Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Patent
1996-07-18
1998-05-26
Tsang, Cecilia J.
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
530324, 530858, A61K 3816, C07K 1400
Patent
active
057564620
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
This invention relates to polypeptides found in the venom of the Geolycosa riogrande spider and to polypeptides having substantially the same amino acid sequence and substantially the same activity as said polypeptides. The polypeptides and the pharmaceutically acceptable salts thereof form pores in the plasma membrane of cells and are effective in mobilizing intracellular calcium ions in cells.
Compounds which are pore formers have a variety of utilities. For example, pore forming compounds can find clinical application in drug delivery, cancer chemotherapy (e.g. immunoconjugates) and immunotherapy (e.g. immunoconjugates; complement-and cell-mediated lysis). Compounds which mobilize intracellular calcium ions have a variety of utilities, for example, in the study of the physiology of cells such as neuronal and muscle cells.
SUMMARY OF THE INVENTION
This invention concerns polypeptides found in the venom of the Geolycosa riogrande spider. The polypeptides of this invention and the fractions in which they are present according to this invention are as follows.
Geolycosa peptide 15a has the amino acid sequence, SEQ ID NO: 1. Geolycosa peptide 15b has the amino acid sequence, SEQ ID NO: 2. Geolycosa peptide 17 has the amino acid sequence, SEQ ID NO: 3.
The polypeptides of this invention form pores in the plasma 3 membrane of cells and mobilize intracellular Ca.sup.2+ in cells. The main biological consequence of pore formation is cell lysis. Accordingly, therapeutants utilizing this effect may find utility in such areas as drug delivery, cancer chemotherapy (e.g. immunoconjugates) and immunotherapy (e.g. immunoconjugates; complement-and cell-mediated lysis).
Also within the scope of this invention are polypeptides which have substantially the same amino acid sequence and substantially the same pore forming and/or Ca.sup.2+ mobilizing activity as the polypeptides described above.
This invention also concerns pharmaceutical compositions comprising said polypeptides and methods of administering said polypeptides.
DETAILED DESCRIPTION OF THE INVENTION
Venom is obtained from the Geolycosa riogrande spider through the process of milking by electrical stimulation according to standard methods well known to those skilled in the art. It is preferred that the method employed is one which safeguards against contamination of the whole venom by abdominal regurgitant or hemolymph. such methods are well known to those skilled in the art. The whole venom so obtained is stored in a frozen state at about -78.degree. C. until used for purification as described below. Purification of the constituents from the whole venom is accomplished by reverse phase high performance liquid chromatography (HPLC) on a variety of preparative and semi-preparative columns such as C4 and C-18 Vydac.RTM. columns (Rainin Instrument Co. Inc., Mack Road, Woburn Mass. 01801). Peak detection is carried out monochromatically at 220-230 nm. Further analysis of the fractions can be accomplished with, for example, polychrome UV data collected with a Waters 990 diode array detector (Millipore Corporation, Waters Chromatography Division, 34 Maple Street, Milford, Mass. 01757). The fractions from the columns are collected by known methods such as through the use of an SCO/"IFOXY" fraction collector and an ISCO 2159 peak detector (ISCO, 4700 Superior, Lincoln, Nebr. 68504). The fractions are collected in appropriately sized vessels such as sterile polyethylene laboratory ware. Concentration of the fractions is then accomplished by lyophilization from the eluant followed by lyophilization from water. Purity of the resulting constituent fractions then can be determined by chromatographic analysis using an analytical column with a gradient system which is more isocratic than the system used in the final purification of the fractions.
The polypeptides of the invention can be sequenced according to known methods. A general strategy for determining the primary structure includes, for example, the following steps. 1) Controlled cleava
REFERENCES:
patent: 5073542 (1991-12-01), Zasloff
Kelbaugh Paul R.
Saccomano Nicholas A.
Volkmann Robert A.
Celsa Bennett
Ginsburg Paul H.
Pfizer Inc.
Richardson Peter C.
Tsang Cecilia J.
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