JP2009221159A - Medicinal preparation containing pegylated lactoferrin as main component - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a preparation form for realizing LF (lactoferrin) having very high utility as a safe and effective medicine for various kinds of diseases and symptoms, especially a pegylated LF as the medicine having excellent portability and preservation stability, and enabling various kinds of administration forms. <P>SOLUTION: The medicinal preparation is prepared by subjecting an aqueous solution containing the pegylated lactoferrin to freeze drying. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a pharmaceutical preparation containing PEGylated lactoferrin as an active ingredient.

  Lactoferrin (hereinafter sometimes referred to as “LF”) is an iron-binding glycoprotein having a molecular weight of about 80,000 and is commonly contained in mammalian milk. Among them, the content in human breast milk is outstanding, and it is known that the concentration is 10 times higher than that of milk. From this, it is said that humans have a particularly high dependence on LF.

  LF is continuously synthesized in the body, and in adults, 3 to 5 g of LF is made in the body per day. These LFs are distributed in a large amount in the portion in contact with the outside world, such as tears, saliva, digestive juices, and mucus, and are particularly contained in a large amount in neutrophils, which are a type of white blood cell that has an immune function.

  In addition, receptors for LF have been found on the surface of all tissues throughout the body, such as the heart, brain, skeletal muscle, liver, adrenal gland, and spleen. LF is a multifunctional physiologically active protein and has various physiological functions in the body. It is strongly suggested that the phenomenon is controlled.

  Functions that have been clarified from previous studies on LF include anti-inflammatory, iron absorption, anti-virus, antibacterial, immunostimulation, antioxidant, anti-cancer, analgesic, anti-stress, lipid metabolism improvement, etc. Applications of LF include viral hepatitis, cancer (treatment, secondary prevention), arthritis (rheumatoid arthritis, osteoarthritis), analgesia (cancer pain, other chronic pain), oral care (periodontal disease) , Stomatitis, dry mice), atopy, hay fever, and the treatment, amelioration or prevention of various diseases or symptoms. Therefore, these functions are very interesting from the viewpoint of drug discovery, and analysis of the functions is being promoted at many research institutions.

  Currently, commercially available products are mainly natural bovine lactoferrin separated and purified from milk. For pharmaceutical use, solution preparations of human-type LF by gene recombination have been developed. In the case of a solution formulation, all the storage and transportation of the formulation must be performed at a low temperature, and since the solution is also heavy, it is considered very inconvenient in terms of transportation costs.

  Since LF is a protein, it has a drawback of being easily decomposed by heat, acid, or enzyme. Orally ingested LF has been reported to be taken up from the intestinal tract via a receptor present in small intestinal epithelial cells and transferred into the blood via the lymphatic system (Non-patent Document 1). In order to exhibit physiological activity in vivo, it is considered important that undigested LF molecules reach the receptor. That is, in order to exert the effect of LF, it is necessary to deliver LF as it is to the small intestine.

  However, when orally ingesting LF, which is a protein, LF is transported to the stomach through the esophagus, where it is degraded by pepsin, a proteolytic enzyme, and cannot efficiently reach the small intestine.

Thus, in order to overcome the drawbacks of LF, PEGylated LF in which LF is modified with polyethylene glycol (hereinafter sometimes referred to as “PEG”) has been developed. A method of chemically modifying a protein using PEG is called “PEGylation” (PEGylation), and is one of important techniques in drug development (Non-patent Document 2). PEG is a water-soluble polymer having a molecular weight of several thousand to several tens of thousands, and PEG itself is hydrophilic, but its basic structure (CH ₂ CH ₂ O) exhibits both hydrophobic and hydrophilic properties. And although it is a water-soluble polymer, it also shows affinity for organic solvents. Therefore, when a protein is PEGylated into a pharmaceutical product, it is expected that if it can be made into a lyophilized formulation, it will be easier to distribute and use in clinical settings, but there are no reports of PEGylated lactoferrin lyophilized formulations. It is known what physicochemical properties, storage stability, solubility when re-dissolved, dispersibility when used as an inhalant, etc. when PEGylated lactoferrin is dried Not.

JP 2007-70277 A Special table 2006-509825 gazette T. Takeuchi et al., Exp. Physiol., 89 (3): 263-270 (2004) M.J.Roberts et al., Advanced Drug Delivery Reviews, 54: 459-476 (2002)

  The present invention puts LF, particularly PEGylated LF, which is very useful as a safe and effective medicine for various diseases and symptoms, into practical use as a pharmaceutical that has excellent transportability and storage stability and can be administered in various ways. The purpose is to develop a dosage form for this purpose.

The present invention
[1] A pharmaceutical preparation prepared by lyophilizing an aqueous solution containing PEGylated lactoferrin;
[2] The pharmaceutical preparation of the above-mentioned [1], which is for transpulmonary, nasal, transmucosal or oral administration;
[3] The pharmaceutical preparation according to [1] or [2], which does not contain an excipient;
[4] The pharmaceutical preparation according to the above [1] or [2], which contains an excipient;
[5] The pharmaceutical preparation of the above-mentioned [3] or [4], which is administered by spray inhalation after redissolving into a solution;
[6] The pharmaceutical preparation according to the above [3] or [4], which is finely powdered and administered by a powder inhalation method,
I will provide a.

  The pharmaceutical preparation of the present invention has a simple manufacturing process and is easy to manufacture, and is space-saving and lightweight compared to conventional solution preparations, etc., and has thermal stability and can be stored at room temperature for a long period of time. This is advantageous for storage and transportation. Moreover, since it is excellent in solubility, it can be easily used by dissolving at the time of use in the medical field. Furthermore, various excipients can be used, but even when no excipient is added, in addition to being excellent in solubility and storage stability, it is also excellent in disintegration. Therefore, as an administration route, in addition to injection, infusion, oral administration, etc., there is a remarkable merit that it can be transpulmonary administered as an aerosol or fine powder by a nebulizer or a powder inhaler.

  “Lactoferrin” (LF) contained in the PEGylated lactoferrin, which is an active ingredient in the preparation of the present invention, is a natural or natural lactoferrin molecule itself, as well as a genetically modified type (modified type with some amino acids substituted). It may be a functional equivalent of lactoferrin such as lactoferrin and an active fragment of lactoferrin, regardless of the presence or absence of iron ions, the content thereof, the species of origin. Preferably, it is bovine lactoferrin derived from milk or recombinant human lactoferrin.

  PEGylation of lactoferrin can be performed by a known method. As a suitable method, the method described in Unexamined-Japanese-Patent No. 2007-70277 is mentioned, for example. The PEG used for PEGylation may be linear or branched, and various commercially available derivatives can be used. The PEG may be polyethylene glycol or a modified product thereof (for example, a methoxy compound), and is most preferably linear PEG or branched PEG.

  In the PEGylated lactoferrin contained in the pharmaceutical preparation of the present invention, the number of covalently bonded PEG derivatives per lactoferrin molecule is preferably 1-5.

  The pharmaceutical preparation of the present invention can be prepared by lyophilizing an aqueous solution containing PEGylated LF. This aqueous solution preferably contains biocompatible salts and / or buffers in addition to PEGylated LF. Examples of aqueous solutions containing such salts and / or buffers include pharmaceutically acceptable salt solutions and buffers such as physiological saline and phosphate buffered saline (PBS). Thus, for example, saline or buffer can be added to an aqueous solution containing PEGylated LF to produce an aqueous solution that can be lyophilized. The aqueous solution can further contain various additives known in the field of excipients and formulations.

  As the excipient, a general substance known in the pharmaceutical field can be used, but any one of mannitol, dextran, and human serum albumin, and a mixture thereof can be preferably used. None of these excipients significantly affected the residual physiological activity of lactoferrin after formulation. Moreover, even when no excipient is added, in addition to being excellent in solubility and storage stability, it is also excellent in disintegration. Does not occur.

  Examples of other additives that may be contained in the aqueous solution or the pharmaceutical preparation of the present invention include sugars, amino acids, organic and inorganic acids and salts thereof, peptides / proteins, nucleic acids, and the like.

  The lyophilization conditions are such that the pH of the aqueous solution containing PEGylated lactoferrin is pH 4-9, preferably pH 6-7.

  Although the pharmaceutical preparation of the present invention can be stored frozen, it has high thermal stability and can be refrigerated or stored at room temperature for a long time.

  The pharmaceutical preparation of the present invention can be used in a solid state or as a solution after redissolving. In the case of a solid, it may be in a state as it is after drying, but it may be further processed by further pulverizing it into a fine powder or molding it into a tablet or the like. In the case of a re-dissolved liquid, it can be administered orally as it is, but it can be administered as an aerosol by a spray administration method using a nebulizer. Further, the finely divided preparation can be administered by an inhalation administration method using a powder inhaler. Therefore, the pharmaceutical preparation of the present invention can be administered by various administration routes such as injection, oral, nasal, transmucosal, and pulmonary by appropriately selecting the dosage form.

  The effective dosage of the pharmaceutical preparation of the present invention varies depending on the kind or degree of the disease or symptom to be treated or prevented, the condition of the administration target, the dosage form, etc., and can be appropriately selected based on the known effective lactoferrin amount. In general, the dose can be made significantly smaller than the known effective lactoferrin amount (for example, 1/2 to 1/20 amount in terms of lactoferrin amount), and if used at an equivalent dose, the number of administrations can be reduced. Is possible.

1. Production of PEGylated LF Based on the method described in Japanese Patent Application Laid-Open No. 2007-70277, PEGylated LF in which a branched PEG chain having a molecular weight of 20,000 (20K) is bound to natural bovine LF (hereinafter referred to as “20K-PEG-LF”) A solution (7.5 mg / ml) was prepared. Specifically, it is as follows.

1-1. PEGylation reaction 3.1 g of commercially available bovine lactoferrin (TB 905 007, TATUA, New Zealand) and 0.78 g of branched PEGylation reagent (GL2-200GS2, molecular weight 20,000, manufactured by NOF Corporation) were added to PBS. (Phosphate buffered saline solution, pH 7.4) was reacted at 25 ° C. for 24 hours at a molar ratio of 1: 1 and a protein concentration of 20 mg / ml.

1-2. Purification on the column The column used for the purification was filled with 93 ml of a cation exchanger (trade name “MacroCAP SP”, manufactured by GE Healthcare Bioscience), and 10 mM sodium phosphate buffer (pH 7.3). And equilibrated.

The following eluate was used.
Starting buffer: 10 mM sodium phosphate buffer (pH 7.4)
Elution buffer: An elution solution having a target salt concentration was prepared by mixing an initiation buffer (pH 7.4) containing 5M NaCl with the initiation buffer. The elution salt concentration was 0.2M and 0.3M.

Elution was performed in two steps as follows.
Purification step-1: Applying the PEGylation reaction solution to the purification column and monitoring the eluted protein with a UV detector, the non-adsorbed fraction (about 2.7 times the column volume) and the 0.2M NaCl elution fraction The fraction eluted with 0.3M NaCl (approximately 3.3 times the column volume) was collected (approximately 3.3 times the column volume).
Purification step-2: The non-adsorbed fraction was adsorbed to a purification column of about 1/3 volume of the purification step-1, and then the fraction eluted with 0.3 M NaCl was collected.

  The 0.2M NaCl elution fraction and 0.3M NaCl elution fraction of purification step-1 and the 0.3M NaCl elution fraction of purification step-2 were collected, and an ultrafiltration membrane (trade name “Centricon YM50”, Desalted and concentrated using Millipore).

  The protein concentration of the obtained purified 20K-PEG-LF was 7.5 mg / ml. It was confirmed by electrophoresis that unreacted lactoferrin was not included. 5 ml of this PEGylated LF solution was dispensed into tubes and stored frozen at -30 ° C.

2. Production of PEGylated LF lyophilized preparation PEGylated LF solution (15 ml in total) was thawed and stirred, and 3 ml each was dispensed into 5 new tubes.

Using D-mannitol, dextran 40 and BSA as excipients, 10% D-mannitol, 20% dextran 40 and 2% BSA solutions (concentrations are both W / V) were prepared in advance. The following five types of PEGylated LF-containing aqueous solutions (hereinafter referred to as “protein solutions”) were prepared by adding to the modified LF solution and stirring well.
(A) PEGylated LF 3 ml + 10% D-mannitol solution 3 ml
(Final concentrations are PEGylated LF: 3.75 mg / ml, D-mannitol: 5%)
(B) 3 ml of PEGylated LF 3 ml + 20% dextran 40 solution
(Final concentration is PEGylated LF: 3.75 mg / ml, dextran 40: 10%)
(C) 3 ml of PEGylated LF 3 ml + 2% BSA solution
(Final concentration is PEGylated LF: 3.75 mg / ml, BSA: 1%)
(D) PEGylated LF 3 ml + 10% D-mannitol solution 1.5 ml + 20% dextran 40 solution 1.5 ml
(Final concentrations are PEGylated LF: 3.75 mg / ml, D-mannitol: 2.5%, dextran 40: 5%)
(E) 3 ml of PEGylated LF + 3 ml of physiological saline (final concentration is PEGylated LF: 3.75 mg / ml; no excipient)

  The prepared 5 types of protein solutions were dispensed in 1.95 ml (total of 15) into 3 x 10 ml vials, and rubber stoppers were half-plugged. A vial into which the protein solution was dispensed was placed in a vat of a freeze dryer (FDU-2100, manufactured by Tokyo Rika Kikai Co., Ltd.) that had been pre-frozen at -40 ° C in advance, and frozen overnight by manual operation. The next morning, the freeze-drying process was completed by confirming freezing and running the program with the following program. The program was at -40 ° C for 20 minutes, -20 ° C for 9 hours, 0 ° C for 9 hours, and 20 ° C for more than 24 hours under reduced pressure.

  After the program operation was completed, the pressure was restored with nitrogen gas, immediately plugged, and tightened with an aluminum cap. Then, it preserve | saved at -30 degreeC.

  In order to confirm various performances, in addition to a sample produced as described above and stored at −30 ° C. for 2 months after freeze-drying (hereinafter referred to as “freeze-stored product”), after freeze-drying, 40 ° C. and relative humidity 75% A sample (hereinafter referred to as “heat-treated product”) that was heat-treated under conditions for 2 months was prepared. Heat treatment for 2 months at 40 ° C. and 75% relative humidity was carried out with a constant temperature and humidity chamber, and the temperature and humidity were checked every week. There were no abnormalities in temperature or humidity.

  Hereinafter, a lyophilized PEGylated LF-containing aqueous solution is a “sample”, and an excipient solution or physiological saline added to the PEGylated LF solution when preparing each sample is the “sample solution” of the sample. A solution obtained by dissolving each sample in water and reconstituting the sample is referred to as a “restoration solution” of the sample.

3. 3. Observation of appearance and solubility test 3-1. Appearance (1) Cryopreserved product Samples A to E all looked like white powdery cakes were cake-like. Sample C (excipient: 1% BSA) was smaller in size than other conditions. In the case of Sample E (without excipient), there were many places that looked like crystals than the others. Overall no major problems were seen.

(2) Heat-treated product As a whole, no major abnormality was observed, and all the samples were white and powdery. Overall, no significant change was seen compared to the frozen product. Specifically, it was as follows.
<Sample A (Excipient: 5% D-mannitol)>
There were no major changes and the surface was clean.
<Sample B (Excipient: 10% Dextran 40)>
The size was shrunk compared with the cryopreserved product. The center of the surface was like a crystal, and the surrounding area was in a powdery state.
<Sample C (Excipient: 1% BSA)>
Compared with the cryopreserved product, it was shrunk more than sample B. The surface was in the same state as Sample B.
<Sample D (excipient: 2.5% D-mannitol, 5% dextran 40)>
There was no significant change. The surface was clean.
<Sample E (without excipient)>
The whole surface was in a crystal-like state, and it seemed to be easy to collapse.

3-2. Solubility test 1.95 ml of distilled water for injection was added to a vial containing 5 types of each of the cryopreserved product and the heat-treated product, and the original sample solution was restored (each restored solution was PEG). 7.3 mg of modified LF).

(1) Cryopreserved product Except for Sample B (excipient: 10% dextran 40), the others were dissolved quickly. Sample B seemed to be relatively difficult to dissolve, but none of the samples finally dissolved and no undissolved material was produced. Specifically, it was as follows.
<Sample A (Excipient: 5% D-mannitol)>
It melted instantly. There was no particular problem and the solution was clean.
<Sample B (Excipient: 10% Dextran 40)>
It did not melt instantaneously, and it was difficult to melt when compared with others. The solution was a little cloudy.
<Sample C (Excipient: 1% BSA)>
It melted immediately. The solution was clear.
<Sample D (excipient: 2.5% D-mannitol and 5% dextran 40)>
It melted instantly. Some bubbles remained.
<Sample E (without excipient)>
It melted instantly. The solution was clean and clear.

(2) Heat-treated product No abnormality was observed in solubility compared to the cryopreserved product, and no problem was found in any sample.

4). Measurement of anti-inflammatory activity of lyophilized PEGylated LF For each sample of the lyophilized PEGylated LF produced above, the inflammatory cytokine IL- produced when the inflammation-inducing substance LPS was allowed to act on THP-1 cells. The activity of LF to suppress the production of 6 (anti-inflammatory effect) was measured.

  1.95 ml of distilled water for injection was added to the vial containing each sample to restore the original sample solution. These reconstitution solutions contained 7.3 mg of PEGylated LF.

Human monocyte-derived cells (THP-1) used in this experiment were cultured at 37 ° C. and 5% carbon dioxide in 10 ml of RPMI1640 medium containing 10% FBS, 2 mM Glutamax (trade name; manufactured by Invitrogen). Passage was performed every 4 days. Cells were seeded in 75 cm ² T-flasks at a concentration of 5 × 10 ⁴ cells / ml and cultured for 72 hours. Then, it centrifuged for 5 minutes at 400xG and 4 degreeC. The supernatant was aspirated and 10 ml of fresh medium was added to suspend the cells. This was a single cell wash and was performed a total of 3 times. After washing, cells were seeded in a new flask so that the number of cells was 5 × 10 ⁴ cells / ml, and cultured for 24 hours. After culturing for 24 hours, interferon-γ (IFN-γ) and calcitriol were added to the culture solution to a final concentration of 40 ng / ml and 20 ng / ml, respectively, and cultured for 48 hours. Since THP-1 cells adhere to the bottom of the flask, the cells were removed by cell scraper and collected, and a cell suspension of 1 × 10 ⁶ cells / ml was finally prepared. This cell suspension was added to a 24-well plate at 400 μl / well, and reagents and the like were further added to each well as follows, followed by incubation for 24 hours.

For sample A,
"Medium" = medium only (no reagents added)
“LF” = 4 μl of 10 mg / ml LF solution (aqueous solution of bovine lactoferrin used for the production of PEGylated LF)
“LPS” = 4 μl of 10 μg / ml LPS solution “LPS + LF” = 4 μl of 10 μg / ml LPS solution and 4 μl of 10 mg / ml LF solution “Sample solution A” = 16.5 μl of sample solution A
“Restoration solution A” = 10.7 μl of restoration solution A (containing 3.75 mg / ml PEGylated LF)
“LPS + sample solution A” = 4 μl of 10 μg / ml LPS solution and 16.5 μl of sample solution A
“LPS + sample solution A + LF” = 4 μl of 10 μg / ml LPS solution, 16.5 μl of sample solution A and 4 μl of 10 mg / ml LF solution “LPS + restoration solution A” = 4 μl of 10 μg / ml LPS solution and 10.7 μl of reconstitution Liquid A (containing 3.75 mg / ml PEGylated LF)
It carried out similarly about sample BE.

  The amount of IL-6 secreted into the culture solution was measured using a “human IL-6 ELISA kit” (Kamakura Technoscience Co., Ltd., product number KTS302), and the anti-inflammatory activity of the reconstituted solution was examined.

(1) Cryopreserved product The results are shown in FIGS. The measured values in FIGS. 1 to 5 are average values measured for two wells. Table 1 shows the residual activity of the anti-inflammatory activity of each reconstitution solution expressed as a relative value when the activity of unmodified LF of the same weight is defined as 100%.

  When only the medium used, only the LF solution, only the sample solution, and only the reconstitution solution are added, there is almost no IL-6 production from THP-1 cells, so there is no endotoxin contamination in these solutions. it is conceivable that. In addition, since various sample solutions used for lyophilization did not change the amount of IL-6 produced from THP-1 when LF and LPS were added, the method for measuring the anti-inflammatory activity used here was used. There is no impact. As can be seen from Table 1, about 40 to about 60% residual activity was confirmed for the reconstitution liquid.

(2) Heat-treated product The results are shown in FIGS. 6 to 10 (average value of measured values for each two wells). Table 2 shows the anti-inflammatory activity of each reconstitution solution.

  Regarding the heat-treated product, when only the medium used, only the LF solution, and only the reconstitution solution were added, IL-6 production from THP-1 cells was hardly observed. It is thought that there is not. As can be seen from Table 2, a residual activity of about 70% was confirmed.

  In summary, the anti-inflammatory activity retained about 40 to about 60% of activity in the cryopreserved product and about 70% of activity in the heat-treated product. It has been found that the anti-inflammatory activity of normal PEGylated LF that has not been lyophilized is about 60% that of native LF. Accordingly, it has been clarified that no activity is significantly reduced by freeze-drying treatment, and that relatively high activity can be maintained even when heat treatment is performed at a temperature of 40 ° C., a humidity of 75%, and two months.

FIG. 1 is a diagram showing the anti-inflammatory activity of the reconstituted solution of Sample A (freezer preserved product) in terms of IL-6 production. FIG. 2 is a diagram showing the anti-inflammatory activity of the reconstituted solution of sample B (frozen product) in terms of IL-6 production. FIG. 3 is a diagram showing the anti-inflammatory activity of the reconstituted solution of sample C (a cryopreserved product) in terms of IL-6 production. FIG. 4 is a diagram showing the anti-inflammatory activity of the reconstituted solution of sample D (a cryopreserved product) in terms of IL-6 production. FIG. 5 is a diagram showing the anti-inflammatory activity of the reconstituted solution of sample E (frozen product) in terms of IL-6 production. FIG. 6 is a diagram showing the anti-inflammatory activity of the reconstituted solution of sample A (heat treated product) in terms of IL-6 production. FIG. 7 is a diagram showing the anti-inflammatory activity of the reconstituted solution of sample B (heat treated product) in terms of IL-6 production. FIG. 8 is a diagram showing the anti-inflammatory activity of the reconstituted solution of sample C (heat treated product) in terms of IL-6 production. FIG. 9 is a diagram showing the anti-inflammatory activity of the reconstituted solution of sample D (heat treated product) in terms of IL-6 production. FIG. 10 is a diagram showing the anti-inflammatory activity of the reconstituted solution of sample E (heat treated product) in terms of IL-6 production.

Claims (6)

  A pharmaceutical preparation prepared by lyophilizing an aqueous solution containing lactoferrin that has been PEGylated.   The pharmaceutical preparation according to claim 1, which is for transpulmonary, nasal, transmucosal or oral administration.   The pharmaceutical formulation of Claim 1 or 2 which does not contain an excipient | filler.   The pharmaceutical formulation of Claim 1 or 2 containing an excipient | filler.   The pharmaceutical preparation according to claim 3 or 4, which is administered by a spray inhalation method after redissolving into a solution.   The pharmaceutical preparation according to claim 3 or 4, which is finely powdered and is administered by a powder inhalation method.

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