nervous system, such as hallucinations, visual hallucinations, REM sleep disorders, and olfactory disorders.
[0258] In still another embodiment, the neurodegenerative disease is stroke. Stroke may be associated with loss of neurons caused by ischemia, where ischemia may be caused by blockage (e.g., thrombosis or arterial embolism) or hemorrhage.
The specification defines prevention and treatment as follow:
[0242] As used herein, the term “prevention” refers to any action that inhibit or delay the occurrence of a neurodegenerative disease by administration of the above peptide (e.g., the peptide itself or a long-acting conjugate form in which a biocompatible substance is bound to the peptide) or a composition containing the peptide, while the term “treatment” refers to any action that alleviates or advantageously change the symptoms of a neurodegenerative diseases by administration of the above peptide (e.g., the peptide itself or a long-acting conjugate form in which a biocompatible substance is bound to the peptide) or a composition containing the peptide.
Regarding long-acting conjugate of Formula 1, the specification discloses:
[0150] Herein, the term “long-acting conjugate of Formula 1” refers to a form in which a peptide containing an amino acid sequence of any one of SEQ ID NOS: 1 to 102 is linked to an immunoglobulin Fc region via a linker, and the conjugate may exhibit an increase in the duration of efficacy compared with a peptide containing an amino acid sequence of any one of the amino acid sequences of SEQ ID NOS: 1 to 102, to which the immunoglobulin Fc region is not bound.
Regarding Fc region, the specification discloses:
[0193] Meanwhile, F may be an immunoglobulin Fc region and, more specifically, the immunoglobulin Fc region may be derived from IgG, but is not particularly limited thereto.
[0194] In a specific embodiment of the present invention, F (the immunoglobulin Fc region) is a dimer consisting of two polypeptide chains and has a structure in which one end of L is linked to only one polypeptide chain of the two polypeptide chains, but is not limited thereto.
[0195] In the present invention, the term “immunoglobulin Fc region” refers to a region that includes heavy chain constant region 2 (CH2) and/or heavy chain constant region 3 (CH3) portions, excluding heavy chain and light chain variable regions in an immunoglobulin. The immunoglobulin Fc region may be one element constituting a moiety of the conjugate of the present invention. The immunoglobulin Fc region may be used interchangeably with the term “immunoglobulin Fc fragment”.
[0196] Herein, the Fc region includes not only native sequences obtained by papain digestion of immunoglobulins, but also derivatives thereof, for example, variants, such as sequences in which one or more amino acid residues in the native sequence have been altered through deletion, insertion, non-conservative or conservative substitution, or a combination of these and is thus different from that of the native form. The derivatives, modifications, and variants are based on the assumption that these retain the ability to bind to FcRn. In the present invention, F may be a human immunoglobulin region, but is not limited thereto. Herein, the “biocompatible substance” or “carrier” may mean the Fc region.
[0197] F (immunoglobulin Fc region) has a structure in which two polypeptide chains are linked by a disulfide linkage, with the two chains being linked through only a nitrogen atom of one of the chains, but is not limited thereto. The linking through a nitrogen atom may be performed on the epsilon amino group or the N-terminal amino group of lysine through reductive amination.
[0223] Meanwhile, the immunoglobulin Fc region may be originated from humans, or other animals including cows, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, and in a more specific embodiment, the immunoglobulin Fc region is originated from humans.
[0224] In addition, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In a still more specific embodiment, the immunoglobulin Fc region is derived from IgG or IgM, which is most abundant in the human blood, and in a still more specific embodiment, the immunoglobulin Fc region is derived from IgG, which is known to increase the half-lives of ligand-binding proteins. In a still more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the immunoglobulin Fc region is an aglycosylated Fc region derived from human IgG4, but is not limited thereto.
[0225] In a specific embodiment, the immunoglobulin Fc region, which is a fragment of human IgG4 Fc, may be in the form of a homodimer in which two monomers are linked through a disulfide bond (inter-chain form) between cysteines, which are the third amino acids of the monomers, respectively. In particular, each monomer of the homodimer independently have/may have an inter-disulfide bond between cysteines at positions 35 and 95 and an inter-disulfide bond between cysteines at positions 141 and 199, that is, two inter-disulfide bonds (intra-chain form). With respect to the number of amino acids, each monomer may consist of 221 amino acids, and the number of the amino acids forming the homodimer may be a total of 442, but the number of amino acids is not limited thereto. Specifically, in the immunoglobulin Fc fragment, two monomers having the amino acid sequence of SEQ ID NO: 123 (consisting of 221 amino acids) form a homodimer through an inter-disulfide bond between cysteines, which are the 3rd amino acid of each monomer, wherein the monomers of the homodimer independently form an inter-disulfide bond between the cysteines at positions 35 and 95 and an inter-disulfide bond between the cysteines at positions 141 and 199, respectively, but the immunoglobulin Fc fragment is not limited thereto.
The specification discloses
[0303] Triple agonists showing activities for all of GLP-1, GIP, and glucagon receptors were prepared, and amino acid sequences thereof are shown in Table 1 (SEQ I D NO: 1 to 102).
Example 2: Preparation of Long-Acting Conjugates of Triple Agonists
a conjugate in which a triple agonist, obtained by amidation of the C-terminus of the triple agonist of SEQ ID NO: 42, is bound to the immunoglobulin Fc via PEG was named “conjugate including SEQ ID NO: 42 and immunoglobulin Fc” or “long-acting conjugate of SEQ ID NO: 42”, and these may be used interchangeably herein.
[0322] The therapeutic effect of the triple agonist of the present invention was examined in acute and chronic animal models of Parkinson’s disease, one of the typical neurodegenerative diseases. The long-acting conjugate of the triple agonist of SEQ ID NO: 42 (long-acting conjugate of SEQ ID NO: 42) was selected as a representative example of a long-acting conjugate of a triple agonist and then experimented.
[0323] An acute Parkinson’s disease animal model was constructed by administering 30 mg/kg 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneally to C57BL/6 mice once a day for 7 days to induce the loss of dopaminergic neurons. During the experiment, the mice were housed in groups and had free access to water. Test groups were assigned into Group 1 (vehicle control group), Group 2 (MPTP-vehicle control group), and Group 3 (MPTP-long acting conjugate of SEQ ID NO: 42, 5.03 nmol/kg), with 10 mice per group. The vehicle and the long-acting conjugate of SEQ ID NO: 42 were subcutaneously administered once, 30 minutes after the first day of administration of MPTP. The experiment was terminated on the 7th day. Statistical analysis was performed by one-way ANOVA.
1) Behavioral Test (Rotarod Test)
[0324] After the experiment was completed, the symptoms of ataxia were evaluated using the Rotarod test. The mouse was allowed to move on a rotating cylinder for up to 180 seconds. The rotation speed of the cylinder was set to start at 10 rpm and gradually increase to 25 rpm after 100 seconds. After 100 seconds, the rotation speed of the cylinder was fixed at 25 rpm. The time until the mouse fell off the cylinder rotating in the above manner was measured, and a total of three trials were performed to calculate the average of the fall latency.
[0325] As a result, as can be seen in FIG. 1, the fall latency was reduced due to toxicity of administered MPTP in Group 2, while the fall latency was significantly improved in Group 3 receiving the long-acting conjugate of SEQ ID NO: 42.
2) Dopamine Cell Protective Activity
[0326] After the behavioral test (Rotarod test) was completed, the mice in each group were sacrificed and brain tissue was extracted. The extracted brain tissue was immersed and fixed in a 4% paraformaldehyde solution, freshly prepared, for 24 hours, followed by hydration, and then frozen section slides were made for immunohistological staining. Subsequently, immunostaining was performed using an antibody for tyrosine hydroxylase (TH) involved in dopamine synthesis. For dopamine cell protection activity, the optical density and number of cells for stained TH in the stratum and substantia nigra were determined and quantified using Image J program.
[0327] As a result, as can be seen in FIG. 2, the number of TH-stained cells in the substantia nigra was reduced due to MPTP toxicity in Group 2, while the reduction in the number of TH-stained cells was significantly suppressed in Group 3 receiving the long-acting conjugate of SEQ ID NO: 42.
[0328] A chronic Parkinson’s disease animal model was constructed by administering 25 mg/kg of MPTP and 250 mg/kg probenecid subcutaneously and intraperitoneally, respectively, to C57BL/6 mice twice a week (3.5-day interval) for a total of 5 weeks to induce the death of dopaminergic neurons. Test groups were assigned into Group 1 (vehicle control group), Group 2 (MPTP/Probenecid-vehicle control group), and Group 3 (MPTP/Probenecid-long acting conjugate of SEQ ID NO: 42, 5.03 nmol/kg), with 10 mice per group. The vehicle and the long-acting conjugate of SEQ ID NO: 42 were subcutaneously administered once a week for a total of 6 weeks after the administration of MPTP/Probenecid.
3) Effect of Inhibiting Excessive Immune Action in Parkinson’s Disease
[0329] Microglia, which are responsible for immunity in the brain, regulate immunity in the brain through phagocytosis or cytokine secretion in a situation, such as infection. Microglia in sections of the striatal region of the brain were stained using an antibody for Iba1 protein present in the microglia. The stained area was quantified using the image J program.
[0330] As a result, as can be seen in FIG. 3, the microglia were increased due to the toxicity by the administration of MPTP/Probenecid in Group 2, while the microglia were significantly reduced in Group 3 receiving the long-acting conjugate of SEQ ID NO: 42.
4) Effect of Reducing Parkinson’s Disease Causative Substance
[0331] Alpha-synuclein is a representative protein that causes Parkinson’s disease. The increase and entanglement of alpha-synuclein in the brain causes the death of neurons. Among the proteins in the brain, alpha-synuclein was examined by an enzyme-linked immunoassay (ELISA) method.
[0332] As a result, as can be seen in FIG. 4, the amount of alpha-synuclein was increased due to the toxicity by the administration of MPTP/Probenecid in Group 2, while the amount of alpha-synuclein was significantly reduced in Group 3 receiving the long-acting conjugate of SEQ ID NO: 42.
[0333] It could be therefore confirmed that the long-acting conjugate of SEQ ID NO: 42, a representative triple agonist of the present invention, had an effect of treating Parkinson’s disease as a representative example of neurodegenerative diseases.
Experimental Example 3: Therapeutic Effect of Long-Acting Conjugates of Triplet Agonist for Alzheimer’s Disease
[0334] In order to examine the therapeutic effect of the triple agonist of the present invention on Alzheimer’s disease, a representative neurodegenerative disease, db/db mice were used. The long-acting conjugate of the triple agonist of SEQ ID NO: 42 (long-acting conjugate of SEQ ID NO: 42) was selected as a representative example of a long-acting conjugate of a triple agonist and then experimented.
[0335] The db/db mice are used for a representative animal model of diabetes and known to show representative features of Alzheimer’s disease through may studies.
[0336] Specifically, 6-week-old db/db mice were subcutaneously administered with vehicle or the long-acting conjugate of SEQ ID NO: 42 (1.08 nmol/kg) every 2 days for a total of 12 weeks. As normal controls for the experiment, db/m mice of the same week age and db/db mice of 6 weeks of age were used. During the experiment, the mice were housed in groups and had free access to water. The test groups and control group had 7 animals for each group, and after 12-week administration, the brain tissue was extracted by autopsy and tested. Statistical analysis was performed by one-way ANOVA.
1) Amyloid Beta-Protein Reducing Effect
[0337] Amyloid beta-protein has been known to be a largest cause of Alzheimer’s disease, along with excessive phosphorylation of tau protein. After 12-week drug administration, the amount of amyloid beta 1-42 protein in the cerebral cortex of db/db mice was measured by ELISA.
[0338] As a result, as can be seen in FIG. 5, the amyloid beta 1-42 protein was significantly reduced in the group administered the long-acting conjugate of SEQ ID NO: 42 compared with the control groups administered vehicle.
2) Advanced Glycation End Product Reducing Effect
[0339] Advanced glycation end products (AGEs), which are sugar-modified proteins or lipids, are associated with aging and thus aggravate degenerative diseases, such as diabetes, arteriosclerosis, and Alzheimer’s disease. The advanced glycation end products in the cerebral cortex were quantified by ELISA.
[0340] As a result, as can be seen in FIG. 6, the advanced glycation end products were significantly reduced in the group administered the long-acting conjugate of SEQ ID NO: 42 compared with the control groups receiving vehicle.
3) Anti-Inflammatory Effect
[0341] Upon completion of 12-week administration, the anti-inflammatory effect was evaluated by measuring inflammatory cytokines in the extracted cerebral cortical tissue. Interleukin-1 beta among the inflammatory cytokines was measured by ELISA.
[0342] As a result, as can be seen in FIG. 7, the interleukin-1 beta was significantly reduced in the group administered the long-acting conjugate of SEQ ID NO: 42 compared with the control groups receiving vehicle.
4) Protection Effect Against Oxidative Stress
[0343] 4-Hydroxynonenal (HNE), which is a byproduct produced by lipid peroxidation under oxidative stress, forms a HNE-protein conjugate by conjugation to an in vivo protein. To examine the protection effect against oxidative stress, the amount of HNE-protein conjugate in the cerebral cortex was measured by ELISA.
[0344] As a result, as can be seen in FIG. 8, the HNE-protein conjugate was significantly reduced in the group administered the long-acting conjugate of SEQ ID NO: 42 compared with the control groups receiving vehicle.
However, there is no objective evidence of preventing any and all neurodegenerative diseases, including but not limited to Parkinson’s disease (PD), Alzheimer’s disease, Huntington’s disease (HD) also known as Huntington’s chorea, amyotrophic lateral sclerosis (ALS) also known as Lou Gehrig’s disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), Lewy body dementia, Parkinson’s disease dementia, epilepsy, stroke, Huntington’s chorea, cerebral hypoxia, peripheral neuropathy, memory impairment, memory loss, forgetfulness, Pick’s disease, Creutzfeldt-Jakob disease, and nerve damage caused by complications of diabetes or stroke by administering any peptide such as SEQ ID NO: 42 or s long-acting conjugate thereof comprising X-L-F containing peptide such as SEQ ID NO 42.
Regarding treating any neurodegenerative disease such as the ones recite in claims 14-16, there are no objective of treating such diseases by administering such peptide comprises SEQ ID NO: 42.
Regarding treating Parkinson’s disease, the specification discloses administering a long-acting conjugate comprising the peptide of SEQ ID NO 42 linked to a Fc polypeptide comprising SEQ ID NO 123 via a linker comprising polyethylene glycol of 10 kDa.
However, there are no in vivo working examples of treating neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) also known as Lou Gehrig’s disease, progressive supranuclear palsy (PSP), multiple system atrophy (MSA), Lewy body dementia, Parkinson’s disease dementia, epilepsy, stroke, Huntington’s chorea, cerebral hypoxia, peripheral neuropathy, memory impairment, memory loss, forgetfulness, Pick’s disease, Creutzfeldt-Jakob disease, and nerve damage caused by complications of diabetes or stroke.
Given the broad definition of the neurodegenerative disease and Fc (from humans, or other animals including cows, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, variants, such as sequences in which one or more amino acid residues in the native sequence have been altered through deletion, insertion, non-conservative or conservative substitution, or a combination), the breadth of the position/location of the residues that involved one or more non-conservative or conservative substitution, deletion, addition, and a combination thereof in the Fc, the specification does not teach modified Fc to maintain their binding specificity for FcRn and/or Fcγ Receptors while maintains effector functions, e.g., ADCC, CDC. The disclosure does not provide a representative of such long-acting conjugates to enable one of skilled in the art to make and use without undue experimentation.
Regarding Fc variant, Moore et al (mAbs 2(2): 181-189, 2010; PTO 892) teaches that the triple mutation Ser267Glu, His268Phe, and serine 324 threonine (Ser324Thr) in the Fc has been found to largely improve CDC at the expense of reduced ADCC and ADCP via increasing the affinity to inhibitory FcγRIIb, see entire document, Table 3, p. 186, right col.
Further, Meng et al (ACS Nano 19: 307-321, 2025; PTO 892) teaches that the blood-brain barrier (BBB) remains a major obstacle for effective for effective delivery of therapeutics to treat central nervous system (CNS) disorders. Limited BBB permeation is one of the most important factors that limit the effectivity of CNS drugs, it is not clear large long-acting conjugate where the PEG linker cannot easily cross the blood-brain barrier (BBB). The scope of the claims must bear a reasonable correlation with the scope of enablement. See In re Fisher, 166 USPQ 19 24 (CCPA 1970).
One cannot extrapolate the teaching of the specification to the breadth of claims because it is well known that the art of preventing neurodegenerative disease is highly unpredictable, for example, Chou et al (Faculty Reviews 10(81): 1-7, 2021 PTO 892) teaches that despite progress, therapies to prevent or decrease disease progression and restore neuronal function remain a challenge and an ongoing focus in both research and clinical practice. Thus, further investigation into the neurodegenerative pathways and the identification and development of neuroprotective agents are needed to develop promising disease-modifying therapeutic approaches for the treatment of neurodegenerative disease.
Given the level of unpredictability in the art, it would take undue experimentation to make and use the invention for the full scope of the claims.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to PHUONG HUYNH whose telephone number is (571)272-0846. The examiner can normally be reached on 9:00 a.m. to 6:30 p.m. The examiner can also be reached on alternate alternative Friday from 9:00 a.m. to 5:30 p.m.
If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Misook Yu, can be reached at 571-270-3497. The fax phone number for the organization where this application or proceeding is assigned is 571-272-0839.
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/PHUONG HUYNH/ Primary Examiner, Art Unit 1641