JP2007527870A5 - - Google Patents

Description

AD symptoms develop slowly and early symptoms may remain mild forgetfulness. At this stage, patients may forget the names of recent events, behaviors, family and things, and may not be able to solve simple mathematical problems. As the disease progresses, symptoms become easily visible and become so severe that AD patients or their families seek medical help . Mid-term symptoms of AD include forgetting how to do simple tasks such as dressing up, and problems with speaking, understanding, reading, or writing. Late-stage AD patients may become anxious or aggressive, may be left out of their homes, and may eventually need full nursing.

Management of AD includes treatments that use drugs and treatments that do not use drugs. Treatments aimed at changing the underlying process of the disease (reversing or delaying progression) have so far been unsuccessful. Drugs that restore defects or defects in neuronal chemical messengers (neurotransmitters), such as cholinesterase inhibitors (ChEIs), have been shown to improve symptoms. Drugs are also used to address AD psychiatric symptoms.

Another factor to consider when developing new drugs is ease of use for the target patient. Oral drug delivery (especially tablets, capsules, and soft gels) accounts for 70% of all dosage forms used, as it provides patient convenience. There is consensus among drug developers that patients prefer oral transport over injection and other highly invasive modes of drug administration. Also preferred are formulations with short dosing intervals (ie, administration once a day or by sustained release). The ease of administration of antibiotics in oral dosage forms increases the patient's compliance rate during the treatment period.

What is needed is an effective method and composition that produces highly specific and highly effective antibodies. Such antibodies preferably recognize specific epitopes present on various antigens , such as amyloid protein, prion protein, or _P170 glycoprotein.

Yet another object of the present invention is to provide an immunogenic composition that can be administered intramuscularly, intravenously, transdermally , orally, or subcutaneously.

Typically, amino acids constituting the peptide are numbered in order (starting from amino terminus, Yuku increasingly numerical toward carboxy-terminal direction of the peptide). Thus, when one amino acid is expressed as “following” another amino acid, the amino acid is located closer to the carboxy terminus of the subject peptide than the preceding amino acid.

Alternatively, the immunogenic peptides described herein are synthesized by recombinant nucleic acid methods. In general, the method comprises the steps of generating a nucleic acid sequence encoding a peptide of interest, placing the nucleic acid in an expression cassette under the control of a particular promoter, expressing the peptide in a host, expressing the expressed peptide or polypeptide. Isolating the peptide and, if necessary, regenerating the peptide. For such a procedure, a method of the skilled artisan can be guided enough, are described in the literature.

The expression “antigen” means the whole or fragment of a molecule capable of causing an immune response in a mammal. This expression includes the immunogen and regions or antigenic determinants involved in antigenicity .

The invention further includes antigenic peptides modified with a hydrophobic moiety , such as palmitic acid , that facilitates insertion of the carrier into the hydrophobic lipid bilayer. The hydrophobic moiety of the present invention can be a fatty acid or a triglyceride and phospholipid in which the carbon skeleton of the fatty acid contains at least 10 carbon atoms. Most preferred is a lipophilic moiety having a fatty acid comprising a carbon skeleton containing at least about 14, up to about 24 carbon atoms. The most preferred hydrophobic moiety has a carbon skeleton of at least 14 carbon atoms. Examples of hydrophobic moieties include, but are not limited to, palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, and linolenic acid. The most preferred hydrophobic moiety is palmitic acid.

Formulation
Including all or active portion of a protein or peptide having immunogenic, natural or synthetic protein, peptide, or protein fragment, formulated in a physiologically acceptable, such as pharmaceutically acceptable carrier In addition, it can be prepared by a known method. For example, a therapeutic composition can be obtained by mixing a protein, peptide, or protein fragment with a pharmaceutically acceptable excipient.

As used herein, a sustained release base is a base made from a material (usually a polymer) that degrades by hydrolysis with enzymes or acids / bases or by dissolution. When such a base is inserted into the body, it acts by the action of enzymes and body fluids. The sustained-release base is preferably a liposome, polylactide (polylactic acid), polyglycolide (glycolic acid polymer), polylactide co-glycolide (copolymer of lactic acid and glycolic acid), polyanhydride, poly (ortho) ester , Polypeptide, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, fatty acid, phospholipid, polysaccharide, nucleic acid, polyamino acid, amino acid (phenylalanine, tyrosine, isoleucine, etc.), polynucleotide, polyvinylpropylene, polyvinylpyrrolidone, silicone, etc. Selected from biocompatible materials. A preferred biodegradable base is any one of polylactide, polyglycolide, or polylactide co-glycolide (a copolymer of lactic acid and glycolic acid).

The dose of the composition is determined by the disease being treated, the type of composition used, and other clinical factors such as the patient's weight and condition, and the route of administration.

In one embodiment of the invention, the FRHDSGY (SEQ ID NO: 1) sequence of the amyloid protein is used, but the sequence of any other amyloid protein can be substituted. In addition to the above in vitro characteristics for polyclonal antibodies, monoclonal antibodies obtained from mice immunized with constructor bets described above, biology have you in APP [V717I] FVB transgenic mice, a model of human Alzheimer's disease It shows the activity. In these mice, a significant level of memory recovery and arousal of curiosity is observed. This mAb does not cause intracerebral hemorrhage in immunized transgenic mice.

Without being bound by the following theory, it is based on an in vitro test examining the interaction (mainly fiber solubilization and CD spectra) of anti-amyloid mAbs (antibodies to 1-16 sequences made by the method of the invention). Thus, the antibody is preferentially bound to β amyloid in the α helix structure. For this reason, it is thought that the solubilization effect | action of an amyloid fiber can be demonstrated in a thermodynamic term. The antibody dissociates the α helix amyloid from the following equilibrium equation by preferentially binding to the α helix:
Aβ (α helix) ← → Aβ (β sheet)
As a result, more β-sheet structure β-amyloid present in the, in order to establish equilibrium again, to structural transformations in α helical soluble. Stoichiometric results support the hypothesis that mAbs directly affect structural equilibrium.

The hydrophobic sequences of Aβ _1-42 and Aβ _1-40 contain the motif GXXXGXXXGG known to induce strong oligomerization of hydrophobic sequences (Eilers et al., 2002; Leeds et al., 2001; Lemmon et al., 1994; Russ and Engelmann, 1999; Russ and Engelmann, 2000; Smith and Bormann, 1995). This motif is considered the primary target of therapy. This is because it must play an important role in all pathogenic processes leading to the formation, oligomerization, and accumulation of Aβ _1-42 and Aβ _1-40 . As for the complete sequence of APP, as revealed from the cleavage of SREBP (Ye et al., 2000), the motif requires a downstream sequence that needs to be unraveled to be processed by γ-secretase. There is a high possibility of covering. It has not been pointed out so far that this sequence plays an important role in amyloid oligomerization. As described herein, the modified supramolecular (preferably PEGylated) antigen of the present invention has high antigenicity, and the antibody derived thereby has high affinity. In addition to Aβ _1-16 , the supramolecular constructs of the present invention, when used as vaccines, are Aβ _4-11 (SEQ ID NO: 2), Aβ _22-35 (SEQ ID NO: 3), Aβ _{29 The} peptide represented by _-40 (SEQ ID NO: 4) is also included.

Thus, in summary, the present invention provides new monoclonal antibodies against supramolecular antigens that expose various amyloid sequences. In particular , a unique synthetic route was devised to covalently link two polyethylene glycol (n = 70) chains to selected amyloid sequences. Phosphatidylethanolamine was covalently bonded to the free end of the PEG chain. Without being bound by the following theory, it is thought that its function is to anchor the PEGylated amyloid sequence in the liposome bilayer. PEGylation has been shown in the present invention to increase the immunogenicity of an antigen compared to palmitoylation. Affinity testing, epitope determination, and structural transition induction using these monoclonal antibodies are currently underway in the inventors' laboratory. The inherent modification methods of the present invention are applicable to a variety of peptides, and ultimately to therapeutic formulations and vaccines for diseases and disorders including but not limited to Alzheimer's disease, cancer, and infectious diseases. Can be used.

Multidrug resistance 1 (MDR 1 ) in cancer cells
Multidrug resistance in cancer cells. 1, from a cancer cell, arising by overexpression of P-glycoprotein is a membrane pump for discharging the various mutually unrelated chemotherapeutic agents (P _170).

Based on the above observations and the method developed by the inventors, we developed a “vaccine” for disease by inducing a strong humoral and cellular immune response to neurotoxic PrP 106-126 in mice. Immunized mice were challenged with scrapie mouse brain extracts.

Example 1
We refer to rat hippocampal primary cultures, by exposing the long term to the peptides of the micromolar concentration corresponding to residues 106-126 of the amino acid sequence deduced from human PrP ^c cDNA, concentration neuronal death It was clarified that it occurs dependently. The obtained data is shown in Table 1.

(Table 1) Long-term treatment of hippocampal neurons for 9 days

It was shown that neuronal death induced by PrP 106-126 occurs in a dose-dependent manner through apoptosis. At the end of subacute encephalopathy such as scrapie, PrP ^Sc reaches a total brain concentration 10-20 times higher than PrP ^c , which is very similar to the data in Table 1 for the two concentrations of PrP 106-126.

Inside including Lys residue or His residues, for peptide sequence (4-11,1-16,22-35), orthogonally protected Lys a (ivDde) was added to each end. Additional Gly was added at the C-terminus to facilitate synthesis. The Fmoc group was removed with 20% piperidine in DMF and N-acetylated with acetic anhydride. Selective cleavage of the ivDde group was performed over 1 hour using 3% hydrazine hydrate in DMF. 2-Chlorotrityl resin is preferred over the more widely used Wang resin because it has proven to be more resistant to hydrazine degradation . Furthermore, since 2-chlorotrityl resin electrode Umate acid sensitive, unlike the Wang resin, enables the isolation of protected peptides. In fact, the peptides bound to trees fat, the coupling to the activated pre PEG lipid reagent DSPE-PEG-SPA, because it did not cause any coupling product, the coupling reaction is carried out in the liquid phase There was a need . Thus, selective cleavage from the resin under mild conditions (acetic acid / trifluoroethanol / dichloromethane, 1: 1: 8, 1 hour, room temperature) resulted in an internally protected peptide (Figure 5). .

Example 3
Comparison of immunogenicity of PEGylated and palmitoylated antigens, ELISA and disaggregation assay Liposome antigens were generated as described above. Dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylethanol containing the sequences PEG-Aβ _1-16 , PEG-Aβ _4-11 , and PEG-Aβ _22-35 , monophosphoryl lipid A (40 mg / mM phospholipid) amine (DMPEA), dimyristoyl phosphatidyl glycerol (DMPG), and cholesterol (molar ratio 0.9: 0.1: 0.1: 0.7) was reconstituted constructs containing liposomes made from.

Disaggregation assay Nine types of serum (dilution ratio 1: 100) from animals immunized with liposome-PEG-Aβ _4-11 were incubated with _preformed Aβ _1-42 fibers and antisera Used in the assay. This assay was performed according to literature procedures (Nicolau et al., 2002).

Example 4
Solubilization assay
From the immunized two animals in Pas Rumitoiru of A [beta] _1-16 / liposome / lipid A, it has been found to exhibit specificity for A [beta] _1-42 specific antibodies, recent fabricated hybridoma clones 25 Supernatant was obtained. These were examined in solubilization assays according to the methods and procedures described in PNAS 2002, 99, 2332-2337. The results obtained are summarized in FIG.

Example 5
Investigation of Aβ _1-42 -peptide transition from β-sheet to α-helix by solid-state NMR spectroscopy
^In order to avoid the loss of ¹³ C-labeled amino acids, the synthesis of Aβ _1-42 by Fmoc peptide synthesis was verified by test synthesis without the use of labeled amino acids. Whether or not Aβ _1-42 peptide was obtained could be verified by MALDI mass spectrometry, and a purification procedure using HPLC using a reverse phase column and an ammonia buffered acetonitrile water gradient could be established .

Following the setting step successful regarding the synthesis and purification of amyloid β peptide, 12 a ⁽¹² val) in ¹³ C-labeled valine, and the labeled peptide comprising the ¹³ C-labeled tyrosine at position 10 ⁽¹⁰ tyr) is synthesized It was .

Example 6
Antibodies induced by supramolecular antigenic constructs
Production of mAbs Liposome antigens were produced by literature procedures (Nicolau et al., 2002, PNAS, 99, 2332-37). The sequences , PEG-Aβ _1-15 , PEG-Aβ _1-16 , PEG-Aβ _4-11 , PEG-Aβ _22-35 , and PEG-Aβ _29-40 , monophosphoryl lipid A (40 mg / mM phospholipid) ) containing, dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidyl ethanolamine (DMPEA), dimyristoyl phosphatidyl glycerol (DMPG), and cholesterol (molar ratio 0.9: 0.1: 0.1: containing liposome prepared from 0.7) Reconstructed in the construct. These antigens, and palmitoylated Aβ _1-16 were used for immunization of C57BL / 6 mice at 2-week intervals. Ten to twelve animals were immunized with each antigen. After 3-6 boosts, mice that showed a therapeutic titer (1: 5000 dilution of sera were positive by ELISA) were selected for fusion. Fusion of B lymphocytes derived from the spleen of this mouse and the myeloma cell line SP2-0 was performed. IgG producing hybridoma clones were selected and examined by ELISA for specific binding to Aβ _1-42 peptide.

Example 8
Behavioral tests to assess antibody efficacy. Efficacy of antibodies induced by the methods described herein, ie, antibodies induced by supramolecular antigenic constructs containing modified amyloid peptides (eg, PEGylated amyloid peptides). In order to evaluate, mice are treated and evaluated with the behavioral test outlined below.

Morris Water Maze
Add 20 ° C water to the pool (white, circular container, 1 m in diameter), and use titanium dioxide as an odorless non-toxic additive to hide the escape platform (1 cm below the surface of the water) Use for. Each mouse swimming is analyzed with a video camera (Ethovision, Noldus information Technology, Wageningen, the Netherlands). Prior to training, rather 15 seconds location of each mouse on the platform. For the place navigation test, the mice are trained to find the location of the hidden platform in 5 blocks of 3 trials for 3 consecutive days. Each trial consists of a forced swim test and break 60 seconds subsequent up to 120 seconds. The time taken for each mouse to reach the platform position is measured. A learning curve is obtained from the results of five consecutive trials.

24 hours after the last training, targeting each animal, in a state excluding the platform, we intend rows probe trial. The mouse is allowed to search for 60 seconds and the quadrant search time and crossing of the initial platform position is measured.

Open field A Plexiglas open field box (52 x 52 x 40 cm) with a black vertical wall and a translucent floor is used for the test, and the lamp is struck by a lamp from the bottom of the box. The computer system (Ethovision, Noldus information Technology, Wageningen, the Netherlands) is assigned the following different compartments: corner (9 x 9 cm), four sides of the box (9 cm from the wall), and the center of the open field box (43 x 43 cm). Shooting each mouse with a video camera, distance traveled (cm), moving speed of the mouse (cm / sec), period / time (seconds) required in the center when compared with the boundary (corner + side ) ) And analyze the activity by measuring the number of crossings (N) (Ethovision). Each mouse was placed in the center of the box, to search freely (10 min). Clean the open field box between tests and let it dry before placing a new mouse in the box.