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
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5406—IL-4
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• IL-4 interleukin-4
• IL-4 interleukin-4
• IL-4 is a lymphokine (stimulator of the immune system) that has a broad range of immune cell stimulation as described in Banchereau et al., Lymphokine Res. Vol. 6, No. 1 : U135 (1987); Yokoto et al., Proc. Natl. Acad. Sci. USA, Q2: 5894-5898 (1986); Lee at al., Proc. Natl. Acad. Sci. USA, 2 : 2061-2065 (1986); Coffman et al., J. Immunol.
• IL-4 has also been referred to as B-cell growth factor (BCGF) [Butler et al., J. Immunol.122.: 251-255 (1984)(human BCGF); and Farrar et al., J.
• BCGF B-cell growth factor
• the method of this invention involves administering to mammals a serum cholesterol lowering effective amount of IL-4.
• the IL-4 is administered to mammals diagnosed as having elevated blood serum cholesterol levels (hypercholesterolemia).
• the invention also relates to the use of IL-4 for the manufacture of a medicament for lowering blood serum cholesterol levels.
• the IL-4 employed is derived from a human source.
• the dosage form is one suitable for administration by intravenous injection or intravenous infusion and administration will suitably be in an amount of about 0.5 to about 600 micrograms of IL-4 per kilogram of body weight per day.
• the IL-4 is administered in an amount of about 0.5 to about 125 micrograms of IL-4 per kilogram of body weight per day, and most preferably about 0.5 to about 30 micrograms of IL-4 per kilogram of body weight per day.
• the invention provides a method for lowering blood cholesterol levels in mammals, e.g., mammals with hypercholesterolemia, by administering to said mammals a serum cholesterol lowering effective amount of IL-4.
• the invention also provides for the use of IL-4 for the manufacture of a medicament for lowering blood serum cholesterol levels.
• IL-4 any suitable IL-4 may be employed in the present invention.
• Complementary DNAs (cDNAs) for IL-4 have recently been cloned and sequenced by a number of laboratories, e.g. Yokoto et al., Proc. Natl. Acad. Sci. USA, 22: 5894-5898 (1986) (human); Lee at al., Proc. Natl. Acad. Sci. USA, 22: 2061-2065 (1986)(mouse); Noma et al., Nature 319: 640-646 (1986)(mouse); and Genzyme Corporation, Boston, Massachusetts (human and mouse).
• non-recombinant IL-4 has been purified from various culture supernatants, e.g.
• the IL-4 used in the present invention will be a human IL-4, and most preferably it will be the human version with the sequence described in Yokoto et al., Proc. Natl. Acad. Sci. USA, 22: 5894-5898 (1986) and PCT Patent Application No. 87/02990 published May 21 , 1987 that is expressed in and isolated from £,. coji (U.S Patent Application No. 079,666, filed July 29, 1987 and U.S. Patent Application No. 194,799, filed July 12, 1988).
• the disclosures of the above articles, PCT Application and U.S. Patent Application are hereby incorporated herein by reference.
• mammals are administered a serum cholesterol lowering effective amount of an IL-4.
• a serum cholesterol lowering effective amount is defined as any amount that will significantly lower the cholesterol level, with a lowering of cholesterol by at least 5 percent considered significant.
• IL-4 preferably human IL-4 (hlL-4)
• per kilogram of body weight per day is preferably administered. More preferably, mammals are administered about 0.5 to about 125 micrograms of hlL-4 per kilogram of body weight per day, and most preferably mammals are administered about 0.5 to about 30 micrograms of hlL-4 per kilogram of body weight per day.
• the amount, frequency and period of administration will vary depending upon factors such as the cholesterol level (e.g., the severity of the cholesterol elevation), age of the patient, nutrition, etc. Usually, the administration will be daily initially and it may continue periodically during the patient's lifetime. Dosage amount and frequency may be determined during initial screenings of cholesterol levels and the magnitude of the effect of IL-4 upon the lowering of the cholesterol levels. Dosage will be aimed to lower the cholesterol level to an acceptable level of about 240 milligrams of cholesterol per deciliter of blood serum, preferably about 200 milligrams of cholesterol per deciliter of blood serum.
• GM-CSF granuiocyte-macrophage colony stimulating factor
• IL-4 can be administered in conjunction with thrombolytic agents [e.g., tissue plasminogen activators (tPAs) (for example, those disclosed in U.S. Patent Nos. 4,370,417, 4,752,603; U.K. Patent No. 2,119,804; PCT Patent Application Nos. 87/05934, 87/04722, 84/01786; Australian
• tissue plasminogen activators e.g., tissue plasminogen activators (tPAs) (for example, those disclosed in U.S. Patent Nos. 4,370,417, 4,752,603; U.K. Patent No. 2,119,804; PCT Patent Application Nos. 87/05934, 87/04722, 84/01786; Australian
• tPAs tissue plasminogen activators
• Administration of the dose can be intravenous, nasal, parenteral, oral, subcutaneous, intramuscular, topical, transdermal or any other acceptable method.
• the IL-4 could be administered in any number of conventional dosage forms.
• Parenteral preparations include sterile solutions or suspensions.
• Inhalation administration can be in the form of a nasal or oral spray, or by insuffulation.
• Topical dosage forms can be creams, ointments, lotions, transdermal devices (e.g., of the conventional resevoir or matrix patch type) and the like.
• formulations and pharmaceutical compositions contemplated by the above dosage forms can be prepared with conventional pharmaceutically acceptable excipients and additives, using conventional techniques.
• the IL-4 is administered via the intravenous route.
• the solutions to be administered may be reconstituted lypholized powders and they may additionally contain preservatives, buffers, dispersants, etc.
• IL-4 is reconstituted with 10 millimolar citrate buffer and preservative-free sterile water with the maximum concentration not to exceed 1500 micrograms per milliliter and administered by continuous intravenous infusion or by intravenous injection.
• the daily dose can be added to 5 ml of normal saline and the solution infused by mechanical pump or by gravity.
• the effect of IL-4 on lowering the serum cholesterol levels in mammals was determined by the following test protocol.
• Cynomolgus monkeys (Macaca fascicularis) are administered human IL-4, which is obtained from Chinese Hamster Ovary (CHO) cells as described below.
• the route of administering the human IL-4 is by intravenous injection in the saphenous vein for about 15 seconds and dosing occurs daily for four weeks. Blood samples are taken at the times indicated in the following table by femoral venipuncture following an overnight fast. Blood samples are taken at two times prior to dosing with the human IL-4 (-2 and -1 weeks prior to initial IL-4 dosing) and control animals (receiving IL-4 doses of 0.0 mg/kg/day in Table 1 ) are maintained that never received any dose of the human IL-4.
• the blood samples are checked for blood cholesterol levels using an enzymatic DACOS analyzer which is commercially available from Coulter Electronics, Inc., 600 West 20 Street, Hialeah, FL 33010.
• plasmid pcD-SRa-205 was constructed by the trimoiecular ligation of the 373bp Ncol-Xhol SRa promoter fragment from pSRa-CAT196, the 434bp Xhol-Sstl splice junction (SJ) and 5' murine IL- 4 (mlL-4) fragment from pcD137 and the 3221bp Sstl-Ncol fragment, also from pcD137, containing the 3' murine IL-4 cDNA, SV40 polyadenylation region and the pBR322 derived plasmid backbone containing the origin of replication and ampicillin resistance gene.
• SJ 434bp Xhol-Sstl splice junction
• mlL-4 5' murine IL- 4
• the G-C tail was deleted from pcD-hlL-4 clone 46 (Yokoto et al., Proc. Natl. Acad. Sci. USA, 22: 5894-5898)) as follows.
• the Okayama-Berg plasmid pL1 (Okayma and Berg, Molecular and Cellular Biology, 2: 280-289 (1983)) was restricted with Pstl and the four nucleotide overhang removed by the 3'-5' exonuclease activity of T4 polymerase.
• Bglll linkers were ligated to the flush DNA ends followed by restriction with Bglll and
• Hindlll Bglll fragment containing the SV40 sequence of pL1 was isolated and inserted into Bglll/Hindlll restricted pcD-MCGF (Yokoto et al., Proc. Natl. Acad. Sci. USA, fll: 1070-1074 (1984)) to yield intermediate plasmid 101.
• the purified 3llbp Pst fragment from plasmid pcD-hlL-4 clone 46 was restricted with Sau3A-l which releases a 163bp fragment with overhangs compatible with Bglll.
• the 162bp fragment was ligated to Bglll restricted p101 to yield intermediate 112.
• Hindlll/Nhel fragment of p112 containing SV40 and human IL-4 cDNA sequences was ligated to Hindlll/Nhel restricted clone 46 DNA to produce pcD-hlL-4 clone 125 containing an SV40 early promoter, SV40 splice junction and complete human IL-4 cDNA with the G-C tail deleted.
• plasmid pcD-Sra224 was constructed by replacing the small Xhol fragment of pcD-SRa205 (containing the SJ and mlL4 cDNA) with the small Xhol fragment of pcDhlL-4 clone 125 containing the SJ and HIL-4 cDNA with G-C tail deleted as described above.
• a Sail site was introduced into pMTVdhfr (Lee et al., Nature, 294: 228-232 (1981)) by EcoRI/BamHI restriction, Klenow polymerase fill in of the overhang and ligation to an octanucleotide Sail linker as shown in Figure 1c. Plasmid pMTVdhfr259, then, lacks restriction sites for EcoRI and BamHI and the region between the two is replaced with a Sail linker.
• the Sail fragment of pcD-SRa224 containing the Sra promoter, SV40 SJ, human IL 4 cDNA and SV40 polyadenylation signals was inserted into the unique Sail site of pMTVdhfr259 as shown in Figure 1d.
• the final human IL-4 expression plasmid, pdhfr-SRalpha263 contains the following elements, counterclockwise from the Sail site:
• SRalpha promoter 4. SV40 derived splice junction.
• the human IL-4 cDNA sequence present in the vector is the same as in pcD-HIL-4 clone 46 given in Yokoto et al., Proc. Natl. Acad. Sci. USA, 22: 5894-5898(1986).
• CHO-dhfr Chinese hamster ovary cell mutants deficient in d ⁇ hydrofolate reductase activity
• CHO-dhfr mutant cells have an auxotrophic requirement for hypoxhantine, thymidine and glycine.
• Expression vectors incorporating a dhfr marker may be used to complement this mutation; selection is achieved by growing cells in the absence of the required media cofactors described above.
• Gene amplification (increase in copy number up to 1000X) may be accomplished by growing cells in increasing concentration of the folate analog methotrexate (MTX).
• MTX folate analog methotrexate
• the plasmid DNA having the coding sequence for dhfr and human IL-4 (pdhfr-SRa263) was constructed as described above.
• Transfection of pdhfr-SRa263 into DXB-II CHO-dhgr cell line was carried out by the calcium phosphate precipitation method.
• Transformants were selected in a selection medium (DMEM, Dulbecco's Modified Eagle's Medium) that lacks hypoxhantine and thymidine.
• DMEM Dulbecco's Modified Eagle's Medium
• a clone designated 3B12 was chosen for the first cycle of amplification.
• the 3B12 clone was cultured in a-MEM medium (Eagle's minimum essential medium) containing 40 nM MTX until resistant clones were selected.
• a clone designated 3B12-A26 was used for further amplification with 1 mm MTX. After the second cycle of drug selection, a clone designated 3B12-A26-19 was chosen for further development. This clone was adapted to growth in a suspension mode with 10% NU SerumTM V and a subclone designated IL-4 SI was selected for the large scale propagation.
• MBC Master Cell Bank
• 100 ml spinner flasks containing the IL-4 SI cells were used.
• the cells were carried through two additional growth medium exchanges and grown in 100 ml spinner flasks (growth medium is basal medium plus 0 to 10% serum, e.g., NU SerumTM V).
• growth medium is basal medium plus 0 to 10% serum, e.g., NU SerumTM V.
• Cells from each flask were collected, washed, resuspended in 10 ml of freezing medium, pooled and aseptically dispensed in about 2.0 ml sterile cell storage vials (freezing medium is basal medium plus 20% serum, e.g., NU SerumTM V plus 10% dimethylsuifoxide).
• the vials were slowly frozen at -70 ⁇ C and stored in liquid nitrogen.
• the cells from three frozen vials were thawed and propagated by suspension in growth medium for 4 to 6 generations in spinner flasks of increasing volume from 100 ml up to 3 liters. Cells were collected by centrifugation, washed, and resuspended in freezing medium. The cell suspension was aseptically dispensed in about 2.0 ml sterile cell storage vials. The vials were slowly frozen at -70 ⁇ C and stored in liquid nitrogen to constitute the Master Cell Bank (MCB).
• MCA Master Cell Bank
• a Master Working Cell Bank (MWCB) was prepared from the MCB by thawing 1 to 3 vials of the MCB and propagating the cells in T- flasks and in suspension for 4 to 6 generations in increasing volumes up to 3 liters. Cells were collected, washed, resuspended in freezing medium and aliquoted and frozen as described for the MCB. The MWCB was stored in liquid nitrogen as well. IL-4 production was carried out in bioreactors of 50 to 200 liters in volume, To start production, one frozen vial from the MWCB was thawed and inoculated into a T-75 flask.
• cells were trypsinized and inoculated into two T-75 flasks (optionally, a T-160 flask can be used). These flasks were again incubated until 100% confluency and the trypsinized cells were used to inoculate a 100 ml spinner flask.
• the 100 ml spinner flask was incubated until adequate cell growth was obtained and was used as inoculum for a 250 ml spinner flask.
• a similar step was repeated in a 1 liter and a 3 liter flask and a 10 to 20 liter bioreactor. Cells from the 10 to 20 liter reactor were used as inoculum for a 50 to 100 liter reactor. This reactor is initially grown batchwise and upon achieving adequate cell concentration, a continuous media perfusion was initiated.
• the media used for growth and continuous perfusion was modified Iscove's medium, which may be supplemented with up to 10% (e.g., NU SerumTM V). No methoxtrexate was used throughout the production process.
• the fermentation stages were carried out under sterile conditions and in closed systems.
• the key fermentation parameters such as temperature, pH, agitation and aeration were monitored and controlled as appropriate throughout the growth and continuous perfusion stages.
• Aseptic samples were taken periodically to measure pH, cell density and to check for sterility (absence of bacteria and fungi). Upon collection of an adequate volume of conditioned media
• the broth was filtered to remove any cells that may be present, and concentrated via ultrafiltration.
• the concentrate which contains crude CHO IL-4, was forwarded to the final purification stages.
• Purification of IL-4 from the crude CHO IL-4 concentrate was carried out by performing a cation exchage chromatography on a sulphonate column (e.g., S-Sepharose). This step was typically repeated. Selected pooled fractions from the sulphonate column were then forwarded to a chelate chromatography step (e.g., cobalt-chelate Sepharose). The selected pooled chelate fractions were then diafiltered and concentrated via membrane filtration. The concentration was chromatographed in a gel filtration column (e.g., HR S-200). The pooled fractions which constitute the purified bulk IL-4 were then filtered and stored at -20 ⁇ C or lower.
• a sulphonate column e.g., S-Sepharose
• the bulk IL-4 was then prepared for injection by thawing and diluted with sterilized water and/or 10 mm citrate buffer.
• a cholesterol amounts expressed as milligrams per deciliter b measurements taken at 2 weeks after dosing

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• 1989
  • 1989-12-18 EP EP90901347A patent/EP0449949A1/de active Pending
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  • 1989-12-18 JP JP2501573A patent/JPH04506339A/ja active Pending
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