WO1995027786A1 - Formulations pharmaceutiques utilisees dans le traitement de la pollinose du cedre du japon - Google Patents

Formulations pharmaceutiques utilisees dans le traitement de la pollinose du cedre du japon Download PDF

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Publication number
WO1995027786A1
WO1995027786A1 PCT/US1995/004249 US9504249W WO9527786A1 WO 1995027786 A1 WO1995027786 A1 WO 1995027786A1 US 9504249 W US9504249 W US 9504249W WO 9527786 A1 WO9527786 A1 WO 9527786A1
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WIPO (PCT)
Prior art keywords
seq
cji
peptide
peptides
gly
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PCT/US1995/004249
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English (en)
Inventor
Henry M. Franzén
Stephen Palmer Powers
Mei-Chang Kuo
Sean Evans
Ze'ev Shaked
Xian Chen
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Immulogic Pharmaceutical Corporation
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Application filed by Immulogic Pharmaceutical Corporation filed Critical Immulogic Pharmaceutical Corporation
Priority to AU22419/95A priority Critical patent/AU2241995A/en
Priority to EP95915576A priority patent/EP0756629A1/fr
Priority to JP52644095A priority patent/JP4179422B2/ja
Publication of WO1995027786A1 publication Critical patent/WO1995027786A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • Japanese cedar (Sugi; Cryptomeria japonic ⁇ ) pollinosis is one of the most important allergic diseases in Japan. The number of patients suffering from this disease is on the increase and in some areas, more than 10% of the population are affected.
  • SBP Sugi basic protein
  • Cryj I has been cloned and the full length nucleic acid sequence and the full length amino acid sequence of the Cryj I protein have been identified (WO 93/01213)
  • the Cryj I allergen found in Cryptomeria japonica has also been found to be cross-reactive with allergens in the pollen from other species of trees, including Cupressus sempervirens.
  • Panzani et al. (Annals of Allergy 57: 26-30 (1986)) reported ⁇ at cross reactivity was detected between allergens in the pollens of Cupressus sempervirens and Cryptomeria japonica in skin testing, RAST and RAST inhibition.
  • a 50 kDa allergen isolated from Mountain Cedar (Juniperus sabinoides, also known as Juniperus ashei) has an NH2-terminal sequence (Gross et al., (1978) Scand. J. Immunol.
  • the Cryj I allergen has also been found to be allergenically cross-reactive with the following species of trees: Cupressus arizonica, Cupressus macrocarpa, Juniperus virginiana, Juniperus communis, Thuya orientalis, and Chamaecyparis obtusa.
  • Desensitization using Japanese cedar pollen extract has drawbacks in that it can elicit anaphylaxis if high doses are used, whereas when low doses are used to avoid anaphylaxis, treatment must be continued for several years to build up a tolerance to the extract.
  • WO 94/01560 discloses improved compositions and methods for treatment of sensitivity to Japanese cedar pollen or to an immunologically cross reactive pollen allergen which greatly minimizes the potential adverse side effects associated with desensitization therapy using Japanese cedar pollen extract.
  • WO 94/01560 discloses isolated antigenic fragments or peptides derived from Cryj I which when administered to a Japanese cedar pollen-sensitive individual, or an individual allergic to an allergen cross-reactive with Japanese cedar pollen allergen, are capable of down regulating the allergic response of the individual to Japanese cedar pollen or such cross reactive allergen. Such down regulation of the allergic response of the individual results in diminution or alleviation of the classic symptoms of allergy including asthmatic symptoms induced by Japanese cedar pollen.
  • Such antigenic fragments are disclosed as being capable of eliciting a T cell response such as stimulation (i.e.
  • WO 94/01560 discloses that the most preferred Cryj I peptides suitable for therapeutic use do not bind IgE specific for Cryj I, or bind IgE to a substantially lesser extent than the native Cry; I allergen, thereby reducing or eliminating the possibility of anaphylaxis in a treatment regimen which includes such peptides.
  • WO 94/01560 discloses peptides which possess the characteristics described above.
  • the present invention provides novel modified Cryj I peptides and novel combinations and formulations of modified Cry j I peptides which are optimal for preparation of a drug product suitable for use in treating Japanese cedar pollen allergy in humans and other mammals.
  • Such Cryj I peptides and formulations thereof for use as an optimized human drug product have not previously been disclosed or contemplated.
  • the present invention provides novel peptides of Cry j I which have been modified as a part of a preformulation scheme to develop an optimized drug product for therapeutic treatment of humans suffering from allergy to Japanese cedar pollen allergen or a pollen allergen which is immunologically cross reactive Japanese cedar pollen allergen.
  • modified peptides possess-certain unique characteristics which render diem particularly suitable for drug product formulation.
  • the present invention further provides therapeutic compositions and multipeptide formulations which have been optimized to accommodate and maintain the unique characteristics of the modified Cryj I peptides and at the same time provide maximum therapeutic effect when used in therapeutic regimens for the treatment of Japanese cedar pollen allergy in humans.
  • Fig. 1 shows the complete cDNA sequence for Cryj I (SEQ. ID. NO: 39) which is composed of 1312 nucleotides, including 66 nucleotides of 5' untranslated sequence, an open reading frame starting with the codon for an initiating methionine of 1122 nucleotides, and a 3' untranslated region.
  • Fig. 1 also shows the deduced amino acid sequence of Cryj I (SEQ. ID. NO: 40);
  • Fig. 2 shows 35 overlapping 20mer peptides which overlap by 10 derived from Cryj I.
  • Fig. 3 shows the amino acid sequences of the novel "unique" peptides of the invention.
  • Fig. 4 is a graphic representation depicting responses of T cell lines from twenty- five patients primed in vitro with purified native Cry; I and analyzed for response to the 35 overlapping 20 mer Cryj I peptides shown in Fig. 2, by percent of responses (positive) with an S.I of at least two (shown over each bar), the mean stimulation index of positive response for the peptide (shown over each bar in parenthesis) and the positivity index (Y axis);
  • Fig. 5 is a graphic representation depicting T cell responses to the overlapping Cry j I peptides shown in Fig. 2, and the novel "unique" peptides of the invention shown in Fig. 3.
  • Fig. 6a is a pH solubility profile of CJI-24.5 (SEQ. ID. NO: 36) (desalted) in 50mM phosphate buffer with 5% mannitol and 22°C, with 95.2% estimated peptide content and 2.7% acetate.
  • Fig. 6b is a pH solubility profile of CJI-43.39 (SEQ. ID. NO: 37) in 50mM phosphate buffer with 5% mannitol and 22°C, with 83.5% estimated peptide content and 1.9% acetate.
  • Fig. 6c is a pH solubility profile of CJI-44.8 (SEQ. ID. NO: 38)in 50mM phosphate buffer with 5% mannitol and 22°C, with 86.% estimated peptide content and 1.5% acetate.
  • the present invention provides novel peptides of Cryj I which have been modified as a part of a preformulation scheme to develop an optimized drug product for therapeutic treatment of humans suffering from allergy to Japanese cedar pollen allergen or an allergen which is immunologically cross reactive with Japanese cedar pollen allergen (e.g. Cupressus sempervirens, Juniperus sabinoides (also known as Juniperus ashei) Cupressus arizonica, Cupressus macrocarpa, Juniperus virginiana, Juniperus communis, Thuya orientalis, and Chamaecyparis obtusa).
  • Such modified peptides possess certain unique characteristics which render diem particularly suitable for drug product formulation, and may be referred to herein as "unique" peptides.
  • preformulation is the process of optimizing a drug through determination and/or definition of those physical and chemical properties considered important in the formulation of a stable, effective, and safe dosage form.
  • the possible interactions with the various components intended for use in the final drug product are also considered.
  • Preformulation is an intensive effort that includes the study of such parameters as solubility, pH profile of stability, and drug-excipient interactions, which may have a profound effect on a drug's physiological availability and physical and chemical stability.
  • the data obtained from such studies are integrated with those obtained from preliminary pharmacological and biochemical studies of the active drug component thus providing information that permits the selection of the best drug form, and the most desirable excipients for use in its development.
  • the active component a peptide or candidate peptide
  • the active component in such formulation should possess the following characteristics which would render such peptides "unique" among all of the possible peptides derived from the Cryj I sequence.
  • a unique peptide should alone or in combination with other unique peptides comprise a sufficient percentage of the T cell reactivity of the Cry j I protein allergen to induce T cell nonresponsiveness or reduced T cell responsiveness in a substantial percentage of the individuals sensitive to Cryj I protein allergen.
  • the candidate peptide should possess the characteristic of "superior solubility" which is defined herein as solubility of greater than 5 mg/ml at a pH in the pH range of pH 6 to pH 8 in an aqueous buffer.
  • the peptide is stable in an aqueous buffer at a pH in the pH range of pH 6 to pH 8.
  • Candidate peptides which have been determined to be "unique" peptides of the invention are CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39 (SEQ. ID. NO: 36), and CJI- 44.8 (SEQ. ID. NO: 38), all as shown in Fig. 3.
  • those peptides found to elicit a T cell response such as T cell proliferation or lymphokine secretion (i.e. comprise at least one T cell epitope), or induce T cell non-responsiveness or reduced T cell responsiveness are understood to have T cell reactivity.
  • T cell epitopes are believed to be involved in initiation and perpetuation of the immune response to a protein allergen which is responsible for the clinical symptoms of allergy. These T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropriate HLA molecule on the surface of an antigen presenting cell and stimulating the relevant T cell subpopulation.
  • T cell proliferation lymphokine secretion, local inflammatory reactions, recruitment of additional immune cells to the site, and activation of the B cell cascade leading to production of antibodies.
  • IgE is fundamentally important to the development of allergic symptoms and its production is influenced early in the cascade of events, at the level of the T helper cell, by the nature of the lymphokines secreted.
  • a T cell epitope is the basic element or smallest unit of recognition by a T cell receptor, where the epitope comprises amino acids essential to receptor recognition.
  • T cell non-responsiveness of appropriate T cell subpopulations such that they become unresponsive or have reduced responsiveness to the protein allergen and do not participate in stimulating an immune response upon such exposure for example, via anergy, tolerance, or apoptosis, the ability to modify the lymphokine secretion profile as compared with exposure to the naturally occurring autoantigen; and /or the ability to cause induction of T suppresser cells.
  • isolated peptides are tested by, for example, T cell biology techniques, to determine whether the peptides elicit a T cell response or induce T cell non-responsiveness.
  • human T cell stimulating activity can be tested by culturing T cells obtained from an individual sensitive to Japanese cedar pollen allergen, (i.e., an individual who has an IgE mediated immune response to Japanese cedar pollen allergen) with a peptide or modified peptide derived from Cryj I and determining whether proliferation of T cells occurs in response to the peptide as measured, e.g., by cellular uptake of tritiated thymidine.
  • Stimulation indices for responses by T cells to peptides can be calculated as the maximum counts per minute (CPM) in response to a peptide divided by the control CPM.
  • a stimulation index (S.I.) equal to or greater than two times the background level is considered "positive". Positive results are used to calculate the mean stimulation index for each peptide for the group of patients tested.
  • Peptides suitable as candidates for formulation into a final drug product have a mean T cell stimulation index of greater than or equal to 2.0 and preferably higher, (e.g. at least 2.5, more preferably at least 3.5, more preferably at least 4.0, more preferably at least 5, even more preferably at least 7 and most preferably at least about 9).
  • candidate peptides are recognized by at least 10%, more preferably at least 20%, more preferably at least 30% and even more preferably at least 40% or more of individuals in a population of individuals sensitive to Japanese cedar pollen.
  • preferred candidate peptides have a positivity index (P.I.) of at least about 100, more preferably at least about 250 and most preferably at least about 350.
  • the positivity index for a peptide is determined by multiplying the mean T cell stimulation index by the percent of individuals, in a population of individuals sensitive to Japanese cedar pollen (e.g., preferably at least 15 individuals, more preferably at least 30 individuals or more), who have a T cell stimulation index to such peptide of at least 2.0.
  • the positivity index represents both the strength of a T cell response to a peptide (S.I.) and the frequency of a T cell response to a peptide in a population of individuals sensitive to Japanese cedar pollen.
  • S.I. the strength of a T cell response to a peptide
  • candidate peptides the frequency of a T cell response to a peptide in a population of individuals sensitive to Japanese cedar pollen.
  • a set of overlapping peptides is produced by systematically dividing Cryj I into at least two overlapping peptide regions of desired lengths (e.g., of about 12-30 amino acid residues in length, preferably not longer than about 25 amino acid residues in length with about 5-15 amino acid residues of overlap). For example, see Fig. 2.
  • This division into peptide regions can be arbitrary, can be made according to an algorithm, or can be wholly or partially based on regions of Cry; I known to comprise at least one T cell epitope.
  • at least 50% of the entire Cryj I protein sequence and more preferably, the entire protein sequence of Cryj I is divided into two or more peptides.
  • a human T cell stimulation index is determined for each of the peptides in an in vitro T cell proliferation assay as described herein for each individual tested in a population of individuals sensitive to the protein antigen. For example, see Fig. 4.
  • a candidate peptide or combination of candidate peptides is selected based, at least in part, on the mean human T cell stimulation index of the candidate peptide in the set of peptides tested and the positivity index of the candidate peptide in the set of peptides tested. For example see, Fig. 5.
  • the human T cell stimulation index for the candidate peptide(s) is summed.
  • die human T cell stimulation index for the candidate peptide(s) is divided by the sum of the human T cells stimulation indices of die remaining peptides in the set of peptides tested to determine a percent of T cell reactivity as shown below:
  • the presence of T cell epitopes in the candidate peptide dependent on amino acids residues in an overlapping peptide located at either d e N-terminus or C-terminus of the candidate peptide in the amino acid sequence of the protein antigen, but which epitopes are not present in the candidate peptide can be considered in calculating the percent of T cell reactivity in the candidate peptide by use of the following formula:
  • Nj flanking peptide refers to a peptide which comprises amino acid residues which overlap with amino acid residues located at d e N- terminus of the candidate peptide in the amino acid sequence of the protein antigen from which the peptide is derived;
  • C flanking peptide refers to a peptide which comprises amino acid residues which overlap with amino acid residues located a the C-terminus of the candidate peptide in the amino acid sequence of the protein antigen from which the peptide is derived.
  • stimulation indices for the candidate peptide the N-terminal flanking peptide and the C-terminal flanking peptide are added and divided by the sum total of the stimulation indices for the entire set of overlapping peptides obtain a percent of T cell reactivity for the candidate peptide. If a combination of two or more candidate peptides is selected each of which contains amino acid residues which overlap, this calculation cannot be used to determine a percent of T cell reactivity for each candidate peptide separately. However, a total percent of T cell reactivity for the combination of candidate peptides can be obtained. In this situation, the stimulation indices for all of the candidate peptides which overlap is included in the calculation.
  • the values obtained for the percentage of T cell reactivity for the candidate peptide or combination of peptides in each individual tested can be expressed as a range of the lower and higher values of the results of the above described calculations.
  • the percent is obtained for at least about twenty (20) and preferably at least about thirty (30) individuals sensitive to the protein antigen and a mean percent is determined.
  • the candidate peptide or combination of candidate peptides has the following criteria: (1) the candidate peptide or combination of candidate peptides has a mean percent of at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40% and more preferably at least about 50-60% or greater; and (2) in the population of individuals tested at least about 60%, preferably at least about 75%, and more preferably at least about 90-100% have positive T call responses (S.I. equal to or greater than 2.0) in response to the candidate peptide or combination of candidate peptides.
  • T call responses S.I. equal to or greater than 2.0
  • a candidate peptide or combination of candidate peptides meeting the above criteria is likely to comprise a sufficient percentage of the T cell reactivity to Cryj I to induce T cell non-responsiveness or reduced T cell responsiveness in a substantial percentage of a population of individuals sensitive to Cryj I.
  • a set of overlapping peptides and candidate peptides, CJI-24.5, CJI-23.39 and CJI-44.8, derived from Cryj I were produced and tested.
  • Secondary T cell cultures determined to be reactive with Cry I protein antigen were derived from 36 Cryj I-allergic subjects and analyzed for reactivity to the overlapping set of peptides in an in vitro T cell proliferation assay as described herein. The results are shown in Fig. 5. The highest stimulation index greater than or equal to 2.0 in response to each peptide was recorded for each subject tested. The data were then analyzed by the equations above. The results and calculations of die percent of T cell reactivity for a single Cry; I-allergic subject are shown below using formulas (1) and (2).
  • CJl-1 (SEQ. ID. NO: 1) CJ1-2 (SEQ. ID. NO: 2) CJ1-3(SEQ. ID. NO: 3) CJ1-4 (SEQ. ID. NO: 4) CJ1-5 (SEQ. ID. NO: 5) CJ1-6 (SEQ. ID. NO: 6) CJ1-7(SEQ. ID. NO: 7) CJ1-8(SEQ. ID. NO: 8) CJl-41(SEQ.ID.NO:41 CJl-ll(SEQ.ID.NO: 11 CJl-12(SEQ.JX>.NO: 12 CJ1-13 (SEQ. ID. NO: 13 CJ1-14 (SEQ. ID. NO: 14 CJ1-15 (SEQ. ID.
  • the combination of the three candidate peptides of the invention are well within the desired range for possessing, in combination, sufficient T cell reactivity of Cryj I, and therefore, meet the first characteristic of a "unique" peptide of the invention.
  • a peptide used in conjunction tiierewith does not bind immunoglobulin E (IgE) or binds IgE to a substantially lesser extent (i.e. at least 100-fold less binding and more preferably, at least 1, 000-fold less binding) than the Cryj I protein allergen from which the peptide is derived binds IgE.
  • Immunoglobulin E is a mediator of anaphylactic reactions which result from the binding and cross-linking of antigen to IgE on mast cells or basophils and the release of mediators (e.g., histamine, serotonin, eosinophil chemotacic factors) in allergic ("atopic") patients.
  • mediators e.g., histamine, serotonin, eosinophil chemotacic factors
  • anaphylaxis in a substantial percentage of a population of individuals sensitive to the allergen being treated could be avoided by the use in immunotherapy of a peptide or peptides which do not bind IgE in a substantial percentage (e.g., at least about 75%) of a population of individuals sensitive to Cryj I, or if the peptide binds IgE, such binding does not result in the release of mediators from mast cells or basophils.
  • the risk of anaphylaxis could be reduced by the use in immunotherapy of a peptide or peptides which have reduced IgE binding. IgE binding may be tested, for example by direct ELISA or capture ELISA.
  • Minimal IgE stimulating activity refers to IgE production that is less than the amount of IgE production and/or IL-4 production stimulated by the native Cry; I protein allergen. If a peptide binds IgE, it is preferable that such binding does not result in the release of mediators (e.g. histamines) from mast cells or basophils.
  • mediators e.g. histamines
  • a histamine release assay can be performed using standard reagents and protocols obtained for example, from Amac, Inc. (Westbrook, ME).
  • a buffered solution of a peptide to be tested is combined with an equal volume of whole heparinized blood from an allergic subject. After mixing and incubation, the cells are pelleted and the supernatants are processed and analyzed using a radio immunoassay to determine the amount of histamine released.
  • candidate peptides of the invention 24.5 (SEQ. ID. NO: 37), 43.39 (SEQ. ID. NO: 36), and 44.8 (SEQ. ID. NO: 38), do not appear to bind IgE .
  • the second characteristic for a unique peptide is that of "superior solubility" which was defined earlier as being solubility of greater than 5 mg/ml at a pH in the pH range of pH 6 to pH 8.
  • Solubility at a pH in a physiologically acceptable pH range e.g. pH 6 to pH 8
  • Administration of a soluble drug product in a physiologically acceptable pH range by intravenous or subcutaneous injection provides 100% bioavailability of the drug component to the physiological system into which the drug is being introduced.
  • a drug product intended for injection be fluid to the extent that easy syringability exists, and the active component be soluble as well if maximum therapeutic effect is to be achieved.
  • Solubility is also useful when formulating compositions to be administered via other modes of administration such as by oral administration (tablet, aerosol, sublingual), or sustained release preparations and formulations.
  • Proteins and peptides may be difficult to formulate into soluble compositions as a peptide may not be soluble in any desirable pH range or may be soluble in only a narrow pH range. It is particularly difficult when multiple peptides are being formulated togetiier into a single multipeptide formulation, as each peptide may be soluble in a pH range which does not overlap with those of the other peptides in die formulation.
  • the unique peptides of the invention are the product of multiple amino acid modifications of the original targeted candidate peptide sequence ("parent") from which the modified unique peptides of the invention were originally derived for die purpose of finding a peptide which fits the characteristic of "superior solubility".
  • parent the amino acid sequence of CJI-44.8 (SEQ. ID. NO: 38) was derived from the protein sequence of I (SEQ. ID. NO: 40) by first identifying those regions of the parent protein (Fig.
  • CJI- 31 SEQ. ID. NO: 31
  • CJI-32 SEQ. ID. NO: 32
  • CJI-44 was synthesized to capture the total T cell reactivity of both peptides.
  • CJI-44 is a peptide 30-mer having the amino acid sequence DEEGAYI ⁇ SSGKYEGGNIYTKKEAEFNVE (SEQ.
  • CJI-31 SEQ. ID. NO: 31
  • CJI- 32 SEQ. ID. NO: 32
  • CJI-44 SEQ. ID. NO: 43
  • CJI-31 SEQ. ID. NO: 31
  • CJI-32 SEQ. ID. NO: 32
  • solubility of CJI-44 SEQ. ID. NO: 43
  • CJI-44.8 was very soluble in the "single pH point protocol” and achieved “superior solubility" in the "pH range protocol procedure" (i.e. wherein solubility is measured as a function of pH in 50 mM sodium phosphate containing 5% mannitol with no agitation after initial mixing).
  • CJl -44.8 (SEQ. ID. NO: 38) was stable and soluble at greater than 5 mg/ml over the pH range pH6-pH8 in an aqueous buffer (see, Example 4 and Fig. 6c).
  • Peptide CJI-44.8 (SEQ. ID. NO: 38) was determined to be a "unique" peptide after confirmation that it also retained a T-cell reactivity similar to its parent peptides, CJI-31 (SEQ. ID. NO: 31), CJI-32 (SEQ. ID. NO: 32)and CJI-44 (SEQ. ID. NO: 43) (see Example 2), and confirmation of its stability as is discussed below. Development of the other "unique" peptides, CJI-24.5 (SEQ. ID. NO: 37) and CJI-43.39 (SEQ. ID. NO: 36), followed a process similar to that described above for CJI-44.8 (SEQ. ID. NO: 38).
  • the third criteria which the unique peptides of this invention must meet is stability at a physiologically acceptable pH in the range of pH 6 to pH 8. It must be stable under the conditions of manufacture and storage, and under conditions of reconstitution if necessary. Stability testing establishes the time period for which the integrity, quality and purity of the drug product is preserved in its finished dosage form. Stability testing may be performed concurrently with solubility studies as discussed in Example 4. Each of the candidate peptides of d e invention remained stable (e.g. no significant degradation) at a physiologically pH in a pH range from pH 6-pH 8, at room temperature for at least 24 hours. Therefore, candidate peptides CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39
  • peptides of this invention possess each of the three required "unique" characteristics outlined above, indicating that this combination of peptides is suitable for formulation as an optimized therapeutic drug product for administration to humans for treatment of allergy to Japanese cedar pollen allergen.
  • Highly purified peptides of this invention may be produced synthetically by chemical synthesis using standard techniques. Various methods of chemically synthesizing peptides are known in the art such as solid phase synthesis which has been fully or semi automated on commercially available peptide synthesizers. Synthetically produced peptides may then be purified to homogeneity (i.e. at least 90%, more preferably at least 95% and even more preferably at least 97% purity), free from all other polypeptides and contaminants using any number of techniques known in the literature for protein purification.
  • homogeneity i.e. at least 90%, more preferably at least 95% and even more preferably at least 97% purity
  • a peptide produced by synthetic chemical means may be purified by preparative reverse phase chromatography.
  • the syn etically produced peptide in "crude” form is dissolved in an appropriate solvent (typically an aqueous buffer) and applied to a separation column (typically a reverse phase silica based media, in addition, polymer or carbon based media may be used).
  • Peptide is eluted from the column by increasing the concentration of an organic component (typically acetonitrile or methanol) in an aqueous buffer (typically TFA, triethylamine phosphate, acetate or similar buffer).
  • an organic component typically acetonitrile or methanol
  • an aqueous buffer typically TFA, triethylamine phosphate, acetate or similar buffer.
  • Fractions of the eluate will be collected and analyzed by appropriate analytical methods (typically reverse phase HPLC or CZE chromatography). Those fractions having the required homogeneity will be pooled.
  • the counter ion present may be changed by additional reverse phase chromatography in the salt of choice or by ion exchange resins.
  • the peptide may then be isolated as its acetate or other appropriate salt.
  • the peptide is then filtered and the water removed (typically by lyophilization) to give a homogenous peptide composition containing at least 90%, more preferably at least 95% and even more preferably at least 97 " • of the required peptide component.
  • purification may be accomplished by affinity chromatography, ion exchange, size exclusion, counter current or normal phase separation systems, or any combination of these methods.
  • Peptide may additionally be concentrated using ultra filtration, rotary evaporation, precipitation, dialysis or other similar techniques.
  • the highly purified homogenous peptide composition is then characterized by any of the following techniques or combinations thereof: a) mass spectroscopy to determine molecular weight to check peptide identity; b) amino acid analysis to check the identity of the peptide via amino acid composition; c) amino acid sequencing (using an automated protein sequencer or manually) to confirm the defined sequence of amino acid residues; d) HPLC (multiple systems if desired) used to check peptide identity and purity (i.e.
  • peptide impurities identifies peptide impurities); e) water content to determine the water concentration of the peptide compositions; f) ion content to determine the presence of salts in the peptide composition; and g) residual organics to check for the presence of residual organic reagents, starting materials, and/or organic contaminants.
  • a peptide of the invention may also be produced by recombinant DNA techniques in a host cell transformed with a nucleic acid sequence coding for such peptide.
  • host cells transformed with nucleic acid encoding the desired peptide are cultured in a medium suitable for the cells and isolated peptides can be purified from cell culture medium, host cells, or both using techniques known in the art for purifying peptides and proteins including ion-exchange chromatography, ultra filtration, electrophoresis or immunopurification with antibodies specific for the desired peptide.
  • Peptides produced recombinantly may be isolated and purified to homogeneity, free of cellular material, other polypeptides or culture medium for use in accordance with the methods described above for synthetically produced peptides.
  • peptides of this invention may also be produced by chemical or enzymatic cleavage of a highly purified full length or native protein of which the sites of chemical digest or enzymatic cleavage have been predetermined and d e resulting digest is reproducible.
  • Peptides having defined amino acid sequences can be highly purified and isolated free of any other poly peptides or contaminants present in the enzymatic or chemical digest by any of the procedures described above for highly purified, and isolated synthetically or recombinantly produced peptides.
  • compositions of the invention may comprise one or more of the unique peptides of the invention which may be administered simultaneously or sequentially as single treatment episode for treatment of allergy to Japanese cedar pollen allergen in a human or other mammal.
  • a treatment regimen may not necessarily be a physical mixture of more than one peptide of die invention, but does comprise a combination of such peptides administered simultaneously or sequentially as a single treatment episode in order to achieve the maximum therapeutic effect the combination of the unique peptides, CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39 (SEQ. ID. NO: 36), and CJT-44.8 (SEQ. ID. NO: 38), provide (e.g. solubility and stability at a pH in an acceptable physiological pH range (preferably pH 7.0 to pH 7.5) as well as covering 36-53% T cell reactivity in 97% of the patients tested).
  • compositions of the invention comprise one or more of peptides CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39 (SEQ. ID. NO: 36), and CJI-44.8 (SEQ. ED. NO: 38) and also comprise one or more pharmaceutically acceptable carriers such as excipients which are compatible with peptide or peptides present in a single composition.
  • the pharmaceutically acceptable carrier When the composition is a multipeptide formulation, the pharmaceutically acceptable carrier must be compatible with all of the peptides in the multipeptide formulation.
  • Preferred excipients include but are not limited to sterile water, sodium phosphate, mannitol, or both sodium phosphate and mannitol or any combination tiiereof.
  • excipients include but are not limited to sorbitol, sucrose, dextrose, lactose dextran and PVP.
  • pharmaceutically acceptable counter ions may be added during the preparation of the multipeptide formulation. Examples of pharmaceutically acceptable counter ions include acetate, HC1, and citrate.
  • a therapeutic composition of the invention should be sterile, stable under conditions of manufacture, storage, distribution and use and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • a preferred means for manufacturing a therapeutic compositions of the invention in order to maintain the integrity of the composition is to prepare the formulation of peptide and pharmaceutically acceptable carrier(s) such that the composition may be in the form of a lyophilized powder which is reconstituted in a pharmaceutically acceptable carrier, such as sterile water, just prior to use.
  • a preferred multipeptide formulation comprises unique Cry; I peptides CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39 (SEQ. ID. NO: 36) and CJI-44.8 (SEQ. ID. NO: 38) and sodium phosphate and mannitol.
  • a suitable counter ion such as acetate may be added during the preparation of the formulation, and the formulation is preferably prepared in the form of a lyophilized powder which is reconstituted in a physiologically acceptable carrier, such as sterile water, prior to use.
  • a physiologically acceptable carrier such as sterile water
  • a suitable multipeptide formulation of the invention is described below.
  • the Cryj I peptides CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39 (SEQ. ID. NO: 36) and CJI-44.8 (SEQ. ID. NO: 38) will preferably be combined during manufacturing with the appropriate counter ion to produce a vial containing a sterile, pyrogen free, lyophilized powder having the following composition:
  • Cryj I peptides CJI-24.5 (SEQ. ID. NO: 37), CJI-43.39 (SEQ. ID. NO: 36) and CJI-44.8 (SEQ. ID. NO: 38)
  • the multipeptide formulation of the invention may also be provided in the form of a kit, including instructions for use.
  • Administration of me therapeutic compositions and multipeptide formulations described above to an individual, preferably in non-immunogenic form, can be carried out using known procedures at dosages and for periods of time effective to cause down regulation of die immune response to Japanese cedar pollen or an allergen immunologically cross reactive with Japanese cedar pollen allergen (i.e., reduce the allergic symptoms caused by Japanese cedar pollen or related allergen including Japanese cedar pollen induced asthma) of the individual.
  • Down regulation of the allergic immune response to Japanese cedar pollen in humans may be determined clinically whenever possible (see e.g., Varney et al, British Medical Journal, 302:265-269 (1990), or may be determined subjectively (i.e. the patient feels as if some or all of the allergic symptoms caused by Japanese cedar pollen have been alleviated).
  • compositions and multipeptide formulations of the invention are particularly suitable for administration by injection (e.g. subcutaneous, or intravenous).
  • optimized compositions and multipeptide formulations of the invention may be administered in any convenient manner wherein solubility of the active drug component is either desirable or acceptable, such as by injection (subcutaneous, intravenous, etc.), oral administration, sublingual, inhalation, transdermal application, rectal administration, or any other route of administration known in the art for administering soluble therapeutic agents.
  • compositions of the invention may be desirable to administer simultaneously or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode.
  • Each of such compositions for administration simultaneously or sequentially as a single treatment episode may comprise only one unique peptide of the invention or may comprise an optimized multipeptide formulation in accordance with the invention.
  • Unit dosage form refers to physically discrete units suited as unitary dosages for human subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the desired pharmaceutical carrier.
  • the specification for the novel unit dosage forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and die particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of human subjects.
  • a composition of the inver n by other than parenteral administration, (i.e. by oral administration) it may oe necessary to coat the composition with, or co-administer the composition with, a material to prevent its inactivation or enhance its absorption and bioavailability.
  • a peptide formulation may be co-administered with enzyme inhibitors or in liposomes.
  • Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) and trasylol.
  • Liposomes include water-in-oil- in-water CGF emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol..
  • the peptide may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • the peptide and other ingredients may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the individual's diet.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, solutions, gels, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 1 % by weight of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between about 5 to 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the active compound may be incorporated into sustained-release or controlled release (steady state or pulsatile release) preparations and formulations.
  • Effective amounts of the optimized drug compositions of the invention will vary according to factors such as the degree of sensitivity of the individual to the antigen, the age, sex, and weight of die individual, and the ability of peptide to cause down regulation of the antigen specific immune response in the individual.
  • Dosage regimen may be adjusted to provide the optimum dierapeutic response. For example, several divided doses may be administered over die course of days, weeks, months or years, or the dose may be proportionally increased or reduced with each subsequent injection as indicated by the exigencies of the dierapeutic situation.
  • subcutaneous injections of therapeutic compositions are given once a week for 3-6 weeks. The dosage may remain constant for each injection or may increase or decrease with each subsequent injection.
  • a booster injection may be administered at intervals of about three months to about one year after initial treatment and may involve only a single injection or may involve another series of injections similar to that of the initial treatment.
  • PBMC Peripheral blood mononuclear cells
  • LSM lymphocyte separation medium
  • T cell Unes were established by stimulation of 2 X 10'*-' PBlVml in bulk cultures of complete medium (RPMI-1640, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, 5x1 O ⁇ M 2-mercaptoethanol, and 10 mM HEPES supplemented with 5% heat inactivated human AB serum) with 20 ⁇ g/ml of partially purified native Cryj I (75% purity containing three bands similar to the three bands in Fig. 2) for 7 days at 37°C in a humidified 5% CO2 incubator to select for Cry; I reactive T cells. This amount of priming antigen was determined to be optimal for the activation of T cells from most cedar pollen allergic patients.
  • Viable cells were purified by LSM centrifugation and cultured in complete medium supplemented with 5 units recombinant human IL-2/ml and 5 units recombinant human _L-4/ml for up to three weeks until the cells no longer responded to lymphokines and were considered "rested”.
  • the abihty of the T cells to proliferate to selected peptides, recombinant Cry j I ( ⁇ Cryj I), purified native Cry; I, or recombinant Amb a l.1 ( ⁇ Amb al.X) or a positive control, phyto- hemaglutinin (PHA) was then assessed.
  • 2 X 10 * re sted cells were restimulated in the presence of 2 X 10 * ⁇ autologous Epstein-Barr virus (EBV)- trarsformed B cells (prepared as described below) (gamma-irradiated with 25,000 RAD5) with 2-50 ⁇ g/ml of selected peptides, Cry; I, purified native Cryj I or xAmb a 1.1 or PHA, in a volume of 200 ⁇ l complete medium in duplicate or triplicate wells in 96-well round bottom plates for 2-4 days. The optimal incubation was found to be 3 days. Each well then received 1 ⁇ Ci tritiated thymidine for 16-20 hours.
  • EBV Epstein-Barr virus
  • the counts incorporated were collected onto glass fiber filter mats and processed for liquid scintillation counting. Data collected (not shown) indicated the effect of varying antigen dose in assays with recombinant Cry; I, purified native Cryj I, and recombinant Amb a l.l and several antigenic peptides syntiiesized as described above. Some peptides were found to be inhibitory at high concentrations in these assays. The titrations were used to optimize the dose of peptides in T cell assays. The maximum response in a titration of each peptide is expressed as the stimulation index (S.I.). The S.I.
  • T cell lines also responded at a significantly lower level to ⁇ Amb a l.l indicating that the Amb a I allergens share a degree of homology with Cry; I and that "shared" T cell epitopes might exist between Cryj I and Amb a I.
  • EBV-transformed cell lines were ⁇ -irradiated with 25,000 Rad and used as antigen presenting cells in secondary proliferation assays and secondary bulk stimulations. These EBV-transformed cell lines were made by incubating 5 X 10 6 PBL with 1 ml of B-59/8 Marmoset cell line (ATCC CRL1612, American Type Culture Collection, Rockville, MD) conditioned medium in the presence of 1 ⁇ g/ml phorbol 12-myristate 13-acetate (PMA) at 37°C for 60 minutes in 12 X 75 mm polypropylene round-bottom Falcon snap cap tubes (Becton Dickinson Labware, Lincoln Park, NJ).
  • B-59/8 Marmoset cell line ATCC CRL1612, American Type Culture Collection, Rockville, MD
  • PMA phorbol 12-myristate 13-acetate
  • the frequency of response at 97% represents reactivity to at least one of the candidate peptides, indicating that this combination of peptides fits the first criteria for "unique" peptides of the invention in that in combination peptides CJI-24.5 (SEQ ID NO: 37), CJI-43.39 (SEQ ID NO: 36), and CJI-44.8 (SEQ ID NO: 38), comprising a sufficient percentage of the total T cell reactivity of Cryj I in a substantial percentage of the population tested.
  • a direct ELISA format was used.
  • ELISA wells were coated with the candidate peptides and then assayed for IgE binding.
  • the binding results were generated using two different pools of Cryj allergic patient plasma.
  • Patient plasma pool A (denoted PHP- A) was formulated by mixing equal volumes of plasma from 22 patients that were all shown to be positive for direct IgE binding to native purified Cry; I by ELISA.
  • the second pool (PHP-D) was formulated by the combination of equal plasma volumes from 8 patients that had IgE binding by direct ELISA to botii native and denatured purified Cry; I.
  • Stock solution B 0.71 g (0.05 mol) of dibasic sodium phosphate was disolved in 100ml WFI. The solution was filtered through a 0.2 micron filter.
  • Dispersion A 3.0 mg of each peptide CJI-24.5 (SEQ. ID. NO: 37), CJI- 44.8 (SEQ. ID. NO: 38)and CJI-43.39 (SEQ. Ii ) . NO: 36) was weighed out separately and placed in separate 1.5 mL eppendorf vials with 600 ⁇ L of stock solution A. The composition was agitated for 5 seconds to mix well.
  • Dispersion B 3.0 mg of each peptide, CJI-24.5 (SEQ. ID. NO: 37), CJI-
  • Dispersions A and B were sonicated for 2 minutes for good homogeneity. A small volume was pipetted from each dispersion into a labeled eppendoff vial according to the following volume ratio:
  • the resultant solutions/suspensions were stored in the dark at 22°C for 24 hours without agitation.
  • 11 micro centrifuge filters were labeled on the retentate cup. The weight of each cup with the cap excluding the filter reservoir was measured. The equilibrated solution/suspension was pipetted from each eppendoff vial into the filer reservoir, and placed into micro centrifuge and spun for 10 minutes. After centrifugation, the filter reservoir was discarded and the weight of each cup/cap with the collected filtrate was recorded. The net weight of the filtrate in each cup was calculated by subtracting the weight of the empty cup/cap from the weight of the cup/cap containing the filtrate. The pH of the filtrates in each cup was measured using a micro combination pH electrode.
  • the electrode tip was rinsed with 1.0 mL of the selected dilution buffer into each cup. The total of diluted solution in each cup/cap was recorded. The dilution factors were calculated by dividing the weight of the diluted solution by the weight of the filtrate.
  • the concentration of peptide in the diluted solution was determined by HPLC analysis.
  • the solubility of peptide at each pH was obtained by multiplying the measured concentration with the dilution factor.
  • the extent of degradation of peptides was estimated by calculating the percent of total degradant peak area over the total peak area.
  • peptides CJI-24.5 SEQ. ID. NO: 37
  • CJI-44.8 SEQ. ID. NO: 38
  • CJI-43.39 SEQ. ID. NO: 36
  • MOLECULE TYPE cDNA to mRNA
  • ORIGINAL SOURCE
  • ORGANISM Crytpomeria japonica
  • AAAAA ATG GAT TCC CCT TGC TTA GTA GCA TTA CTG GTT TTC TCT TTT 107
  • MOLECULE TYPE peptide
  • FRAGMENT TYPE internal
  • SEQUENCE DESCRIPTION SEQ ID NO : 41 :

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Abstract

La présente invention se rapporte à de nouveaux peptides de Cry j I qui ont été modifiés dans le cadre d'un système de préformulation afin de développer un médicament optimisé destiné au traitement des personnes souffrant d'allergie due à l'allergène du pollen du cèdre du Japon ou à un allergène qui présente une réaction croisée immunologique avec l'allergène du cèdre du Japon. Ces peptides modifiés possèdent certaines caractéristiques uniques qui les rendent particulièrement aptes à être utilisées dans la formulation de médicaments. La présente invention se rapporte également à des compositions thérapeutiques et à des formulations multipeptidiques qui ont été optimisées pour adapter et conserver des caractéristiques uniques des peptides modifiés Cry j I, et en même temps, afin d'obtenir un effet thérapeutique maximum lorsqu'ils sont utilisés dans des régimes thérapeutiques s'appliquant au traitement de la pollinose du cèdre du Japon chez l'homme.
PCT/US1995/004249 1994-04-08 1995-04-06 Formulations pharmaceutiques utilisees dans le traitement de la pollinose du cedre du japon WO1995027786A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU22419/95A AU2241995A (en) 1994-04-08 1995-04-06 Pharmaceutical formulations for treating japanese cedar pollen allergy
EP95915576A EP0756629A1 (fr) 1994-04-08 1995-04-06 Formulations pharmaceutiques utilisees dans le traitement de la pollinose du cedre du japon
JP52644095A JP4179422B2 (ja) 1994-04-08 1995-04-06 スギ花粉アレルギーを治療するための医薬組成物

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US22624894A 1994-04-08 1994-04-08
US35022594A 1994-12-06 1994-12-06
US08/350,225 1994-12-06
US08/226,248 1994-12-06

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Cited By (13)

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Publication number Priority date Publication date Assignee Title
WO1998012219A1 (fr) * 1996-09-23 1998-03-26 Astra Aktiebolag Peptides contenant des analogues de methionine, de penicillamine et de cysteine et exerçant un effet immunomodulateur
WO1998012216A1 (fr) * 1996-09-23 1998-03-26 Astra Aktiebolag Peptides contenant methionine et exerçant un effet immunomodulateur
US5760184A (en) * 1995-03-31 1998-06-02 Immulogic, Inc. Hapten-carrier conjugates for use in drug-abuse therapy and methods for preparation of same
JPH1192497A (ja) * 1997-09-17 1999-04-06 Sankyo Co Ltd ペプチド及びその用途
EP0960887A1 (fr) * 1996-06-14 1999-12-01 Meiji Milk Products Company Limited PEPTIDE ANTIGENE Ly
US6054127A (en) * 1995-03-31 2000-04-25 Immulogic Pharmaceutical Corporation Hapten-carrier conjugates for use in drug-abuse therapy and methods for preparation of same
US6232082B1 (en) 1998-12-01 2001-05-15 Nabi Hapten-carrier conjugates for treating and preventing nicotine addiction
JP2006045230A (ja) * 1996-06-14 2006-02-16 Meiji Milk Prod Co Ltd Ｔ細胞エピトープペプチド
JP2007056036A (ja) * 2006-10-23 2007-03-08 Sankyo Co Ltd ペプチド及びその用途
JP2007091745A (ja) * 2006-10-23 2007-04-12 Sankyo Co Ltd ペプチド及びその用途
CN100364611C (zh) * 1996-11-13 2008-01-30 明治乳业株式会社 肽基免疫治疗剂
CN102793919A (zh) * 2011-05-20 2012-11-28 日东电工株式会社 医药组合物及医药组合物的制造方法
WO2016209959A2 (fr) 2015-06-22 2016-12-29 Alk-Abelló A/S Combinaisons de peptides et leur utilisation dans le traitement de l'allergie aux pollen de cèdre

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WO2005027774A2 (fr) * 2003-09-17 2005-03-31 Baby Boost, Inc. Procede et appareil de prevention d'allergies
JP2012240978A (ja) * 2011-05-20 2012-12-10 Nitto Denko Corp 医薬組成物及びゼリー状製剤
JP2012240975A (ja) * 2011-05-20 2012-12-10 Nitto Denko Corp 医薬組成物及びゼリー状製剤
JP6200630B2 (ja) * 2012-02-10 2017-09-20 オーストリッチファーマ株式会社 医薬組成物

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WO1994001560A1 (fr) * 1991-07-12 1994-01-20 Immulogic Pharmaceutical Corporation Proteines et peptides allergenes tires du pollen du cedre du japon

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T.SONE ET AL.: "Cloning and sequencing of a cDNA clone coding for Cry j I, a major allergen of Japanese cedar pollen", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 199, no. 2, 15 March 1994 (1994-03-15), ORLANDO, FL US, pages 619 - 625 *

Cited By (25)

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Publication number Priority date Publication date Assignee Title
US5760184A (en) * 1995-03-31 1998-06-02 Immulogic, Inc. Hapten-carrier conjugates for use in drug-abuse therapy and methods for preparation of same
US6054127A (en) * 1995-03-31 2000-04-25 Immulogic Pharmaceutical Corporation Hapten-carrier conjugates for use in drug-abuse therapy and methods for preparation of same
US7547440B2 (en) 1996-06-14 2009-06-16 Meiji Dairies Corporation T-cell epitope peptides
JP2006045230A (ja) * 1996-06-14 2006-02-16 Meiji Milk Prod Co Ltd Ｔ細胞エピトープペプチド
US7112329B1 (en) 1996-06-14 2006-09-26 Meiji Milk Products Co. Ltd. T cell epitope peptide
CN102286077A (zh) * 1996-06-14 2011-12-21 明治乳业株式会社 T细胞表位肽
EP0960887A4 (fr) * 1996-06-14 2002-09-18 Meiji Milk Prod Co Ltd PEPTIDE ANTIGENE Ly
JP3734040B2 (ja) * 1996-06-14 2006-01-11 明治乳業株式会社 Ｔ細胞エピトープペプチド
US7407657B2 (en) 1996-06-14 2008-08-05 Meiji Dairies Corporation T-cell epitope peptides
EP0960887A1 (fr) * 1996-06-14 1999-12-01 Meiji Milk Products Company Limited PEPTIDE ANTIGENE Ly
WO1998012216A1 (fr) * 1996-09-23 1998-03-26 Astra Aktiebolag Peptides contenant methionine et exerçant un effet immunomodulateur
WO1998012219A1 (fr) * 1996-09-23 1998-03-26 Astra Aktiebolag Peptides contenant des analogues de methionine, de penicillamine et de cysteine et exerçant un effet immunomodulateur
CN100364611C (zh) * 1996-11-13 2008-01-30 明治乳业株式会社 肽基免疫治疗剂
JPH1192497A (ja) * 1997-09-17 1999-04-06 Sankyo Co Ltd ペプチド及びその用途
US6518031B2 (en) 1998-12-01 2003-02-11 Nabi Hapten-carrier conjugates for treating and preventing nicotine addiction
US7247502B2 (en) 1998-12-01 2007-07-24 Nabi Biopharmaceuticals Hapten-carrier conjugates for treating and preventing nicotine addiction
US6773891B2 (en) 1998-12-01 2004-08-10 Nabi Biopharmaceuticals, Inc. Hapten-carrier conjugates for treating and preventing nicotine addiction
US7776620B2 (en) 1998-12-01 2010-08-17 Nabi Biopharmaceuticals Hapten-carrier conjugates for treating and preventing nicotine addiction
US8026109B2 (en) 1998-12-01 2011-09-27 Nabi Biopharmaceuticals, Inc. Hapten-carrier conjugates for treating and preventing nicotine addiction
US6232082B1 (en) 1998-12-01 2001-05-15 Nabi Hapten-carrier conjugates for treating and preventing nicotine addiction
JP2007091745A (ja) * 2006-10-23 2007-04-12 Sankyo Co Ltd ペプチド及びその用途
JP2007056036A (ja) * 2006-10-23 2007-03-08 Sankyo Co Ltd ペプチド及びその用途
CN102793919A (zh) * 2011-05-20 2012-11-28 日东电工株式会社 医药组合物及医药组合物的制造方法
EP2524701A3 (fr) * 2011-05-20 2012-12-19 Nitto Denko Corporation Composition pharmaceutique et son procédé de production
WO2016209959A2 (fr) 2015-06-22 2016-12-29 Alk-Abelló A/S Combinaisons de peptides et leur utilisation dans le traitement de l'allergie aux pollen de cèdre

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CA2187336A1 (fr) 1995-10-19
JPH09511747A (ja) 1997-11-25

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