CA2097932A1 - Antibody containing composition - Google Patents
Antibody containing compositionInfo
- Publication number
- CA2097932A1 CA2097932A1 CA 2097932 CA2097932A CA2097932A1 CA 2097932 A1 CA2097932 A1 CA 2097932A1 CA 2097932 CA2097932 CA 2097932 CA 2097932 A CA2097932 A CA 2097932A CA 2097932 A1 CA2097932 A1 CA 2097932A1
- Authority
- CA
- Canada
- Prior art keywords
- antibody
- complex
- inhalation
- fab
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/007—Pulmonary tract; Aromatherapy
- A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
- A61K9/0075—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a dry powder inhaler [DPI], e.g. comprising micronized drug mixed with lactose carrier particles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/007—Pulmonary tract; Aromatherapy
- A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
- A61K9/008—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy comprising drug dissolved or suspended in liquid propellant for inhalation via a pressurized metered dose inhaler [MDI]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
Abstract Disclosed herein is a composition containing an antibody or its complex, which is obtained by freeze-drying a solution of the antibody or its complex followed by milling the freeze-dried product so that it has an effective particle diameter of more than 0.5 µm and less than 10 µm. Because the antibody containing composition of the present invention has high stability, it is suitable as a composition for the inhalation of the antibody or its complex which is pharmaceutically effective.
Description
DESCRIPTION
ANTIBODY CONTAINING COMPOSITION
Field ~f the Invention The present invention relates to a novel antibody containing medicine which is pharmaceutically useful. More particularly, it relates to a composition containing the antibody or its complex such as an immunotoxin complex and the like.
Backgrou~d of the Invention Recently, an application of the antibodies to the medicines is extensively studied. The research and development of pharmaceutical formulations containing the antibodies in various fields including the fields of tumor, cardiac and circulatory system, immunity, allergy and infection have been advanced extensively. For example, the research for treating cancer with monoclonal antibodies against tumor-associated antigens is most active. Such antibodies will be developed as the pharmaceutical formulations in the near future. At present, some formulations are subjected to clinical experiments wherein the tumor sites to I -be treated are covered with a wide range, including blood, hematopoietic organ, lung, liver, digestive organ, ovarium and prostate. As a method for treating the cancer with the monoclonal antibody, an immunotherapy with only the antibody so that macrophagus and lymphocytes having receptors against Fc fragments of the antibody are collected at the tumor sites, thereby the cancer is attacked and a target therapy with the ' .. ~ ~ ', ' ' .
ANTIBODY CONTAINING COMPOSITION
Field ~f the Invention The present invention relates to a novel antibody containing medicine which is pharmaceutically useful. More particularly, it relates to a composition containing the antibody or its complex such as an immunotoxin complex and the like.
Backgrou~d of the Invention Recently, an application of the antibodies to the medicines is extensively studied. The research and development of pharmaceutical formulations containing the antibodies in various fields including the fields of tumor, cardiac and circulatory system, immunity, allergy and infection have been advanced extensively. For example, the research for treating cancer with monoclonal antibodies against tumor-associated antigens is most active. Such antibodies will be developed as the pharmaceutical formulations in the near future. At present, some formulations are subjected to clinical experiments wherein the tumor sites to I -be treated are covered with a wide range, including blood, hematopoietic organ, lung, liver, digestive organ, ovarium and prostate. As a method for treating the cancer with the monoclonal antibody, an immunotherapy with only the antibody so that macrophagus and lymphocytes having receptors against Fc fragments of the antibody are collected at the tumor sites, thereby the cancer is attacked and a target therapy with the ' .. ~ ~ ', ' ' .
- 2 - 20979~2 immunotoxin complex comprising the monoclonal antibody to which a toxic substance such as a toxic protein, a radioactive substance (RI) and an anticancer agent i5 bound are known.
Dosage forms for administrating the monoclonal antibodies are all injections, because the monoclonal antibodies are macromolecular protein. Most of the injections are the injections for intravenous administration. Because the injection is a troublesome and imposes a burden to a patient, simpler dosage form is desired. One is an inhalation By the inhalation having a dosage form for general administration, the burden to the patient can be lightened and the patient can be treated at home. On the other hand, when the immunotoxin complex wherein the monoclonal antibody is bound with, for example, the toxic protein is intravenously administered, it circulates through major organs including liver and kidney and as the result, these organs may be seriously damaged. Thus, the dosage form in place of the injection is desired. One is the inhalation for a topical administration in order to treat lung cancer. It is expected to be effectively used. ~owever, the inhalation has a defect because of a drug used being macromolecular.
One of the inhalations is an aqueous inhalation, wherein the drug in the form of water-soluble microparticles is administered using a suitable device such as a jet nebulizer and an ultrasonic nebulizer. The other is a solid inhalation, wherein the drug in the form o~ solid particulate powder is administered using a metered-dose inhaler. The drugs in both inhalations are mainly low molecular compounds such as ~2-adrenergic antagonists, steroids and antibiotics.
An attempt for formulating polypeptides as aerosol preparations is relatively new. For example, a solid aerosol preparation containing insulin (Lee and Sciarra, J. Pharm., 65, 567, 1976), a solid aerosol preparation containing recombinant 1~antitrypsin (Hubbard et al., Proc. Natl. Acad. Sci. USA, 86, 680, 1989), the administration of an aqueous solution of human leukocyte interferon- through the jet nebulizer (Kin-nula et al., J. Interferon Res., 9, 419, 1989) and the administration of an aqueous solution of human growth hormone through the nebulizer (JP-A-63-51868) are reported. They describe that the polypeptide having the molecular weight of 50,000 or less is used. And, they describe that the preferable particle diameter is 0.5 to 10 ~m, particularly 5 ~m or less, for adsorbing the -polypeptides by the inhalation. The effectiveness of certain drugs as the dosage form for the topical administration has been also examined.
In case of the aerosol preparation, it is considered that the absorption sites are determined depending on the particle size. It is said that smaller particles can reach to alveoli pulmonum, while larger particles deposit in a nasal or oral cavity. For reaching to the alveoli pulmonum, the particle diameter should be 0.5 to 10 ~. (Porush et al., J. Amer. Pharm.
Ass. Sci, Ed., 49, 70, 1960), preferably 5 ~m or less in diameter (Newman and Clarks, Thorax, 3~, 881, 1983).
At present, the inhalation of the antibody is pharmaceutically desired as a simple general administration and as a topical administration to the alveoli pulmonum. However, the macromolecular substance such as the monoclonal antibody having the molecular weight of 150,000 among the polypeptides is hardly adsorbed through the inhalation. Nevertheless, the absorption of the macromolecular substance through the inhalation and the retention thereof in the lung through the topical administration are important and strongly desired.
Unfortunately, the technic for obtaining the stable preparation containing the antibody while maintaining its physiological activities is not well established.
The monoclonal antibody having the physiological activities such as a binding capacity to cancer cells is easily affected in a solution system, with respect to its stability and pharmaceutical activities. For example, it is frequently deactivated by oxidation, coagulation and thermal denaturation in the solution system. When its aqueous solution is nebulized through the jet nebulizer or the ultrasonic nebulizer, a shear stress is applied to the monoclonal antibody. Therefore, the powdery preparation is preferable and excellent. Irrespective of the form (aqueous or solid) of the preparation, the ': 1 '- '.',:' ' . ., ., :
- . ~ -.~:
selections of a stabilizer and stabilizing conditions are essential in order to obtain the stable preparation containing the monoclonal antibody which has higher pharmaceutical activity.
An object of the present invention is to obtain an antibody containing composition which is expected to have a pharmaceutical applicability, as a stable solid aerosol preparation capable of being inhaled.
Summa~yQ f th~ Invention The above object will be attained by the present invention.
The present invention relates to a composition containing an antibody or its complex, which is obtained by freeze-drying a solution of an antibody or its complex followed by milling and which has an effective particle diameter of more than 0.5 ~m and less than 10 ~m.
Br~ gPl~a~ion of Drawings Fig. 1 shows a serum concentration after inhaling to rabbits each of the MDI preparations a) and c) which were prepared in Example 1, as a function of time. - O - is the serum concentration of human Fab in the rabbits to which the MDI
preparations a) were administered, and - - is that of human IgG
in the rabbits to which the the MDI preparations c) were administered.
- 6 ~ 20 97 9 3 2 Fig. 2 shows a serum concentration after inhaling to the rabbits the MDI preparation e) which was prepared in Example 2, as a function of time.
~est Mode for car~ying out the InventiQ~
The antibody herein includes immunoglobulins (IgGs), their fragments or their functionally equivalent molecules, and their products modified by a genetic engineering technic such as a chimera antibody in which constant regions in an amino acid sequence are replaced with other amino acid sequences. As the antibody fragments, F(ab')2, Fab', Fab and Fv which are produced by conventional methods are exemplified. The antibody usable in the present invention is not limited to either polyclonal or monoclonal antibody. And, an animal species from which the antibody is derived is not limited.
The antibody having the physiologically activities such as the binding capacity to the cancer cells can be applied, for example, to the cancer treatment. When the antibody is systemicaly administered for treating the cancer, the antibody used is not limited. When the antibody ls topically administered to lung, however, only the antibody specific to the pulmonary cancer is used. For example, SF25 [Cancer Res., 48, 6573-6579 (1988)], XF8, AF20 [Hepatology, ~, 625-634 (1989)], SWA11 [Br. J. Cancer, 59, 174-178 (1989)], SM1 [Cancer Res., ~, 26S ~1984)] and TFS-4 [Cancer ~es., 47, 826 (1987)] can be used.
As the method for treating the cancer, the immunotherapy is ~' ' ' : , ' .
'' , ~ . .,, ~ 7 ~ 209~ 932 mentioned, in which the antibody is singly administered and thereby, the macrophagus or the lymphocytes which have the receptors against Fc regions of the antibody are collected on the tumor sites so as to attack the tumor. Alternatively, the target therapy is mentioned, in which the complex comprising the antibody to which the toxic substance (for example, the toxic protein, the radioactive substance (RI) and anticancer agent) is bound is used. In the complex comprising the antibody and the toxic substance, the toxic protein as one of the usable toxic substances includes A chains of plant toxins (type II) such as ricin and abrin and active components of plant toxins (type I) such as luffin, momordin and PAP-S and of bacterial protein toxin such as P~eudomonas toxin and diphtheria toxin, which irreversibly terminates a protein synthesis and thereby a high toxicity is given to the cells. The other preferable toxic protein includes an enzyme derived from human such as human RNase, which is incorporated into the cells and thereby lethal damages can be given to the cells. In the preparation of the immunotoxin complex by binding the antibody with the toxic protein, a crosslinking agent is used [Tanpakushitsu-Kakusan-Koso (PROTEIN NUCLEIC ACID ENZYME), supplement No. 31, p.335-343 (1987)].
For stabilizing the antibody, the addition of a sugar such as lactose, maltose, sorbitose, tolehalose and xylose and/or a sugar alcohol such as mannitol, sorbitol and xylitol is - 8 ~ 2097 932 effective in the present invention. The addition of the protein such as human serum albumin is also effective for stabilizing the antibody. The added amount of the sugar or sugar alcohol is preferably 0.01 to 200 %, preferably 1 to 50 % based on the total weight of the antibody and the human serum albumin.
The milling of the freeze-dried product is conducted using a suitable apparatus such as a jet milling apparatus (for example "Micronizer Mill") and a ball milling apparatus. For the inhalation, the particles have a particle size distribution such that more than 50 % or more, preferably 75 % or more of the total particles have the particle diameter of 0.5 to 5 ~m. The particle size distribution can be determined by a standard particle size distribution analyzer (for example, model CAPA-700 of Horiba Ltd. and QCM-Cascade Impactor model PC-2 of California Measurement Inc.).
It is preferable to add any surfactant to the freeze-dried and milled product for improving the dispersibility of the particles. The surfactant used in the present invention is at least one of sorbitan trioleate (Span 85), oleyl alcohol, soya lecitin and hydrogenated castor oil derivatives (HCO60). The addition amount of the surfactant is 0.001 to 5 %, preferably 0.05 to 2 % based on the total weight of the antibody and the human serum albumin.
Optionally, mineral ions which are necessary to keep an osmotic pressure intrinsically present in an organism, such as . " . ' ,, 20~7932 calcium, magnesium, sodium, potassium, chloride and phosphate ions may be added. If necessary, a surfactant derived from alveoli pulmonum may be added for controlling a stimulating property to the organism.
The thus-prepared antibody containing composition is as such or dispersed in a nebulizing gas such as compressed air and compressed carbon oxide gas or in a suitable propellant in order to inhale through the oral or nasal cavity. The usable .
propellant includes chlorofluorocarbon (furon 11, 12, 114 etc.), hydrochlorofluorocarbon (furon 123, 124, 141b) and fluorocarbon (furon 125, 134a).
Exa~les The following examples will more fully illustrate the present invention, but they are not intended to be limiting of the present invention.
Exam.~?le 1 Powders were prepared by freeze-drying 20 ml of a solution con~aining 0.75 mg/ml of a freeze-dried powder of human immunoglobulin IgG (ex. PAESEL) or human immunoglobulin Fab (ex.
CAPPEL), 15.0 mg/ml of human serum albumin, 2.0 mg/ml of sorbitol, 0.375 mg/ml of phosphate buffer and 1.5 mg/ml of sodium chloride. Then, each of the freeze-dried solid powders was collected followed by milling in Sturtevant jet milling apparatus (500 ml capacity), thereby a milled powder was obtained. The resultant milled powder was analyzed using the :
.~ . :
.
,.
particle size distrlbution analyzer (model CAPA-700 of Horiba Ltd.). The milled powder comprising IgG had the average particle diameter of 5.20 i 1.86 ~m, the proportion of the particles with the particle diameter of 5.45 ~m or less being 55.0 %. While, the milled powder comprising Fab had the average particle diameter of 1.95 i 1.05 ~m, the proportion of the particles with the particle diameter of 4.00 ~m or less being 97.9 %.
Next, 50 to 100 mg of the powder was placed in a glass vial (10 ml capacity), to which oleyl alcohol or sorbitan trioleate (Span 85) was added such that it is contained in an amount of 0.25 % under nitrogen atmosphere. After a metered-dose valve was attached on the glass vial, furon 12 was introduced therein so that the total amount was 10 ml. Thus, four MDI (metered -dose inhalation) preparations a), b), c) and d) were obtained.
A composition of each preparation was shown in Table 1.
'; ~- ~ . -. , ~, .
.. , -Table 1MDI preparations comprising human immunoglobulin IgG and human immunoglobulin Fab' .. ~ ..
com~ o~ition and d~ ~e _ _ MDI preparation compo~ition amount weight do~e . (w/v ~) (~) (Uq/time) a) Fab powder* 0.5 0.050 9.55 Fab oleyl alcohol 0.25 0.025 (oleyl alcohol) CFC_12 99.25 13.27 b~ Fab powder*1.0 0.10 19.1 Fab SPAN 85 O.25 O.025 (SPAN 85) CFC 12 98.75 _ 13.20 c) IgG 1.0 0.10 500 IgG oleyl alcohol 0.25 0.025 (oleyl alcohol) CFC 12 9a.25 13.20 d) IgG 1.0 0.10 500 IgG SPAN 85 O.25 O.025 (SPAN 85) CFC 12 98.25 _13.20 * Fab powder contained the following ingredients per 100 mg:
Fab powder : 3.82 mg HSA : 76.4 mg phosphate : 1.91 mg NaCl : 7.6 mg D-sorbitol : 10.2 mg ' ~ ~
.
Using a metered~dose inhaler, the human immunoglobulin Fab or IgG in a dose shown in Table l was inhaled to rabbits. Three NZW male rabbits of body weight of 3.0 to 3.5 kg were used per each group. Before the administration to the rabbit, the MDI
preparation was forcedly sprayed in a bellows-type spacer five times. Then, an adapter was attached, through which the MDI
preparation was administered to the lung of the rabbit by a spontaneous inhalation for one minute. For each of the MDI
preparations c), d), a) and b), the above procedure was repeated ~wo times, four times, six times and six times, respectively.
Accordingly, the expected final dose of the immunoglobulin was 5 mg, lO mg, 287 ~g and 573 ~g, respectively.
After 0.5, l, 3, 6, 9 and 12 hours from the administration of the MDI preparation, a blood was sampled from an ear auricular vein of each rabbit. A serum in a blood sample was separated so as to prepare a sample for determining a serum concentration of human immunoglobulin IgG or Fab. The determination is conducted according to the EIA method.
Firstly, an immunoplate (ex. NUNC) was coated with lO0 ~l of a lO ~g/ml solution of a sheep anti-human F(ab')2 antibody (ex.
CAPPEL) in PBS(-) at 4C overnight. After washing with 250 ~l of PBS(-), the plate was blocked with 1% BSA/PBS(-) at 4C
overnight followed by washing with each of 250 ~l of a washing solution (0.05% Tween 20/PBS(-)) three times. Then, a human immunoglobulin IgG standard (ex. ZYMED), a human immunoglobulin .
- '. , ~ .:. '' , ~ ' ' ' .: . , ~ :
Fab standard (ex. CAPPEL) and a sample were added and the reaction was continued at room temperature for 1 hour. After washing with each of 250 ~1 of the washing solution three times, 100 ~1 of a horseraddish peroxidase (HRP) standard goat anti-human IgG (H + L) chains antibody (ex. ZYMED) which was previously diluted 12,000 times was reacted at room temperature for 1 hour. Thereafter, the washing was repeated. A substrate for HRP was added in order to develop a color. After the reaction was terminated, an absorption at 490 nm was detected using an immuno reader.
The results showed that in both preparations comprising the human immunoglobulins IgG and Fab, the preparations comprising oleyl alcohol showed higher absorption efficiency, as compared with those comprising SPAN 85. Thus, the change in serum concentration after the inhalation of the preparation (a) or (c) comprising oleyl alcohol was determined. The results are shown in Fig. 1. After 30 minutes from the inhalation, the serum concentration was reached to a maximum value in both preparations a) and c). In the preparation a), it was 1.18 % of the dose. In the preparation c), it was 0.98 % of the dose.
Further, Fab showed the higher serum concentration as compared with IgG.
xample 2 A IgG-Abrin A chain complex as the immunotoxin complex was prepared from human immunoglobulin IgG (ex. PAESEL) and abrin A
.. -' 'i" ~: , - 1~- 2097932 chain using SPDP (N-succinimidyl 3-(2-pyridyl-dithio)propionate) of the crosslinking agent. For the purification of the complex, blue Sepharose column and a gel filtration column were used.
A powder was prepared by freeze-drying 20 ml of a solution containing 0.7 mg/ml of IgG-abrin A chain complex, 15.0 mg/ml of human serum albumin, 2.0 mg/ml of sorbitol, 0.75 mg/ml of phosphate buffer and 1.5 mg/ml of sodium chloride. The freeze-dried powder was milled in Sturtevant jet milling apparatus (500 ml capacity), thereby a milled powder was obtained. The resultant milled powder was analyzed using the particle size distribution analyzer (model CAPA-700 of Horiba Ltd.). The milled powder had the average particle diameter of 4.65 i 1.58 ~m, the proportion of the particles with the particle diameter of 5.00 ~m or less being 63.6 %.
One hundred mg of the powder was placed in a glass vial (10 ml capacity), to which oleyl alcohol was added such that it is contained in an amount of 0.25 % under nitrogen atmosphere.
After a metered-dose valve was attached on the glass vial, furon 12 was introduced therein so that the total amount was 10 ml.
Thus, MDI preparations e) was obtained.
Using the metered-dose inhaler, the complex in a dose of 500 mg/time was inhaled to three NZW male rabbits of body weight of 3.0 to 3.5 kg per each group. Before the administration to the rabbit, the MDI preparation was forcedly sprayed in a bellows-type spacer five times. Then, an adapter was attached, .
.. .
' ' ..
, - 1S- 2~97932 through which the MDI preparation was a~ninistered to the lung of the rabbit by a spontaneous inhalation for one minute. The ,,above procedure was repeated two times. Accordingly, the expected final dose of the complex was 5 mg.
After 0.5, 1, 3, 6, 9 and 12 hours from the administration of the MDI preparation, a blood was sampled from an ear auricular vein of each rabbit. A serum in a blood sample was separated so as to prepare a sample for determining a serum concentration of the complex. The determination is conducted according to the EIA method.
The change in serum concentration after the inhalation is shown in Fig. 2. After 30 minutes from the inhalation, the serum concentration was reached to a maximum value. It was confirmed to be 0.42 % of the dose.
Utiliz,~a,,,bility in Industries Because the antibody containing composition of the present invention is the stable solid composition, it is suitable as the composition for the inhalation of the àntibody or its complex which is pharmaceutically effective.
Dosage forms for administrating the monoclonal antibodies are all injections, because the monoclonal antibodies are macromolecular protein. Most of the injections are the injections for intravenous administration. Because the injection is a troublesome and imposes a burden to a patient, simpler dosage form is desired. One is an inhalation By the inhalation having a dosage form for general administration, the burden to the patient can be lightened and the patient can be treated at home. On the other hand, when the immunotoxin complex wherein the monoclonal antibody is bound with, for example, the toxic protein is intravenously administered, it circulates through major organs including liver and kidney and as the result, these organs may be seriously damaged. Thus, the dosage form in place of the injection is desired. One is the inhalation for a topical administration in order to treat lung cancer. It is expected to be effectively used. ~owever, the inhalation has a defect because of a drug used being macromolecular.
One of the inhalations is an aqueous inhalation, wherein the drug in the form of water-soluble microparticles is administered using a suitable device such as a jet nebulizer and an ultrasonic nebulizer. The other is a solid inhalation, wherein the drug in the form o~ solid particulate powder is administered using a metered-dose inhaler. The drugs in both inhalations are mainly low molecular compounds such as ~2-adrenergic antagonists, steroids and antibiotics.
An attempt for formulating polypeptides as aerosol preparations is relatively new. For example, a solid aerosol preparation containing insulin (Lee and Sciarra, J. Pharm., 65, 567, 1976), a solid aerosol preparation containing recombinant 1~antitrypsin (Hubbard et al., Proc. Natl. Acad. Sci. USA, 86, 680, 1989), the administration of an aqueous solution of human leukocyte interferon- through the jet nebulizer (Kin-nula et al., J. Interferon Res., 9, 419, 1989) and the administration of an aqueous solution of human growth hormone through the nebulizer (JP-A-63-51868) are reported. They describe that the polypeptide having the molecular weight of 50,000 or less is used. And, they describe that the preferable particle diameter is 0.5 to 10 ~m, particularly 5 ~m or less, for adsorbing the -polypeptides by the inhalation. The effectiveness of certain drugs as the dosage form for the topical administration has been also examined.
In case of the aerosol preparation, it is considered that the absorption sites are determined depending on the particle size. It is said that smaller particles can reach to alveoli pulmonum, while larger particles deposit in a nasal or oral cavity. For reaching to the alveoli pulmonum, the particle diameter should be 0.5 to 10 ~. (Porush et al., J. Amer. Pharm.
Ass. Sci, Ed., 49, 70, 1960), preferably 5 ~m or less in diameter (Newman and Clarks, Thorax, 3~, 881, 1983).
At present, the inhalation of the antibody is pharmaceutically desired as a simple general administration and as a topical administration to the alveoli pulmonum. However, the macromolecular substance such as the monoclonal antibody having the molecular weight of 150,000 among the polypeptides is hardly adsorbed through the inhalation. Nevertheless, the absorption of the macromolecular substance through the inhalation and the retention thereof in the lung through the topical administration are important and strongly desired.
Unfortunately, the technic for obtaining the stable preparation containing the antibody while maintaining its physiological activities is not well established.
The monoclonal antibody having the physiological activities such as a binding capacity to cancer cells is easily affected in a solution system, with respect to its stability and pharmaceutical activities. For example, it is frequently deactivated by oxidation, coagulation and thermal denaturation in the solution system. When its aqueous solution is nebulized through the jet nebulizer or the ultrasonic nebulizer, a shear stress is applied to the monoclonal antibody. Therefore, the powdery preparation is preferable and excellent. Irrespective of the form (aqueous or solid) of the preparation, the ': 1 '- '.',:' ' . ., ., :
- . ~ -.~:
selections of a stabilizer and stabilizing conditions are essential in order to obtain the stable preparation containing the monoclonal antibody which has higher pharmaceutical activity.
An object of the present invention is to obtain an antibody containing composition which is expected to have a pharmaceutical applicability, as a stable solid aerosol preparation capable of being inhaled.
Summa~yQ f th~ Invention The above object will be attained by the present invention.
The present invention relates to a composition containing an antibody or its complex, which is obtained by freeze-drying a solution of an antibody or its complex followed by milling and which has an effective particle diameter of more than 0.5 ~m and less than 10 ~m.
Br~ gPl~a~ion of Drawings Fig. 1 shows a serum concentration after inhaling to rabbits each of the MDI preparations a) and c) which were prepared in Example 1, as a function of time. - O - is the serum concentration of human Fab in the rabbits to which the MDI
preparations a) were administered, and - - is that of human IgG
in the rabbits to which the the MDI preparations c) were administered.
- 6 ~ 20 97 9 3 2 Fig. 2 shows a serum concentration after inhaling to the rabbits the MDI preparation e) which was prepared in Example 2, as a function of time.
~est Mode for car~ying out the InventiQ~
The antibody herein includes immunoglobulins (IgGs), their fragments or their functionally equivalent molecules, and their products modified by a genetic engineering technic such as a chimera antibody in which constant regions in an amino acid sequence are replaced with other amino acid sequences. As the antibody fragments, F(ab')2, Fab', Fab and Fv which are produced by conventional methods are exemplified. The antibody usable in the present invention is not limited to either polyclonal or monoclonal antibody. And, an animal species from which the antibody is derived is not limited.
The antibody having the physiologically activities such as the binding capacity to the cancer cells can be applied, for example, to the cancer treatment. When the antibody is systemicaly administered for treating the cancer, the antibody used is not limited. When the antibody ls topically administered to lung, however, only the antibody specific to the pulmonary cancer is used. For example, SF25 [Cancer Res., 48, 6573-6579 (1988)], XF8, AF20 [Hepatology, ~, 625-634 (1989)], SWA11 [Br. J. Cancer, 59, 174-178 (1989)], SM1 [Cancer Res., ~, 26S ~1984)] and TFS-4 [Cancer ~es., 47, 826 (1987)] can be used.
As the method for treating the cancer, the immunotherapy is ~' ' ' : , ' .
'' , ~ . .,, ~ 7 ~ 209~ 932 mentioned, in which the antibody is singly administered and thereby, the macrophagus or the lymphocytes which have the receptors against Fc regions of the antibody are collected on the tumor sites so as to attack the tumor. Alternatively, the target therapy is mentioned, in which the complex comprising the antibody to which the toxic substance (for example, the toxic protein, the radioactive substance (RI) and anticancer agent) is bound is used. In the complex comprising the antibody and the toxic substance, the toxic protein as one of the usable toxic substances includes A chains of plant toxins (type II) such as ricin and abrin and active components of plant toxins (type I) such as luffin, momordin and PAP-S and of bacterial protein toxin such as P~eudomonas toxin and diphtheria toxin, which irreversibly terminates a protein synthesis and thereby a high toxicity is given to the cells. The other preferable toxic protein includes an enzyme derived from human such as human RNase, which is incorporated into the cells and thereby lethal damages can be given to the cells. In the preparation of the immunotoxin complex by binding the antibody with the toxic protein, a crosslinking agent is used [Tanpakushitsu-Kakusan-Koso (PROTEIN NUCLEIC ACID ENZYME), supplement No. 31, p.335-343 (1987)].
For stabilizing the antibody, the addition of a sugar such as lactose, maltose, sorbitose, tolehalose and xylose and/or a sugar alcohol such as mannitol, sorbitol and xylitol is - 8 ~ 2097 932 effective in the present invention. The addition of the protein such as human serum albumin is also effective for stabilizing the antibody. The added amount of the sugar or sugar alcohol is preferably 0.01 to 200 %, preferably 1 to 50 % based on the total weight of the antibody and the human serum albumin.
The milling of the freeze-dried product is conducted using a suitable apparatus such as a jet milling apparatus (for example "Micronizer Mill") and a ball milling apparatus. For the inhalation, the particles have a particle size distribution such that more than 50 % or more, preferably 75 % or more of the total particles have the particle diameter of 0.5 to 5 ~m. The particle size distribution can be determined by a standard particle size distribution analyzer (for example, model CAPA-700 of Horiba Ltd. and QCM-Cascade Impactor model PC-2 of California Measurement Inc.).
It is preferable to add any surfactant to the freeze-dried and milled product for improving the dispersibility of the particles. The surfactant used in the present invention is at least one of sorbitan trioleate (Span 85), oleyl alcohol, soya lecitin and hydrogenated castor oil derivatives (HCO60). The addition amount of the surfactant is 0.001 to 5 %, preferably 0.05 to 2 % based on the total weight of the antibody and the human serum albumin.
Optionally, mineral ions which are necessary to keep an osmotic pressure intrinsically present in an organism, such as . " . ' ,, 20~7932 calcium, magnesium, sodium, potassium, chloride and phosphate ions may be added. If necessary, a surfactant derived from alveoli pulmonum may be added for controlling a stimulating property to the organism.
The thus-prepared antibody containing composition is as such or dispersed in a nebulizing gas such as compressed air and compressed carbon oxide gas or in a suitable propellant in order to inhale through the oral or nasal cavity. The usable .
propellant includes chlorofluorocarbon (furon 11, 12, 114 etc.), hydrochlorofluorocarbon (furon 123, 124, 141b) and fluorocarbon (furon 125, 134a).
Exa~les The following examples will more fully illustrate the present invention, but they are not intended to be limiting of the present invention.
Exam.~?le 1 Powders were prepared by freeze-drying 20 ml of a solution con~aining 0.75 mg/ml of a freeze-dried powder of human immunoglobulin IgG (ex. PAESEL) or human immunoglobulin Fab (ex.
CAPPEL), 15.0 mg/ml of human serum albumin, 2.0 mg/ml of sorbitol, 0.375 mg/ml of phosphate buffer and 1.5 mg/ml of sodium chloride. Then, each of the freeze-dried solid powders was collected followed by milling in Sturtevant jet milling apparatus (500 ml capacity), thereby a milled powder was obtained. The resultant milled powder was analyzed using the :
.~ . :
.
,.
particle size distrlbution analyzer (model CAPA-700 of Horiba Ltd.). The milled powder comprising IgG had the average particle diameter of 5.20 i 1.86 ~m, the proportion of the particles with the particle diameter of 5.45 ~m or less being 55.0 %. While, the milled powder comprising Fab had the average particle diameter of 1.95 i 1.05 ~m, the proportion of the particles with the particle diameter of 4.00 ~m or less being 97.9 %.
Next, 50 to 100 mg of the powder was placed in a glass vial (10 ml capacity), to which oleyl alcohol or sorbitan trioleate (Span 85) was added such that it is contained in an amount of 0.25 % under nitrogen atmosphere. After a metered-dose valve was attached on the glass vial, furon 12 was introduced therein so that the total amount was 10 ml. Thus, four MDI (metered -dose inhalation) preparations a), b), c) and d) were obtained.
A composition of each preparation was shown in Table 1.
'; ~- ~ . -. , ~, .
.. , -Table 1MDI preparations comprising human immunoglobulin IgG and human immunoglobulin Fab' .. ~ ..
com~ o~ition and d~ ~e _ _ MDI preparation compo~ition amount weight do~e . (w/v ~) (~) (Uq/time) a) Fab powder* 0.5 0.050 9.55 Fab oleyl alcohol 0.25 0.025 (oleyl alcohol) CFC_12 99.25 13.27 b~ Fab powder*1.0 0.10 19.1 Fab SPAN 85 O.25 O.025 (SPAN 85) CFC 12 98.75 _ 13.20 c) IgG 1.0 0.10 500 IgG oleyl alcohol 0.25 0.025 (oleyl alcohol) CFC 12 9a.25 13.20 d) IgG 1.0 0.10 500 IgG SPAN 85 O.25 O.025 (SPAN 85) CFC 12 98.25 _13.20 * Fab powder contained the following ingredients per 100 mg:
Fab powder : 3.82 mg HSA : 76.4 mg phosphate : 1.91 mg NaCl : 7.6 mg D-sorbitol : 10.2 mg ' ~ ~
.
Using a metered~dose inhaler, the human immunoglobulin Fab or IgG in a dose shown in Table l was inhaled to rabbits. Three NZW male rabbits of body weight of 3.0 to 3.5 kg were used per each group. Before the administration to the rabbit, the MDI
preparation was forcedly sprayed in a bellows-type spacer five times. Then, an adapter was attached, through which the MDI
preparation was administered to the lung of the rabbit by a spontaneous inhalation for one minute. For each of the MDI
preparations c), d), a) and b), the above procedure was repeated ~wo times, four times, six times and six times, respectively.
Accordingly, the expected final dose of the immunoglobulin was 5 mg, lO mg, 287 ~g and 573 ~g, respectively.
After 0.5, l, 3, 6, 9 and 12 hours from the administration of the MDI preparation, a blood was sampled from an ear auricular vein of each rabbit. A serum in a blood sample was separated so as to prepare a sample for determining a serum concentration of human immunoglobulin IgG or Fab. The determination is conducted according to the EIA method.
Firstly, an immunoplate (ex. NUNC) was coated with lO0 ~l of a lO ~g/ml solution of a sheep anti-human F(ab')2 antibody (ex.
CAPPEL) in PBS(-) at 4C overnight. After washing with 250 ~l of PBS(-), the plate was blocked with 1% BSA/PBS(-) at 4C
overnight followed by washing with each of 250 ~l of a washing solution (0.05% Tween 20/PBS(-)) three times. Then, a human immunoglobulin IgG standard (ex. ZYMED), a human immunoglobulin .
- '. , ~ .:. '' , ~ ' ' ' .: . , ~ :
Fab standard (ex. CAPPEL) and a sample were added and the reaction was continued at room temperature for 1 hour. After washing with each of 250 ~1 of the washing solution three times, 100 ~1 of a horseraddish peroxidase (HRP) standard goat anti-human IgG (H + L) chains antibody (ex. ZYMED) which was previously diluted 12,000 times was reacted at room temperature for 1 hour. Thereafter, the washing was repeated. A substrate for HRP was added in order to develop a color. After the reaction was terminated, an absorption at 490 nm was detected using an immuno reader.
The results showed that in both preparations comprising the human immunoglobulins IgG and Fab, the preparations comprising oleyl alcohol showed higher absorption efficiency, as compared with those comprising SPAN 85. Thus, the change in serum concentration after the inhalation of the preparation (a) or (c) comprising oleyl alcohol was determined. The results are shown in Fig. 1. After 30 minutes from the inhalation, the serum concentration was reached to a maximum value in both preparations a) and c). In the preparation a), it was 1.18 % of the dose. In the preparation c), it was 0.98 % of the dose.
Further, Fab showed the higher serum concentration as compared with IgG.
xample 2 A IgG-Abrin A chain complex as the immunotoxin complex was prepared from human immunoglobulin IgG (ex. PAESEL) and abrin A
.. -' 'i" ~: , - 1~- 2097932 chain using SPDP (N-succinimidyl 3-(2-pyridyl-dithio)propionate) of the crosslinking agent. For the purification of the complex, blue Sepharose column and a gel filtration column were used.
A powder was prepared by freeze-drying 20 ml of a solution containing 0.7 mg/ml of IgG-abrin A chain complex, 15.0 mg/ml of human serum albumin, 2.0 mg/ml of sorbitol, 0.75 mg/ml of phosphate buffer and 1.5 mg/ml of sodium chloride. The freeze-dried powder was milled in Sturtevant jet milling apparatus (500 ml capacity), thereby a milled powder was obtained. The resultant milled powder was analyzed using the particle size distribution analyzer (model CAPA-700 of Horiba Ltd.). The milled powder had the average particle diameter of 4.65 i 1.58 ~m, the proportion of the particles with the particle diameter of 5.00 ~m or less being 63.6 %.
One hundred mg of the powder was placed in a glass vial (10 ml capacity), to which oleyl alcohol was added such that it is contained in an amount of 0.25 % under nitrogen atmosphere.
After a metered-dose valve was attached on the glass vial, furon 12 was introduced therein so that the total amount was 10 ml.
Thus, MDI preparations e) was obtained.
Using the metered-dose inhaler, the complex in a dose of 500 mg/time was inhaled to three NZW male rabbits of body weight of 3.0 to 3.5 kg per each group. Before the administration to the rabbit, the MDI preparation was forcedly sprayed in a bellows-type spacer five times. Then, an adapter was attached, .
.. .
' ' ..
, - 1S- 2~97932 through which the MDI preparation was a~ninistered to the lung of the rabbit by a spontaneous inhalation for one minute. The ,,above procedure was repeated two times. Accordingly, the expected final dose of the complex was 5 mg.
After 0.5, 1, 3, 6, 9 and 12 hours from the administration of the MDI preparation, a blood was sampled from an ear auricular vein of each rabbit. A serum in a blood sample was separated so as to prepare a sample for determining a serum concentration of the complex. The determination is conducted according to the EIA method.
The change in serum concentration after the inhalation is shown in Fig. 2. After 30 minutes from the inhalation, the serum concentration was reached to a maximum value. It was confirmed to be 0.42 % of the dose.
Utiliz,~a,,,bility in Industries Because the antibody containing composition of the present invention is the stable solid composition, it is suitable as the composition for the inhalation of the àntibody or its complex which is pharmaceutically effective.
Claims (2)
1. A composition containing an antibody or its complex, which is obtained by freeze-drying a solution of the antibody or its complex followed by milling the freeze-dried product so that it has an effective particle diameter of more than 0.5 µm and less than 10 µm.
2. The composition as claimed in claim 1, which is a composition for inhalation.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26392691 | 1991-10-11 | ||
| JP263926/1991 | 1991-10-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2097932A1 true CA2097932A1 (en) | 1993-04-12 |
Family
ID=17396185
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2097932 Abandoned CA2097932A1 (en) | 1991-10-11 | 1992-10-09 | Antibody containing composition |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0562125A4 (en) |
| CA (1) | CA2097932A1 (en) |
| WO (1) | WO1993006862A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104127387A (en) * | 2014-02-21 | 2014-11-05 | 杭州长典医药科技有限公司 | Special ultrafine omeprazole sodium powder freeze-dried preparation and preparation method thereof |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2004033499A1 (en) | 2002-10-11 | 2006-02-09 | 中外製薬株式会社 | Cell death inducer |
| TW200530269A (en) | 2003-12-12 | 2005-09-16 | Chugai Pharmaceutical Co Ltd | Anti-Mpl antibodies |
| JP2006063019A (en) * | 2004-08-27 | 2006-03-09 | Daikin Ind Ltd | Harmful substance-catching agent, and spray device and spray method therefor |
| TW200722518A (en) | 2005-03-31 | 2007-06-16 | Chugai Pharmaceutical Co Ltd | Sc(fv)2 structural isomers |
| CA2610987C (en) * | 2005-06-10 | 2013-09-10 | Chugai Seiyaku Kabushiki Kaisha | Stabilizer for protein preparation comprising meglumine and use thereof |
| US9241994B2 (en) | 2005-06-10 | 2016-01-26 | Chugai Seiyaku Kabushiki Kaisha | Pharmaceutical compositions containing sc(Fv)2 |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR7461M (en) * | 1968-06-19 | 1970-01-05 | ||
| JPS5484025A (en) * | 1977-12-16 | 1979-07-04 | Green Cross Corp:The | Immuno-globulin preparation for respiratory tract administration |
| JPS5653622A (en) * | 1979-10-09 | 1981-05-13 | Mochida Pharmaceut Co Ltd | Preventive and remedy for infectious disease of respiratory organ and oral cavity |
| IT1220979B (en) * | 1981-06-04 | 1990-06-21 | Lofarma Farma Lab | CAPSULES CONTAINING AN ALLERGEN AND PROCEDURE FOR THEIR PREPARATION |
| US4676973A (en) * | 1983-04-20 | 1987-06-30 | Best Mark P | Method for diagnosing lung abnormalities using radiolabelled agents |
| US4703002A (en) * | 1985-05-01 | 1987-10-27 | Eastman Kodak Company | Method for preparing coating compositions containing an immunologically reactive species and elements containing same |
| ZA887658B (en) * | 1987-10-15 | 1990-06-27 | Syntex Inc | Intranasal administration of polypeptides in powdered form |
| EP0417193B1 (en) * | 1988-05-27 | 1993-08-04 | Centocor, Inc. | Freeze-dried formulation for antibody products |
| CA2050905A1 (en) * | 1989-02-23 | 1990-08-24 | George R. Felt | Therapeutic aerosol formulations |
| EP0465513A1 (en) * | 1989-03-27 | 1992-01-15 | Centocor, Inc. | FORMULATIONS FOR STABILIZING OF IgM ANTIBODIES |
-
1992
- 1992-10-09 WO PCT/JP1992/001316 patent/WO1993006862A1/en not_active Application Discontinuation
- 1992-10-09 CA CA 2097932 patent/CA2097932A1/en not_active Abandoned
- 1992-10-09 EP EP19930906345 patent/EP0562125A4/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104127387A (en) * | 2014-02-21 | 2014-11-05 | 杭州长典医药科技有限公司 | Special ultrafine omeprazole sodium powder freeze-dried preparation and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0562125A4 (en) | 1994-03-18 |
| WO1993006862A1 (en) | 1993-04-15 |
| EP0562125A1 (en) | 1993-09-29 |
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