Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/20—Interleukins [IL]
  • A61K38/2013—IL-2

Definitions

• BACKGROUND OF THE INVENTION Mammals and birds, particularly commercial poultry flocks, are often at risk due to exposure to various viral or fungal infections. Such exposure can arise in the natural environment, particularly the dairy or poultry farm, or from unusual situations such as surgery, acci ⁇ dents or wounds.
• the mammalian body defends against viral and fungal infections by an immunological sequence of events stimulated by the presence of the invasive entity such as virus, bacteria or fungus, the end result of which is the production of T-cells which act in conjunction with acrophages to promote phagocytosis.
• Some mammals are deficient in this immunological response either because of some metabolic malfunction, or due to medical treatment such as chemotherapy or spleen- ectomy.
• Many domestic animals, particularly poultry seem to be constantly susceptible to such infections and do not respond immunologically with sufficient rapidity to prevent the loss of large numbers of them. Turkey flocks are especially sensitive in this regard. This leads to significant increases in the costs of turkey husbandry.
• a material to enhance the immunological response of mammals to infection would be an important addition to the ar amentarium of the physician or veterinarian.
• Such a substance could be used prophylactically with subjects constantly exposed to infection, defensively with subjects about to undergo surgery, or supportively to improve the immunological response of subjects already suffering from an active infection or recovering from surgery or other trauma.
• This factor has a number of distinguishing character ⁇ istics by which it can be identified and distinguished from other stimulatory factors which have been described, including some which have been found in thymus tissue. Among these characteristics are the following:
• Its isoelectric point is from about 7.0 to 7.8.
• the immunostimulatory factor of this invention is useful to stimulate the resistance of the mammalian or avian body to infection either as a preventative measure or as a curative measure.
• Fig. 1 is a Sephadex G-25 column profile of a calf thymus extract acetone precipitate. Fractions were screened for absorbance at 280 nm ( ) and for activity in a murine MLC ( ). The marker A indicates the elution
• OMPI position of bovine serum albumin B, bacitracin; and C, sodium chloride.
• Fig. 2 is a Sephadex G-50 column profile of a G-25 stimulator pool.
• the Sephadex G-50 column was equilibra ⁇ ted with phosphate buffered saline (PBS).
• PBS phosphate buffered saline
• a 5.0 mg sample of the G-25 stimulator pool was applied in 1 ml of PBS.
• the markers are A, bovine serum albumin; B, cytochrome C; and C, bacitracin. Fractions were screened for absorbance at 280 nm ( ) and for activity in MLC ( ).
• Fig. 3 is a BioGel P-10 column profile of a " G-25 stim ⁇ ulator pool. The BioGel P-10 column was equilibrated with 0.5 M ammonium bicarbonate, pH 8.0.
• a 2.7 mg sample of G-25 stimulator pool was applied in 1 ml of elution buf ⁇ fer.
• the markers are A, bovine serum albumin and B, bacitracin. Fractions were screened for absorbance at 280 nm ( ) and for activity in MLC ( ).
• Fig. 4 shows the effect of the passage of a calf thymus extract-acetone precipitate fraction through anion (Ag-1) or cation (AG-50) exchange columns on the ability to inhibit the MLC.
• AG-1 (o—o) shows the effect ob ⁇ served with the fraction not retained by the AG-1 column;
• AG-50 (•—•) shows the inhibition observed with the frac ⁇ tion not retained by the AG-50 column.
• Control ( — ) shows the inhibition observed with the untreated sample.
• Fig. 5 illustrates ion exchange chromatography of the G-25 stimulator pool.
• Fig. 6 shows the effect of 2-mercaptoethanol concen ⁇ tration on the activity of the G-25 stimulator pool.
• a murine MLC was set up at various concentrations of 2-mer- captoethanol and a constant amount of G-25 stimulator pool, 20 ⁇ g/ml.
• the incorporation of ⁇ H-thymidine into control cultures (o—o) and G-25 stimulator containing cultures (•—•) was determined.
• Fig. 7 shows the column profile of the G-25 stimulator pool on BioGel P-2 column.
• a 10 mg sample of the active pool was applied to a 1 x 45 cm column of BioGel P-2 equi ⁇ librated in 0.05 M NH4OH, pH 10.2 and eluted with the same reagent. Fractions were- screened for absorbance at
• the markers are A, bovine serum albumin and B, sodium chloride.
• the immunostimulatory factor of this invention has been isolated from bovine thymus tissue, specifically frozen calf thymus or dried, defatted calf thymus.
• bovine thymus tissue specifically frozen calf thymus or dried, defatted calf thymus.
• the latter material is available from Viobin Corporation, -Monticello, Illinois.
• thymus tissue from either source is cut into small pieces, e.g. 1 cm 2 and homogenized into 3 to 4 volumes of ice cold buffer at about 0° to 5°C at pH 7 to 9, for example, 50 mM ammonium carbonate, pH 8.5 in a Pol tron homogenizer for two minutes at low speed and two minutes at high speed.
• the homogenate is centrifuged at 26,000 to 30,000 x G for 20 to 40 minutes at about 0°C to 5°C, preferably 28,000 x G for 30 minutes, and the resulting supernatant solution re ⁇ moved.
• the pellet is re-extracted as above and the pooled supernatants lyophilized.
• the lyophilized powder is suspended in water at about 0 ⁇ C to 5°C at a concentration of 0.2 to 0.3 gm/ml and absolute ethanol is slowly added at the same temperature while stirring to a final concen ⁇ tration of 50% to 60% (V/V) .
• the solution is stirred for 16 to 18 hours at 0° to 5°C followed by centrifugation at the same rates and times mentioned above.
• the resulting clear supernatant is slowly added to 5 to 6 volumes of acetone precooled to about -5° to -10°C and maintained at -5°C to -15°C for 3 to 6 days.
• the precipitate is iso ⁇ lated, e.g.
• the material isolated by this procedure is normally a light tan sticky powder which contains the stimulatory factor of this invention as well as a number of inhibitory factors and other materials.
• the desired factor can be isolated from this fraction by gel filtration or by ion exchange chromatography. For convenience, it is referred to herein as the CTE-acetone precipitate.
• CTE-acetone precipi ⁇ tate is passed over Sephadex G-25 in a 90 x 2.2 cm column equilibrated with an acidic lyophilizable buffer at a pH of from 4 to 5 such as 0.1M acetic acid, 100 mM pyridine acetate, pH 4.5 or 0.1M ammonium acetate, pH 5.0.
• an acidic lyophilizable buffer at a pH of from 4 to 5 such as 0.1M acetic acid, 100 mM pyridine acetate, pH 4.5 or 0.1M ammonium acetate, pH 5.0.
• Two milliliter fractions are collected and assayed by MLC. The results with the pyridine acetate are shown in Fig. 1.
• the pooled stim ⁇ ulatory fractions from a G-25 column are passed over a selected BioGel column of the same dimensions at pH 10.2 in 0.05M NH4OH and eluted with the same reagents.
• the Sephadex products are a well known series of cross linked dextrans available from Pharmacia Fine Chemicals, Inc. of Piscataway, N.J. They are widely em ⁇ ployed for the separation of natural materials.
• the selected Sephadex is packed in a column, a solution containing the dissolved material to be ana ⁇ lyzed is passed over the column and eluted with a selected solvent or series of solvents.
• Each specific Sephadex will separate materials of a specific molecular weight range. Dissolved materials above that molecular weight range will pass through the column in solution in the solvent front the so called void volume. Materials which are retained will be successively eluted as more and more solvent is passed through the column. They will elute in order of progressively decreasing molecular weights.
• Sephadex G10 will separate materials with molecular weights of from 200 to 700 Daltons.
• Sephadex G-25 will separate materials with molecular weights of from 5000 to 1000 Daltons.
• Sephadex G-50 will separate materials with molecular weights of from 30,000 to 1,500 Daltons.
• BioGel P is a series of porous polyacrylami ' de beads used for high resolution gel filtration in a manner simi ⁇ lar to Sephadex Gels. Available from Bio-Rad Laboratories, Richmond, CA.
• BioGel P-10 will separate 20,000 to 1,500 Daltons. BioGel P-4 will separate 4,000 to 800 Daltons. BioGel P-2 will separate 1,800 to 100 Daltons.
• Small scale ion exchange columns are prepared using Bio-Rad, AG-50 WX8 and AG-1-X8 resins, ammonia and acetate forms respectively. The resins are washed extensively and equilibrated with 10 mM ammonium acetate, pH 7.0. Small columns of approximately 8 ml volume are prepared in 10 ml plastic syringes fitted with Luer-lok valves. For analy- sis of the ionic nature of the stimulatory factor, 150 mg of the acetone precipitated product described above is dissolved in distilled water such that the conductivity is less than the 10 mM ammonium acetate, and the pH adjust ⁇ ed to 7 to 7.5.
• ion exchange chromatography is performed on a 1 x 30 cm column of AG 50 WX8 resin, ammonia form, equilibrated with distilled water. Pooled samples of stim ⁇ ulatory composition from a G-25 column are dissolved in distilled water and adjusted to low conductivity and a pH of 5 to 6 with 0.01 acetate buffer. The column is eluted with a gradient made by mixing 90 ml each of distilled water and 1 M ammonium hydroxide. Fractions of approxi ⁇ mately 2 ml are collected, lyophilized, and assayed for activity by MLC. DIALYSIS Dialysis is performed using Spectrapor-3 dialysis tubing (Spectrum Medical Industries, Inc.
• the spleen is sterilly removed from C57/BL and Balb/C mice and placed in a Petri dish containing cell collection media (Earl's Balanced Salt solution containing 2.5% heat- inactivated fetal calf or human serum, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and 100 IU/ml heparin).
• the spleen is perfused briefly using a 22- or 25-gauge needle to re ⁇ move red blood cells, placed in a second dish, and teased gently to remove the spleen lymphocytes.
• the large pieces are removed through a 50-mesh screen and the lymphocytes are recovered by centrifugation at 200 G for 10 minutes.
• the cells are washed once with cell collecting media and resuspended in complete assay media (Minimum Essential Media, Gibco), 5% or 10% heat inactivated human or fetal calf serum, and 5 x 10 ⁇ 4 M 2-mercaptoethanol.
• Human peri ⁇ pheral blood lymphocytes are obtained from blood donated by healthy laboratory personnel. Blood is defibrinated by shaking with glass beads and lymphocytes obtained by centrifugation through a Ficole-Hypaque cushion. The lym ⁇ phocytes collect at the interphase, are washed by centri ⁇ fugation with Earl's Balanced Salt Solution (Gibco) and suspended in complete assay media without added 2-mercapto- ethanol.
• Gibco Earl's Balanced Salt Solution
• Two-way MLC are set up by adding 1.25 x 10 5 cells/well from each strain or donor to microwell plates (Falcon No. 3040 or Linbro No. IS-FB-96) . The plates are incubated at 37°C for four days. Tritiated thymidine (0.5 ⁇ Ci, 2Ci/nmole) is adddd for the last six hours of the culture period. At the end of the culture period, the extent of blast cell formation is obtained by microscopic examination of the plates. The cells are harvested using a Satron cell har ⁇ vester, and the amount of radioactive label incorporated determined.
• spleen cell suspensions are prepared as above and 250,000 cells of one strain are placed in each well. Experimentally determined optimum amounts of mitogens are added and the incubations continued for 72 hours " .
• the lectins used are Con A (Difco, 0.1 ⁇ g/well), Phytohema- glutinin-M (PHA-M, Difco, 20 ⁇ g/well) and lipopolysacchar- ide from Salmonella typhosa (LPS, Sigma, 1.5 ⁇ g/well). The extent of blast cell formation and label incorporation are measured by the same procedures as described for MLC. SKIN GRAFTS
• the biological activities present in the CTE-acetone precipitate were resolved by gel filtration on columns of Sephadex G-25.
• the entire effluent of the column was screened by determining the effect of the various frac ⁇ tions on murine MLC in human serum.
• the use of human serum to culture the lymphoid cells was observed to be essential to avoid the strong inhibitory effects of poly- amines present in the tissue extracts.
• Fig. 1 The results of the screening with pyridine acetate buffer at pH 4.5 are shown in Fig. 1 from which it will be seen that the inhibitory factor is eluted just after the bacitracin marker at a K-&y_ of 0.76.
• the stimulatory factor elutes immediately behind the salt at a ⁇ VE of 1-33 near a large 280 nm absorbing peak.
• the yield of stimulatory material obtained by this proceed- ure was about 1.5 mg for each 1000 mg of CTE-acetone preci ⁇ pitate applied to the column.
• the stimulatory factor From the elution position of the stimulatory factor it is clear that it must interact or adsorb strongly to Sephadex G-25 under the elution conditions as described above. As shown in Fig. 2, the stimulatory factor is also strongly bound to Sephadex G-50 equilibrated with PBS at pH 7.4. It was observed in this study that the stimulatory factor eluted well beyond the molecular weight appropriate to other thymus factors which have been reported and to interleukin-2. The factor is also distinguished from these factors since they have not been reported to adsorb to Sephadex under these conditions. These reports appear in: Goldstein, G. : Nature 247: 11-14 (1974).
• this stimulatory factor dis ⁇ plays a property of chromatographic behavior on Sephadex gels which is distinctly different from previously described thymic or lymphokine factors.
• a number of peptides possess the property of cheraab- sorbance on Sephadex gels possess the property of cheraab- sorbance on Sephadex gels.
• One of the techniques for minimizing this is to use a polyacrylamide gel matrix such as BioGel at an alkaline pH as described by Chin and Wold: Anal. Biochem. 16 : 585 (1972).
• a sample of G-25 absorbed material was subjected to chromatography on BioGel P-10 run in 0.5M ammonium bicarbonate, pH 8.0. The elution pattern is shown in Fig. 3.
• the 280 nm absorbing material was held back under these conditions, similar to the re ⁇ sults with the Sephadex G-25 and Sephadex G-50 columns.
• the stimulatory activity was also held back, indicating the strongly absorbing properties of the stimulatory fac ⁇ tor.
• the stimula ⁇ tory factor of this invention strongly adheres to any Sephadex at pH 4.5 to 8.0 and to any BioGel at pH 8.0.
• the technique of Chin and Wold was employed under strongly basic conditions, the stimulatory factor eluted before the salt on a BioGel P-2 column as shown in Fig. 7.
• 10 mg of G-25 stimulatory pool was passed over a 1 x 45 cm column of BioGel P-2 which had been equilibrated with 0.05 M NH4OH, pH 10.2.
• the fractions were eluted with the same reagent.
• One re ⁇ sult of this treatment is that the factor is split into two fractions.
• the pattern of Fig. 7 suggests that one of these fractions has a molecular weight of about 1400 Dal ⁇ tons and the other a molecular weight of less than 1400
• OMPI Daltons but more than 100 Daltons.
• the significance of the appearance of the two fractions under these strongly basic conditions is not completely understood. It may be that the factor of the invention actually contains two fractions, or that it is split into two fractions by the strong alkaline treatment.
• the charge nature of the stimulatory activity has been investigated in several experiments.
• the CTE-acetone precipitated material was subjected to analysis on small columns of ion exchange resins.
• the CTE-acetone precipitated fraction was applied to small columns of either an anion (AG-1) or a cation (AG-50) exchange resin at pH 7.
• the fraction not retained by the column was examined for its effects on the MLC.
• the fraction not retained by the AG-1 resin signi icantly stimulated the 3 H-thymidine incorpora ⁇ tion in the MLC. Microscopic examination revealed obvious increases in the number of blast cells at the end of the culture period compared to the control sample.
• the frac ⁇ tion not retained by the AG-50 column showed an almost threefold increase in total inhibitory activity. This was apparently the result of the removal of stimulatory activity present in the crude CTE-acetone precipitate fraction. This also confirms the results with the G-25 columns which indicated the presence of both stimulatory and inhibitory activity in the extracts.
• the G-25 stimulatory pool was also subjected to treatment on the ion exchange resin, AG-50, equilibrated with distilled water. The elution profile is shown in Fig. 5. The stimulatory activity was clearly separated from the bulk of the 280 nm absorbing material which was not retarded by the column under these conditions. These results indicate that the stimulatory factor does not bind to an anion exchange resin and is held slightly back by a cation exchange resin.
• the stimulatory factor has a neutral or slightly basic isoelectric point, i.e. about 7.0 to 7.8.
• the G-25 stimulatory pool increases the DNA-synthe- tic response of both murine and human lymphocytes to allo- antigens in the two-way MLC.
• the stimulation can be significant, up to five-fold, depending on the culture con ⁇ ditions.
• the average 2-fold stimulatory dose at the G-25 stage is 10 to 20 ⁇ g/ml for a murine MLC in 10% human serum.
• Table 1 the response of murine and human lymphocytes in MLC and of murine lymphocytes ⁇ Q a
• the G-25 stimulatory pool also has an effect on the proliferation of the murine elukemia, L1210, as shown also in Table 1. This indicates that the factor can have a direct mitogenic effect on continuously dividing cells.
• the G-25 stimulator pool seemed to work less well in two-way MLCs in medium contain ⁇ ing 10% fetal calf serum than in medium containing 10% human serum.
• a two-way MLC of murine lymphocytes was conducted in 5% fetal calf serum, under which conditions the total thymidine uptake is about 60% of that which can be demonstrated at 10% fetal calf serum, the stimulator could now increase the total thymidine uptake of these cultures to that observed at 10% serum.
• the G-25 stimulator pool at a concentration of 40 ⁇ g/ml could increase the response to that observed in 20% FCS. In this manner, the stimulator can partially replace the serum re ⁇ quirement for the MLC.
• mouse serum will inhibit the efficacy of T-cell growth factor, Interleukin-2, Hardt et al: 4_2: 363 (1982).
• Various concentrations of pooled mouse serum along with various concentrations of the G-25 stimulator pool have been added to two-way murine mixed lymphocyte culture in 5% human serum.
• mouse serum at any concentration had an inhibitory effect against the stimulatory ability of any concentra ⁇ tion of the stimulatory factor. This serves further to distinguish between the thymic stimulator of this inven ⁇ tion and Interleukin-2.
• An important aspect of the immune stimulatory factor is its activity in vivo.
• mice The ability of the G-25 stimula ⁇ tor pool to accelerate the rejection of histoincompatible skin grafts in mice has been examined. The results are shown in Table 5. At two different doses, the partially purified stimulator significantly accelerated the rejec ⁇ tion of the grafts. In the second experiment, the treated mice rejected the grafts at a rate comparable with pre- immunized controls. At the doses used, there were no apparent toxic effects.
• the reversibility was determined by incubating 1 x 10 ⁇ spleen cells of each type in 1.6 ml standard culture media plus or minus the indicated concentration of G-25 stimu ⁇ lator pool in 3 ml plastic culture tubes. After 24 hr., all the tubes were centrifuged at 200 x g for 10 min. to pellet the cells and the media was aspirated from one set of tubes and replaced with an equal volume of prewarmed media without stimulator. These tubes were centrifuged and the media replaced again. After this procedure, the tubes were returned to the incubator for an additional 72 hr. At the end of the incubation, the cells were re ⁇ suspended and 0.2 ml aliquots placed in microtiter wells for microscopic examination, labeling, and harvesting by the usual procedure.
• the reversibility was determined as described under Table 2 except the cells were washed at the indicated times.
• Standard murine MLC assays were set up and the indicated amount of G-25 stimulator pool was added at the indicated time after initiation of the culture. The cells were labeled after the last addition.
• Skin grafts were performed as described herein. Treated mice received the indicated amounts of the G-25 stimulator pool. Significance was determined using the Students T-test.
• the G-25 stimulator pool had no apparent effect on murine B-lymphocyte response to the polyclonal mitogen LPS.
• the reversibility experiments described above show that the factor acts very late in the MLC, after or during the formation of morphologically recognizable lymphoblasts in the cultures. The stimulatory effects are totally re ⁇ versible if the factor is removed before the expiration of 72 hr. of the MLC, but not after 96 hr.
• the factor will stimulate the MLC if it is added as late as 72 hr. after start up. The stimulation achieved is equal to the level of stimulation obtained if the factor is continuously present.
• the most important biological activity of the factor is the ability to stimulate an ⁇ n vivo immune response.
• the treated mice rejected allogenic grafts at a rate comparable to that of pre-im- munized animals. This is probably the maximal rate ob ⁇ tainable in this type of strongly histoincompatible system. It should be noted that these results were obtained in fully immunocompetent, adult mice, responding to a strong antigenic stimulus.
• MLC 5 stimulates no effect inhibits/ stimulates in stimulates
• the immunostimulatory factor of this invention can be obtained in highly purified form in accordance with the procedures described herein. However, it is not essential that there be complete purity to achieve the desired ther ⁇ apeutic effect.
• Compositions containing the factor can be isolated and used alone or in various pharmaceutical com ⁇ positions to achieve a therapeutic level of the factor In mammalian or avian blood or tissues which is effective to generate an immunostimulatory response.
• Several different commercially available factors with immunostimulatory activity were tested in the MLC system. The results of the studies are shown in Table 7. The dosages were selected so as to cover the range which ap ⁇ peared to be optimal based on study of the publications shown in the table and other standard considerations.
• % increase 100 x (Experimental cpm-saline co ntrol cpm)/saline control cpm.
• the % synergy is defined as 100 x (cpm with G-25 + IL-2 - cpm with G-25 alone)/saline control cpm.
• compositions containing both the immunostimulatory factor of this invention and IL-2 are valuable for the treatment of patients in need of immuno- stimulation.
• Such compositions will typically contain from 10% to 90% by weight of IL-2 based on the total weight of the immunostimulatory factors.
• the composition may contain any of the usual pharmaceutical excipients.
• the products of this invention that is the factor in highly purified form, salts of the factor or isolated fractions containing the factor, are useful mammalian and avian therapeutic agents and are effective for stimulat ⁇ ing an immune response with patients in need of such treat ⁇ ment.
• the immunostimulatory factor of this invention is useful for the treatment of any avian or mammalian disease associated with depression of cell mediated immunity. Severe viral, bacterial and fungal infections are often associated with such depression. The factor can be used to enhance the effects of standard therapy normally employed for such infections.
• the factor is especially valuable to assist in stim ⁇ ulating the immune response of patients undergoing radia ⁇ tion or chemotherapy for the treatment of malignant diseases.
• the dosage will be in the range of from 0.1 to 0.5 mg/Kg of body weight.
• dosage unit forms containing from 0.05 to 0.25 mg of active product can be prepared.
• Dosage unit forms may, as indi ⁇ cated above, additionally contain from 0.005 to 0.225 mg of IL-2.
• the products of this invention may be administered alone but will generally be administered with pharmaceu ⁇ tically acceptable, non-toxic carriers, the proportions of which are determined by the suitability and chemical nature of the particular carrier, the chosen route of ad ⁇ ministration, and standard pharmaceutical practice.
• pharmaceu ⁇ tically acceptable, non-toxic carriers the proportions of which are determined by the suitability and chemical nature of the particular carrier, the chosen route of ad ⁇ ministration, and standard pharmaceutical practice.
• intravenous or intramuscular administration may be used.
• the compositions may be in the form of sterile solu ⁇ tions containing other solutes, for example, enough saline or glucose to make them isotonic. Those skilled in the art will know of many standard procedures for preparing such compositions.
• An advantage arising from the amphoteric nature of the factor of this invention is that it can be utilized in the form of pharmacologically acceptable salts which may be either metallic salts or acid addition salts. These salts have the advantage of water solubility and are particular ⁇ ly useful for parenteral administration.
• the metallic salts, especially. the alkali metal salts are relatively stable and for that reason are preferred over acid addition salts.
• the sodium salts are especially preferred because of their ease of preparation.
• the acids which may be used to prepare the pharma ⁇ cologically acceptable acid addition salts of this inven ⁇ tion are those containing non-toxic anions and include, for example, hydrochloric, sulfuric, phosphoric, acetic, lactic, citric, tartaric, oxalic, succinic, maleic, gluconic and saccharic acids.

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• 1983
  • 1983-09-16 WO PCT/US1983/001439 patent/WO1984001090A1/en not_active Ceased
  • 1983-09-16 EP EP19830903269 patent/EP0122926A4/en not_active Withdrawn
  • 1983-09-16 AU AU20798/83A patent/AU2079883A/en not_active Abandoned
  • 1983-09-16 JP JP58503297A patent/JPS59501786A/ja active Pending
  • 1983-09-16 FI FI842032A patent/FI842032A7/fi not_active Application Discontinuation
• 1984
  • 1984-05-21 NO NO842015A patent/NO842015L/no unknown
  • 1984-05-21 DK DK2494/84A patent/DK249484D0/da not_active Application Discontinuation

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