Patent Application
20050037430
The invention relates generally to biotechnology, and more specifically to methods of generating and screening small molecules, such as di-mer, tri-mer, 4-mer, 5-mer, 6-mers, 7-mers, and a database generated by screening the small molecules and methods of using the database.
U.S. Pat. No. 5,380,668 to Herron (Jan. 10, 1995), the contents of the entirety of which are incorporated by this reference, discloses, among other things, various compounds having the antigenic binding activity of human chorionic gonadotropin (hCG). The oligopeptides disclosed therein are disclosed generally for use in diagnostice methods.
Various patents and patent applications to Gallo et al. (e.g., U.S. Pat. No. 5,677,275 (corresponding to WO 96/04008 A1), U.S. Pat. No. 5,877,148 (also corresponding to WO 96/04008 A1), WO 97/49721 A1, U.S. Pat. No. 6,319,504 (corresponding to WO 97/49373), U.S. Patent Application 2003/0049273 A1 (also corresponding to WO 97/49373), U.S. Pat. No. 5,968,513 (corresponding to WO 97/49418), U.S. Pat. No. 5,997,871 (corresponding to WO 97/49432), U.S. Pat. No. 6,620,416, U.S. Pat. No. 6,596,688, WO 01/11048 A2, WO 01/10907 A2., and U.S. Pat. No. 6,583,109) relate to various oligopeptides and their use in, among other things, “inhibiting HIV infection”, “treating or preventing HIV infection”, “treating or preventing cancer”, “treating or preventing a condition characterized by loss of body cell mass”, “treating or preventing a condition associated with pathological angiogenesis”, “treating or preventing hematopoietic deficiency”, “ex vivo gene therapy”, “expanding blood cells in vitro”, and/or “providing blood cells to a subject”.
The current invention relates to the body's innate way of modulating important physiological processes and builds on insights reported in PCT International Publications WO99/59617 and WO01/72831 and PCT International Application PCT/NLO2/00639, the contents of the entirety of all of which are incorporated herein by this reference. These applications describe small gene-regulatory peptides that are present in pregnant women and are derived from proteolytic breakdown of placental gonadotropins, such as hCG. These breakdown products are often only about 4 to 6 amino acids long and were shown to have unsurpassed immunological activity that is exerted by regulating expression of genes encoding inflammatory mediators such as cytokines. Surprisingly, it was found that breakdown of hCG provides a cascade of peptides that helps maintain a pregnant woman's immunological homeostasis. These peptides balance the immune system to assure that the mother stays immunologically sound while her fetus does not get prematurely rejected during pregnancy, but instead is safely carried until its time of birth.
During pregnancy, the maternal system suppresses maternal rejection responses directed against the fetus. Paradoxically, during pregnancy, often the mother's resistance to infection is increased and she is found to be better protected against the clinical symptoms of various autoimmune diseases such as rheumatism and multiple sclerosis. The protection of the fetus thus cannot be interpreted as only a result of immune suppression. Each of the above three applications has provided insights by which the immunological balance between protection of the mother and protection of the fetus can be understood.
Where it was generally thought that the smallest breakdown products of proteins have no specific biological function on their own, it now emerges that the body may utilize the normal process of proteolytic breakdown to generate important compounds such as gene-regulatory compounds. In particular, certain short breakdown products of hCG (i.e., short peptides which can easily be synthesized, if needed modified, and used as a pharmaceutical composition) exert a major regulatory activity on pro- or anti-inflammatory cytokine cascades that are governed by a family of crucial transcription factors, the NF-κB family, which generally regulate the expression of genes involved in the body's immune response.
In an exemplary embodiment, the invention provides a method for identifying one or more peptides; and/or determining the activity of one or more peptides, comprising, e.g., screening a peptide to determine the activity of the peptide; analyzing the results; and/or identifying one or more proteins having immunoregulatory activity and/or gene regulatory activity. Optionally, the results may be stored in a database, wherein the database may be sorted by one or more desired characteristic, for example, production of nitric oxide (NO). In addition, the database may allow searching (e.g., by means of a computer), form a remote location, such as a second computer, wherein at least part of the search result may be transmitted to the searcher via a network.
In another embodiment, the invention provides compiling and/or organizing data and/or properties for one or more peptides in a database. In another embodiment, software that performs correlative database searching is used.
The invention also provides a method of accessing information from a collection of data, including receiving a query and generating results to the query. Generating a result to the query may include storing hierarchical information generated from the collection of data, and applying search rules to the collection of data to produce a canonical non-terminal representation of the data.
In an exemplary embodiment, generating results includes applying search rules to the query to produce a query canonical form and matching the query canonical form to a canonical non-terminal representation of the data.
In yet another embodiment, the invention provides a method for conducting a drug discovery business, comprising: i) determining the identity of a compound that modulates development of the systemic inflammatory response, release of other inflammatory mediators (e.g., IL-1-α, IL-1-β, IL-6, TNF-α, LIF, IFN-γ, OSM, CNTF, TGF-β, GM-CSF, IL-11, IL-12, IL-17, IL-18, IL-8 and a variety of other chemokines), regulation of members of the nuclear factor-κB (NF-κB) family, accentuation or protection from sepsis, nitrate production, nitric oxide (NO) production, and/or glucose tolerance; ii) conducting therapeutic profiling of the compound identified in step i), or further analogs thereof, for efficacy and toxicity in animals; and, iii) formulating a pharmaceutical preparation including one or more compounds identified in step ii) as having an acceptable therapeutic profile. Such business method can be further extended by including an additional step of establishing a distribution system for distributing the pharmaceutical preparation for sale, and may optionally include establishing a sales group for marketing the pharmaceutical preparation. Optionally, determining the identity of a compound may comprise searching a database of the invention.
The instant invention also provides a business method comprising: i) by suitable methods described herein, determining the identity of a compound that modulates a systemic inflammatory response, release of other inflammatory mediators (e.g., IL-1-α, IL-1-β, IL-6, TNF-α, LIF, IFN-γ, OSM, CNTF, TGF-β, GM-CSF, IL-11, IL-12, IL-17, IL-18, IL-8 and a variety of other chemokines), regulation of members of the nuclear factor-KB (NF-κB) family, sepsis, nitrate production, nitric oxide (NO) production, and/or glucose tolerance ii) licensing, to a third party, the rights for further drug development of one or more compounds identified in step i).
The instant invention also provides a business method comprising: i) by suitable methods determining the identity of the polypeptide and the nature of the treatment or effect associated with the polypeptide, the methods optionally include searching a database of the invention; ii) licensing, to a third party, the rights for further drug development of the polypeptide.
In another embodiment, the invention provides a method of screening a peptide and derivatives thereof, for activity. Optionally, the result of a screen is entered into a database. In another embodiment, the peptide and/or derivative are arranged on a chip or array or in a multiwell format. In yet another embodiment, a peptide array or multiwell format is screened using methods known in the art and/or disclosed herein to determine one or more activities of a peptide.
As described in PCT International Publication No. WO 03/029292 A2 (published Apr. 10, 2003), PCT International Publication No. WO 01/72831 A2 (published Oct. 4, 2001), and U.S. Patent Application Publications 20020064501 Al (published May 30, 2002), 20030119720 A1 (published Jun. 26, 2003), 20030113733 A1 (published Jun. 19, 2003), and 20030166556 A1 (published Sep. 4, 2003), the contents of all of which are incorporated by this reference, compositions containing purified or isolated oligopeptides described therein have immunoregulatory activity useful in, for example, the treatment of sepsis and other disease states and conditions. They also have gene regulatory activities.
The invention includes a method of screening compounds, including a purified or isolated peptide consisting of particular four to eight amino acid segments of the sequence MTRVLQGVLPALPQVVC (SEQ ID NO:44); and derivatives thereof. Immunoregulatory activity may be determined by measuring a peptides's ability to modulate production of NO by a cell. In one embodiment, the compositions have the ability to decrease shock in a subject (e.g., a mammal) undergoing sepsis.
In one embodiment, the amino acid segment includes a tetrameric sequence (e.g., corresponding to the LQVG (SEQ ID NO:1)) portion of SEQ ID NO:44, i.e.,
Xaa1 Xaa2 Xaa3 Xaa4
wherein Xaa1 is a substituted or unsubstituted non-polar amino acid selected from the group consisting of Ala and Leu; Xaa2 is a substituted or unsubstituted amino acid selected from the group consisting of Gln, Pro, and Ala; Xaa3 is a substituted or unsubstituted Gly; and Xaa4 is a substituted or unsubstituted non-polar amino acid selected from the group consisting of Val and Ala. For instance, the peptide could be selected from the group consisting of LQGV (SEQ ID NO:1), the derivative AQGV (SEQ ID NO:2), the derivative LQGA (SEQ ID NO:19), the derivative LAGV (SEQ ID NO:26), and the derivative LPGC (SEQ ID NO:41).
In another embodiment, the segment is six amino acids long, and comprises the sequence
Xaa1 Xaa2 Pro Ala Xaa2 Xaa3
wherein Xaa1 is substituted or unsubstituted Val or Ala, wherein Xaa2 is independently selected from substituted or unsubstituted Leu or Ala, and wherein Xaa3 is a substituted or unsubstituted Pro or Ala.
In such an embodiment, the peptide can have a formula selected from the group consisting of VLPALP (SEQ ID NO:3), the derivative ALPALP (SEQ ID NO:21), the derivative VAPALP (SEQ ID NO:22), the derivative ALPALPQ (SEQ ID NO:23), the derivative VLPAAPQ (SEQ ID NO:24), the derivative VLPALAQ (SEQ ID NO:25), the derivative VLPALA (SEQ ID NO:28), VLPALPQ (SEQ ID NO:29), the derivative VLPALPA (SEQ ID NO:31), the derivative GVLPALP (SEQ ID NO:32), and the derivative VLAALP (SEQ ID NO:117).
In another embodiment, the composition has no more than eight amino acids, and includes an amino acid sequence consisting of: Xaa1 Xaa2 Xaa3 Xaa4
wherein Xaa1 is a substituted or unsubstituted amino acid selected from the group of amino acids consisting of Ala, Leu, and Met, wherein Xaa2 is a substituted or unsubstituted amino acid selected from the group of amino acids consisting of Gln, Thr, Ala, and Pro, wherein Xaa3 is substituted or unsubstituted Gly or Arg, and wherein Xaa4 is a substituted or unsubstituted amino acid selected from the group of amino acids consisting of Cys, Ala, and Val. Immunoregulatory activity may be determined by, for example, measuring the capability to modulate production of NO by a cell or affect sepsis.
In such an embodiment, the sequence may be selected from the group consisting of Leu Gln Gly Val (SEQ ID NO:1), Ala Gln Gly Val (SEQ ID NO:2), Leu Gln Gly Ala (SEQ ID NO:19), Leu Ala Gly Val (SEQ ID NO:26), Leu Pro Gly Cys (SEQ ID NO:41), or Met Thr Arg Val (SEQ ID NO:42), or a derivative thereof.
In another embodiment, the segment may be the tetramer MTRV (SEQ ID NO:42) or QVVC (SEQ ID NO:43) or a derivative thereof.
In one embodiment, a genomic or functional database includes the level of a pro inflammatory cytokine, such as pro inflammatory cytokines that are responsible for early responses, including IL-1-α, IL-1-β, IL-6, and TNF-α. Other pro inflammatory mediators include LIF, IFN-γ, OSM, CNTF, GM-CSF, IL-11, IL-12, IL-17, IL-18, IL-8 and a variety of other chemokines that chemoattract inflammatory cells, and various neuromodulatory factors. It is preferred that the results include the level of a pro inflammatory cytokine that is selected from the group of TNF-α, IFN-γ, IL-1-β and IL-6. Upon determination of pro inflammatory cytokine levels of a compound that may down regulate translocation and/or activity of pro inflammatory cytokine gene expression may be identified. In another embodiment, the relative levels of one or more transcription factor, for example, an NF-κB/Rel protein, are determined, for example, using LPS stimulated RAW264.7 cells using a multiwell format. The pro inflammatory cytokine levels, or other attributes, of the compounds may be compiled in a database and used according to the invention.
Inflammatory cytokines include IL-4, IL-10, and IL-13, IL-16, IFN-α, IL-Ira, G-CSF, and soluble receptors for TNF or IL-6. Information regarding peptides having NF-κB up regulating activity may be assayed using a peptide array or multiwell format and the results compiled in a database. For example, the activity of a peptide may be assayed in a multiwell format for modulation of NF-κB activity or NO production using RAW264.7 cells.
An essentially inflammatory condition is preferably characterized by elevated levels of at least one, but preferably at least two or three pro inflammatory cytokines, for example, produced by circulating polymorph bone marrow cells (PBMCs), such as elevated plasma or serum levels of one or more of the pro inflammatory cytokines that are responsible for early responses such as IL 1 α, IL 1 β, IL 6, and TNF α. Examples of other pro inflammatory mediators include LIF, IFN γ, OSM, CNTF, GM CSF, IL 11, IL 12, IL 17, IL 18, IL 8. As each diagnostician may have his or her own preference for testing an inflammatory mediator, it is preferred that the database be based on the level of a pro inflammatory cytokine is selected from TNF α, IFN γ, IL 1 β and/or IL 6.
Useful tests include, but are not limited to, flow cytometry assays of serum/plasma/supernatant available from BD Biosciences as a Cytometric Bead Array (CBA), for example, a CBA human Th1/Th2 cytokine kit for the measurement of IL 2, IL 4, IL 5, IL 10, TNF α and IFN γ in a single sample or available from Biosource International, the Biosource Multiplex antibody Bead Kit for measurement of IL 1β, IL 2, IL 4, IL 5, IL 6, IL 8, IL 10, IFN γ, TNF α and GM CSF in single sample or, for example, the Biosource cytoset ELISA for measurement of individual cytokines and soluble DR4.
It is also provided herein to determine HLA-DR expression, wherein decreased levels of HLA-DR antigen expression may indicate a counter inflammatory condition.
The tests and/or assays described here in may be used to screen a large number of peptides, for example, derivatives of LQGV (SEQ ID NO:1) may be synthesized and placed in a protein array. Derivatives of LQGV (SEQ ID NO:1) can include peptides produced by Ala-scanning and/or peptides produced by replacement net analysis. Derivatives may also include modified or non-natural amino acids. An array may be prepared with such peptides and used to screen the peptides, which include derivatives, for activity. Such a screen may identify peptides with one or more desired activity and/or produce positional information, for example, identification that position four (e.g., the V of SEQ ID NO:1) is preferably a non-polar amino acid, by alignment of the screen results and/or peptide sequences screened.
Organization, searching and construction of a database may be performed by any of the means known in the art (see, for example, U.S. Pat. Nos. 5,966,712; 6,278,794; 6,379,970; 6,539,102; 6,507,788; and 6,711,563, each of which is hereby incorporated by reference).
The invention further includes a pharmaceutical composition comprising a purified or isolated peptide, or acid addition salt thereof, the purified or isolated peptide identified according to the invention.
The invention provides a method for the identification of a peptide useful in the treatment of bone disease such as osteoporosis comprising administering to a subject believed to be in need of such treatment a compositon comprising a peptide, derivative or functional analogue thereof, the particular molecule capable of modulating production of NO and/or TNF-alpha by a cell.
Such compounds are particularly useful in post-menopausal women that no longer experience the benefits of being provided with a natural source of hCG and its breakdown products. Such a treatment can be achieved by systemic administration of a composition of the invention according to the invention, but local administration in joints, bursae or tendon sheaths is provided as well. The molecule can be selected from Table 6 or identified by a method desribed herein, including searching a database of the invention. The treatment comprises administering to the subject a pharmaceutical composition comprising a peptide or derivative thereof. For example, a peptide capable of reducing production of NO by a cell. In another embodiment, the composition comprises at least two peptides or derivatives thereof. For example, each peptide being capable of reducing production of NO and/or TNF-alpha by a cell. By way of example, the at least two oligopeptides may be selected from the group LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), and VLPALP (SEQ ID NO:3) or peptides identified by a method described herein.
The methods of the invention have been used to assay several peptides according to the invention by ex vivo, in vivo, and animal assays. A beneficial effect of these oligopeptides on LPS-induced sepsis in mice, namely the inhibition of the effect of the sepsis, was observed. Immunomodulatory effects with these oligopeptides have been observed in vitro and ex vivo such as in T-cell assays showing the inhibition of pathological Th1 immune responses, suppression of inflammatory cytokines (MIF), increased production of anti-inflammatory cytokines (IL-10, TGF-beta) and immunomodulatory effects on antigen-presenting cells (APC) like dendritic cells, monocytes and macrophages.
Knowing the gene modulatory effect of the peptides of the invention, allows for the rational design of molecular mixtures that better alleviate the symptoms seen with sepsis. For example, a 1:1:1 mixture of LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2) and VLPALP (SEQ ID NO:3) was administered to primates in a gram-negative induced rhesus monkey sepsis model to test for the prevention of septic shock and found to be effective in this primate model. Accordingly, the invention provides information regarding peptides which may be used to prepare a pharmaceutical composition for the treatment of sepsis in a primate. In addition, a method for the treatment of sepsis in a primate comprising subjecting the primate to a composition of the invention, preferably selected according to the invention, preferably utilizing a mixture of such compositions. Administration of such a composition or a mixture preferably occurs systematically, for example, by intravenous or intraperitoneal administration. In a further embodiment, such treatment also comprises the use of, for example, an antibiotic, however, only when such use is not contra indicated because of the risk of generating further toxin loads due to lysis of the bacteria by those antibiotics.
The invention also provides use of a composition according to the invention for the preparation of a pharmaceutical composition or medicament and methods of treating various medical conditions, such as an immune-mediated disorder.
As used herein, a “purified or isolated” peptide is one that has been purified from a natural or biotechnological source, or, more preferably, is synthesized as described herein.
“Composition,” as used herein, refers to chemical compounds which contain or consist of the peptide. The peptide is preferably isolated before inclusion within the composition. The peptide most preferably consists of three (3) to nine (9) amino acids.
As used herein, a “functional analogue” or “derivative” of a peptide includes variations made with regard to a reference peptide, which retains an identifiable relationship to the reference peptide, including variations made by pepscan, ala-scanning, replacement net analysis, methods disclosed in U.S. patent application Ser. No. 10/456,375 and/or conservative substitutions relative to the reference sequence, for example, SEQ ID NO:44. Derivatives also include compounds having the same or equivalent sidechains as the particular amino acids used in a peptide, and arranged sequentially in the same order as the peptides, but joined together by non-peptide bonds, e.g., by isosteric linkages such as the keto isostere, hydroxy isostere, diketo isostere, or the keto-difluoromethylene isostere. Once a derivative is produced, such a derivative is a peptide for the purposes of screening, identification of activity, inclusion in a database, production of a pharmaceutical and the like.
Also included within derivatives or functional analogues are peptidomimetic compounds that functionally or structurally resemble the original peptide taken as the starting point, but that are, for example, composed of non-naturally occurring amino acids or polyamides. With “conservative amino acid substitution”, one amino acid residue is substituted with another residue with generally similar properties (size, hydrophobicity, or charge), such that the overall functioning of a peptide sequence having such substitution is likely not to be seriously affected. A derivative can also be provided by systematically altering at least one amino acid of the reference peptide. This can, for instance, be done by an Alanine scanning (Ala-scan) and/or replacement net analysis, in which each amino acid is replaced in turn with one of the 19 (or 21, if selenocysteine and pyrrolysine are included) other amino acids. With these methods, many different peptides may be generated, based on an original amino acid sequence but each containing a variation or substitution of at least one amino acid residue. This way, many positional variants of the original amino acid sequence are synthesized and/or enzymatically prepared.
A derivative or analogue can also be, for instance, generated by substitution of an L-amino acid residue with a D-amino acid residue. Such a substitution may improve a property of an amino acid sequence, for example, to provide a peptide sequence of known activity of all D-amino acids in retro inversion format, thereby allowing for retained activity and increased half-life values. By generating many positional variants (derivatives) of an original amino acid sequence and screening for a specific activity, an improved peptide, for example, comprising D-amino acids, can be identified and used according to the invention.
The peptides (which include derivatives and/or functional analogues) may optionally be arranged on a chip, array or in a multiwell format (for array technology see, U.S. Pat. Nos. 6,630,308; 6,610,482; 6,506,558; 6,346,413; and 6,329,143). The peptides may be screened for a specific activity. Optionally, the generated data may further be used to design improved peptide derivatives of a certain amino acid sequence.
For instance, the previously described preferred compound could, in one embodiment be:
NT Xaa1 Xaa2 Xaa3 Xaa4 CT
wherein NT at the N-terminus is selected from the group of H—, CH3—, an acyl group, or a general protective group; and CT at the C-terminus is selected from the group of small (e.g., 1 to 5 amino acids) peptides, —OH, —OR1, —NH2, —NHR1, —NR1R2, or —N(CH2)1-6 NR1R2, wherein R1 and R2, when present, are independently selected from H, alkyl, aryl, (ar)alkyl, and wherein R1 and R2 can be cyclically bonded to one another. Such modifications constitute derivatives of the reference peptide.
“Alkyl” as used herein, is preferably a saturated branched or unbranched hydrocarbon having one to six carbon atoms, e.g., methyl, ethyl, and isopentyl.
“Aryl” as used herein, is an aromatic hydrocarbon group, preferably having 6 to 10 carbon atoms, such as phenyl or naphthyl.
“(Ar)alkyl”, as used herein, is an arene group (having both aliphatic and aromatic portions), preferably having 7 to 13 carbon atoms such as benzyl, ethylbenzyl, n-propylbenzyl, and isobutylbenzyl.
“Peptide,” as used herein, means peptides having from 2 to about 50 amino acids joined together by peptide bonds.
“Composition” also includes, for example, an acceptable salt of the oligopeptide or a labeled peptide. As used herein, “acceptable salt” refers to salts that retain the desired activity of the peptide or equivalent compound, but preferably do not detrimentally affect the activity of the peptide or other component of a system, which uses the peptide. Examples of such salts are acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like. Salts may also be formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, and the like. Salts may be formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel and the like or with an organic cation formed from N,N′-dibenzylethylenediamine or ethylenediamine, or combinations thereof (e.g., a zinc tannate salt).
The composition can be administered or introduced in-vivo systemically, topically, or locally. The composition can be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with an inorganic acid (such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid); or with an organic acid (such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxahc acid, malonic acid, succinic acid, maleic acid, and fumaric acid); or by reaction with an inorganic base (such as sodium hydroxide, ammonium hydroxide, potassium hydroxide); or with an organic base (such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines). A peptide may also be conjugated to sugars, lipids, other polypeptides, nucleic acids and PNA; and function in situ as a conjugate or be released locally after reaching a targeted tissue or organ.
The compounds according to the invention may be prepared by methods known in the art (for example, see, U.S. patent application Ser. No. 10/456,375). For example, by peptide synthesis methods known in the art, including, suitable N alpha protection (and side-chain protection if reactive side-chains are present). The amino acid derivatives or peptides are activated and coupled to suitably carboxylprotected amino acid or peptide derivatives either in solution or on a solid support. Protection of the α-amino group may utilize an acid-labile tertiary-butyloxycarbonyl group (“Boc”), benzyloxycarbonyl (“Z”) group or substituted analogs or the base-labile 9-fluoremyl-methyloxycarbonyl (“Fmoc”) group. The Z group can also be removed by catalytic hydrogenation, other suitable protecting groups include Nps, Bmv, Bpoc, Aloc, MSC, etc. A good overview of amino protecting groups is given in The peptides, Analysis, Synthesis, Biology, Vol. 3 E. Gross and J. Meienhofer, eds., (Academic Press, New York, 1981). Protection of carboxyl groups can take place by ester formation, for example, base-labile esters like methyl or ethyl, acid labile esters like tertiary butyl or, substituted, benzyl esters or hydrogenolytically. Protection of side-chain functions like those of lysine and glutamic or aspartic acid can take place using the aforementioned groups. Protection of thiol, and although not always required, of guanidino, alcohol and imidazole groups can take place using a variety of reagents such as those described in The Peptides, Analysis, Synthesis, Biology, or in Pure and Applied Chemistry, 59(3), 331-344 (1987). Activation of the carboxyl group of the suitably protected amino acids or peptides can take place by the azide, mixed anhydride, active ester, or carbodiimide method especially with the addition of catalytic and racemization-suppressing compounds like 1-N-N-hydroxybenzotriazole, N-hydroxysuccin-imide, 3-hydroxy-4-oxo-3,4-dihydro-1,2,3,-benzotriazine, N-hydroxy-5 norbornene-2,3-dicarboxyimide. Also the anhydrides of phosphorus based acids can be used. See, e.g., The Peptides, Analysis, Synthesis, Biology, supra and Pure and Applied Chemistry, 59(3),331-344 (1987).
It is also possible to prepare the compounds by the solid phase method of Merrifield. Different solid supports and different strategies are known see, e.g. Barany and Merrifield in The Peptides, Analysis, Synthesis, Biology, Vol. 2, E. Gross and J. Meienhofer, eds., (Acad. Press, New York, 1980), Kneib-Cordonier and Mullen Int. J. Peptide Protein Res., 30, 705-739 (1987) and Fields and Noble Int. J. Peptide Protein Res., 35, 161-214 (1990). The synthesis of compounds in which a peptide bond is replaced by an isostere, can, in general, be performed using the previously described protecting groups and activation procedures. Procedures to synthesize the modified isosteres are described in the literature e.g., for the—CH2—NH—isostere and for the—CO—CH2—isostere.
Removal of the protecting groups, and, in the case of solid phase peptide synthesis, the cleavage from the solid support may be performed by means known in the art (see, e.g. volumes 3, 5 and 9 of the series on The Peptides Analysis, Synthesis, Biology, supra).
Another possibility is the application of enzymes in synthesis of such compounds; for reviews see, e.g., H. D. Jakubke in The Peptides, Analysis, Synthesis, Biology, Vol. 9, S. Udenfriend and J. Meienhofer, eds., (Acad. Press, New York, 1987). For example, by modifications such as glycosylation, phosphorylation and other modifications known in the art.
Peptides according to the invention may also be made according to recombinant DNA methods. Such methods involve the preparation of the desired peptide by means of expressing recombinant polynucleotide sequence which codes for one or more of the oligopeptides in a suitable host cell. Generally the process involves introducing into a cloning vehicle (e.g., a plasmid, phage DNA, or other DNA sequence able to replicate in a host cell) a DNA sequence coding for the particular oligopeptide or oligopeptides, introducing the cloning vehicle into a suitable eucaryotic or procaryotic host cell, and culturing the host cell thus transformed. When a eucaryotic host cell is used, the compound may include a glycoprotein portion.
The administration dose of the active molecule may be varied over a fairly broad range. The concentrations of an active molecule which can be administered would be limited by efficacy at the lower end and the solubility of the compound at the upper end. The optimal dose or doses for a particular patient should and can be determined by the physician or medical specialist involved, taking into consideration well-known relevant factors such as the condition, weight and age of the patient, etc.
The invention provides methods for screening and organizing the results from such screens, for example, regarding peptides derived from MTRVLQGVLPALPQVVC (SEQ ID NO:44) having a length of 3, 4, 5, 6, 7, 8, or 9 amino acids. In one exemplary embodiment, a scanning concept may utilize the process of looking at the linear protein sequence through a series of moving windows of a predetermined size. By way of example, peptides of four amino acid lengths (e.g., MTRV, TRVL, RVLQ, VLQG, LQGV, QGVL, GVLP, VLPA, LPAL, PALP, etc. through QVVC (SEQ ID NOs: 176-179, 1, 180-185, respectively)) are produced from MTRVLQGVLPALPQVVC (SEQ ID NO:44). In another embodiment, derivatives of one or more tetramer are produced by an amino acid variation procedure, such as a replacement net analysis or other methods known in the art (see, for example, Ward et al. (1996) Systematic Mapping of Potential Binding Sites for Shc and Grb2 SH2 Domains on Insulin Receptor Substrate-1 and the Receptors for Insulin, Epidermal Growth Factor, Platelet-derived Growth Factor, and Fibroblast Growth Factor, J. Biol. Chem. 271(10):5603-5609), thereby producing a library of tetramers that may be screened for useful characteristics, such as modulation of sepsis and/or the prevention or treatment of diabetes.
The invention alsom provides methods for screening, organizing and using the results from such screens, for example, with peptides derived from a refernce peptide. For example, peptides to be analyzed in this way may be derived from the reference peptide C-Reactive Protein (CRP) (ie.g., human CRP), such peptides include, LTSL, FVLS, NMWD, LCFL, MWDF, FSYA, FWVD, AFTV, and WDFV (SEQ ID NOs:186-194, respectively); peptides derived from Beta-catenin (e.g., human CTNB), such as GLLG, TAPS, VCQV, CLWT, VHQL, GALH, LGTL, TLVQ, QLLG, YAIT, LCEL, GLIR, APSL, ITTL, QALG, HPPS, GVLC, LCPA, LFYA, NIMR, NLIN, LHPP, LTEL, SPIE, VGGI, QLLY, LNTI, LWTL, LYSP, YAMT, LHNL, TVLR, and LFYA (SEQ ID NOs:195-227, respectively); peptides derived from beta-hCG (e.g., human CGHB), such as GLLLLLLLS, MGGTWA, TWAS, TLAVE, RVLQ, VCNYRDV, FESI, RLPG, PRGV, NPVVS, YAVALS, LTCDDP, EMFQ, PVVS, VSYA, GVLP, FQGL, and AVAL (SEQ ID NOs:228-245, respectively); peptides derived from Bruton's tyrosine kinase (e.g., human BTK), such as LSNI, YVFS, LYGV, YVVC, FIVR, NILD, TIMY, LESI, FLLT, VFSP, FILE, TFLK, FWID, MWEI, QLLE, PCFW, VHKL, LYGV, LESI, LSNI, YVFS, IYSL, and NILD (SEQ ID NOs:246-268, respectively); and peptides derived from matrix metalloproteinase-2 (e.g., human MM02), such as FKGA, FFGL, GIAQ, LGCL, YWIY, AWNA, ARGA, PFRF, APSP CLLS, GLPQ, TFWP, AYYL, FWPE, CLLG, FLWC, RIIG, WSDV, PIIK, GLPP, RALC, LNTF, LSHA, ATFW, PSPI, AHEF, WRTV, FVLK, VQYL, KFFG, FPFR, IYSA, and FDGI (SEQ ID NOs:269-301, respectively).
A non-extensive list of relevant oligopeptides useful for application in a method and/or database according to the invention include:
A further non-limiting list includes collagen, PSG, CEA, MAGE (malanoma associated growth antigen), Thrombospondin-1, Growth factors, MMPs, Calmodulin, Olfactory receptors, Cytochrome p450, Kinases, Von Willebrand factor (coagulation factors), Vacuolar proteins (ATP sythase), Glycoprotein hormones, DNA polymerase, Dehydrogenases, Amino peptidases, Trypsin, Viral proteins (such as envelope protein), Elastin, Hibernation associated protein, Antifreeze glycoprotein, Proteases, Circumsporozoite, Nuclear receptors, Transcription actors, Cytokines and their receptors, Bacterial antigens, Nramp, RNA polymerase, Cytoskeletal proteins, Hematopoietic (neural) membrane proteins, Immunoglobulins. HLA/MHC, G-coupled proteins and their receptors, TATA binding proteins, Transferases, Zinc finger protein, Spliceosmal proteins, HMG (high mobility group protein), ROS (reactive oxygen species), superoxidases, superoxide dismutase, Proto-oncogenes/tumor suppressor genes, and Apolipoproteins.
The invention is further explained with the aid of the following illustrative examples.
Material and Methods
PEPTIDE SYNTHESIS: The peptides as mentioned herein such as LQG, AQG, LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2), LQGA (SEQ ID NO:19), VLPALP (SEQ ID NO:13), ALPALP (SEQ ID NO:21), VAPALP (SEQ ID NO:22), ALPALPQ (SEQ ID NO:23), VLPAAPQ (SEQ ID NO:24), VLPALAQ (SEQ ID NO:25), LAGV (SEQ ID NO:26), VLAALP (SEQ ID NO:27), VLPALA (SEQ ID NO:28), VLPALPQ (SEQ ID NO:29), VLAALPQ (SEQ ID NO:30), VLPALPA (SEQ ID NO:31), GVLPALP (SEQ ID NO:32), VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL (SEQ ID NO:35), RPRCRPINATLAVEKEGCPVCITVNTTICAGYCPT (SEQ ID NO:45), SKAPPPSLPSPSRLPGPS (SEQ ID NO:38), LQGVLPALPQVVC (SEQ ID NO:34), SIRLPGCPRGVNPVVS (SEQ ID NO:39), LPGCPRGVNPVVS (SEQ ID NO:40), LPGC (SEQ ID NO:41), MTRV (SEQ ID NO:42), MTR, and VVC were prepared by solid-phase synthesis (R. B. Merrifield, J. Am. Chem. Soc., 85:2149-2165 (1963)) using the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl-based methodology (Atherton, 1985) with 2-chlorotrityl chloride resin (Barlos et al., Int. J. Peptide Protein res., 37:513-520 (1991)) as the solid support.
The side-chain of glutamine was protected with a trityl function. The peptides were synthesized manually. Each coupling consisted of the following steps: (i) removal of the alpha-amino Fmoc-protection by piperidine in dimethylformamide (DMF), (ii) coupling of the Fmoc amino acid (3 eq) with diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) in DMF/N-methylformamide (NMP) and (iii) capping of the remaining amino functions with acetic anhydride/diisopropylethylamine (DIEA) in DMF/NMP. Upon completion of the synthesis, the peptide resin was treated with a mixture of trifluoroacetic acid (TFA)/H2O/triisopropylsilane (TIS) 95:2.5:2.5. After 30 minutes, TIS was added until decolorization. The solution was evaporated in vacuo and the peptide precipitated with diethylether.
The crude peptides were dissolved in water (50-100 mg/ml) and purified by reverse-phase high-performance liquid chromatography (RP-HPLC). HPLC conditions were: column: Vydac TP21810C18 (10×250 mm); elution system: gradient system of 0.1% TFA in water v/v (A) and 0.1% TFA in acetonitrile (ACN) v/v (B); flow rate 6 ml/min; absorbance was detected from 190-370 nm. There were different gradient systems used. For example, for peptides LQG and LQGV (SEQ ID NO:1): 10 minutes 100% A followed by linear gradient 0-10% B in 50 minutes. For example for peptides VLPALP (SEQ ID NO:3) and VLPALPQ (SEQ ID NO:29): 5 minutes 5% B followed by linear gradient 1% B/minute. The collected fractions were concentrated to about 5 ml by rotation film evaporation under reduced pressure at 40° C. The remaining TFA was exchanged against acetate by eluting two times over a column with anion exchange resin (Merck II) in acetate form. The eluate was concentrated and lyphilized in 28 hours. Peptides later were prepared for use by dissolving them in PBS.
Endotoxin Shock Model (Sepsis)
Sepsis. For the endotoxin model, BALB/c mice were injected i.p. with 8-9 mg/kg LPS (E. coli 026:B6; Difco Lab., Detroit, Mich., USA). Control groups (PBS) were treated with PBS i.p. only. To test the effect of NMPF from different sources (synthetic, commercial hCG preparation [c-hCG]), we treated BALB/c mice with a dose of 300-700 IU of different hCG preparations (PG23; PREGNYL™ batch no. 235863, PG25; PREGNYL™ batch no. 255957 from NV Organon of Oss, NL) and with synthetic peptides (5 mg/kg) after two hours of LPS injection. In other experiments, BALB/c mice were injected i.p. either with 10 mg/kg or with 11 mg/kg LPS (E. coli 026:B6; Difco Lab., Detroit, Mich., USA). Subsequently, mice were treated after 2 hours and 24 hours of LPS treatment with NMPF peptides.
Semi-quantitative sickness measurements. Mice were scored for sickness severity using the following measurement scheme:
Sepsis experiments. To determine the effect of synthetic peptides (NMPF) in high-dose LPS shock model, BALB/c mice were injected intraperitoneally with different doses of LPS and survival was assessed daily for 5 days. In this experiment (for the LPS endotoxin model), BALB/c mice were injected i.p. with 8-9 mg/kg LPS (E. coli 026:B6; Difco Lab., Detroit, Mich., USA). Control groups (PBS) were treated with PBS i.p. only. We treated BALB/c mice with a dose of 300-700 IU of different hCG preparations (PG23; PREGNYL batch no. 235863, PG25; PREGNYL batch no. 255957) or with peptides (5 mg/kg) after two hours of LPS injection.
These experiments showed (Table 1) that NMPF peptides 4, 6, 66 and PG23 inhibited shock completely (all mice had in first 24 hours sickness scores not higher than 2; shortly thereafter they recovered completely and had sickness scores of 0), while peptides 2, 3 and 7 accelerated shock (all mice had in first 24 hours sickness scores of 5 and most of them died, while the control mice treated with LPS+PBS had sickness scores of 3-4 in first 24 hours and most of them died after 48 hours with sickness scores of 5; 17% survival rate at 72 hours). In addition, peptides 1, 5, 8, 9, 11, 12, 13, 14 and 64 showed in a number of different experiments variability in effectiveness as well as in the kind (inhibitory vs accelerating) of activity. This variability is likely attributable to the rate of breakdown of the various peptides and the different effects the various peptides and their breakdown products have in vivo. In addition, these experiments also showed the variability in anti-shock activity in c-hCG preparations that is likely attributable to the variation in the presence of anti-shock and shock-accelerating NMPF. Visible signs of sickness were apparent in all of the experimental animals, but the kinetics and obviously the severity of this sickness were significantly different. These data are representative of at least 10 separate experiments.
In Table 2, we see the effect of ALA-replacement (PEPSCAN) in peptide LQG, LQGV (SEQ ID NO:1), VLPALP (SEQ ID NO:3), VLPALPQ (SEQ ID NO:29) in septic shock experiments. The change of even one amino acid by a neutral amino acid can lead to different activity. So, genomic differences as well as polymorphism in these peptides can regulate the immune response very precisely. Derivatives of these peptides, for example (but not limited to) by addition of classical and non-classical amino acids or derivatives that are differentially modified during or after synthesis, for example benzylation, amidation, glycosylation, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. may be assayed to identify the activity of each peptide.
Assay results may be compiled in a database and, optionally, the results may be sorted, for example, by the kinetics of visible signs of sickness.
To determine whether treatment of BALB/c mice with NMPF inhibits septic shock at different stages of disease, synthetic peptides (NMPF) were injected i.p. at 2 and 24 hours after the induction of septic shock with high dose LPS (10 mg/kg).
As shown in Tables 3 and 4, control mice treated with PBS after the shock induction reached a sickness score of 5 at 14 and 24 hours, and remained so after the second injection with PBS. The survival rate in control group mice was 0% at 48 hours. In contrast to control mice, mice treated with NMPF 9, 11, 12, 43, 46, 50 and 60 reached a maximum sickness score of 2-3 at 24 hours after the induction of septic shock and further reached a maximum sickness score of 1-2 at 48 hours after the second injection of NMPF. In addition, mice treated with NMPF 5, 7, 8, 45, 53 and 58 reached a sickness score of 5 and after the second injection with NMPF all mice returned to a sickness score of 1-2 and survival rates in NMPF groups were 100%. Mice treated with NMPF 3 reached sickness scores of 3-4 and the second NMPF injection did save these mice. These experiments show that NMPF peptides have anti-shock activity at different stages of the disease and NMPF have anti-shock activity even at the disease stage when otherwise irreversible damage had been done. This indicates that NMPF have effects on different cellular levels and also have repairing and regenerating capacity.
NOD Experiment
Mice. Female NOD mice at the age of 13-14 weeks were treated i.p. with PBS (n=6) or NMPF peptides (VLPALPQVVC (SEQ ID NO:20), LQGV (SEQ ID NO:1), GVLPALPQ (SEQ ID NO:33), VLPALP (SEQ ID NO:3), VLPALPQ (SEQ ID NO:29), MTRV (SEQ ID NO:42), LPGCPRGVNPVVS (SEQ ID NO:40), CPRGVNPVVS (SEQ ID NO:50), LPGC (SEQ ID NO:41), MTRVLQGVLPALPQVVC (SEQ ID NO:44), VVCNYRDVRFESIRLPGCPRGVNPVVSYAVALSCQCAL (SEQ ID NO:35)) (5 mg/kg, n=6) three times a week for 2 weeks. Every four days urine was checked for the presence of glucose (Gluketur Test; Boehringer Mannheim, Mannheim, Del.). All mice used in these studies were maintained in a pathogen-free facility. They were given free access to food and water. The experiments were approved by the Animal Experiments Committee of the Erasmus University Rotterdam. Diabetes was assessed by measurement of the venous blood glucose level using an Abbott Medisense Precision glucometer. Mice were considered diabetic after two consecutive glucose measurements ≧11 mmol/l (200 mg/dl). Onset of diabetes was dated from the first consecutive reading.
Glucose tolerance test (GTT) was performed at 28 weeks of age in fasted mice (n=5) by injecting 1 g/kg D-glucose intraperitoneally (i.p.). At 0 (fasting), 5, 30 and 60 minutes, blood samples were collected from the tail and tested for glucose content.
NO Experiment
Cell culture. The RAW 264.7 murine macrophage cell line, obtained from American Type Culture Collection (Manassas, Va., USA), were cultured at 37° C. in 5% CO2 using DMEM containing 10% fetal calf serum (FCS), 50 U/ml penicillin, 50 pg/ml streptomycin, 0.2 M Na-pyruvate, 2 mM glutamine and 50 μM 2-mercaptoethanol (Bio Whittaker, Europe). The medium was changed every 2 days.
Nitrite measurements. Nitrite production was measured in the RAW 264.7 macrophage supernatants. The cells (7.5×105/ml) were cultured in 48-well plates in 500 μl of culture medium. The cells were stimulated with LPS (10 microg/ml) and/or NMPF (1 μg/ml, 1 ng/ml, 1 μg/ml) for 24 hours, then the culture media were collected. Nitrite was measured by adding 100 microl of Griess reagent (Sigma) to 100 microl samples of culture medium. The OD540 was measured using a microplate reader, and the nitrite concentration was calculated by comparison with the OD540 produced using standard solutions of sodium nitrite in the culture medium.
Results
NOD Experiment
In order to determine whether NMPF has effect on the disease development in NOD mice, we tested NMPF on pre-diabetic female NOD mice at the age of 13-14 weeks. After only two weeks of treatment (injection of NMPF (5 mg/kg) every other day), glucosuria data of all NOD mice was analyzed at the of 17 weeks. Profound anti-diabetic effect (mice negative for glucosuria) was observed in different NMPF groups as compared to the PBS group, especially in NMPF groups treated with peptide VLPALPQVVC (SEQ ID NO:20), VLPALP (SEQ ID NO:3), MTRV (SEQ ID NO:42), LPGCPRGVNPVVS (SEQ ID NO:40) and LPGC (SEQ ID NO:41). In addition, impairment of the glucose tolerance test was positively correlated to insulitis, but negatively correlated to the number of functional beta cells; also this test showed that NOD mice successfully treated with NMPF were tolerant for glucose as compared to the PBS group. Our results show that PBS treated NOD mice were all diabetic at the age of 23 weeks. Whereas, NOD mice treated three times a week for two weeks with NMPF showed profound inhibition of diabetes development. The strongest anti-diabetic effects were seen with NMPF-1, -4, -5, -6, -7, -65, -66 and commercial hCG preparation (PREGNYL, batch no. 235863). These mice had a low fasting blood glucose level and were tolerant for glucose (data partially shown). However, NMPF-71 showed no effect on the incidence of diabetes, while NMPF-64 and NMPF-11 had a moderate anti-diabetic effect.
NO Experiment
NO production is a central mediator of the vascular and inflammatory response. Our results show that macrophages (RAW 264.7) stimulated with LPS produce large amounts of NO. However, these cells co-stimulated with most of the NMPF peptides (NMPF peptides 1 to 14, 43 to 66 and 69) even in a very low dose (1 pg/ml) inhibited the production of NO.
Results
apoE Experiment
The invention provides a method and a composition of the invention for the treatment of conditions that are associated with dysfunctional LDL receptors such as apoE and other members of the apolipoprotein family. In particular, use of a composition of the invention comprising GVLPALPQ (SEQ ID NO:33) (NMPF-5) and/or VLPALP (SEQ ID NO:3) (NMPF-6) or a functional analogue or derivative thereof is preferred. Groups of apoE deficient mice (n=6 per group) were fed a high cholesterol food and given PBS or NMPF every other day intraperitoneally. After 2.5 weeks, body weight was determined as shown in the Table below.
+ = effects;
− + = variable effect;
no entry is no effect or not yet tested when table was assembled
from − + to + + + + + + + indicates from barely active to very active in modulating
TNF-αexperiment
Cell culture. The RAW 264.7 murine macrophage cell line, obtained from American Type Culture Collection (Manassas, Va., USA), is cultured at 37° C. in 5% CO2 using DMEM containing 10% fetal calf serum (FCS), 50 U/ml penicillin, 50 μg/ml streptomycin, 0.2 M Na-pyruvate, 2 mM glutamine and 50 μM 2-mercaptoethanol (Bio Whittaker, Europe). The medium is changed every 2 days.
Cell transfection. RAW 264.7 cells are transfected with a NF-κB dependent reporter construct.
NF-κB measurements. Output from the NF-κB dependent reporter is measured. The cells (7.5×105/ml) are cultured in 96-well plates in 500 μl of culture medium. The cells are stimulated with LPS (10 μg/ml) and/or a NMPF derivative (e.g., 1 μg/ml, 1 ng/ml, 1 μg/ml) for 24 hours, then the output from the NF-κB reporter is measured as appropriate for the reporter. Alternatively, NF-κB mRNA levels may be quantitated, for example by Northern blot analysis or other mRNA quantitation methods known in the art.
Peptide derivatives. Peptides derived from VLPALP (SEQ ID NO:3) are synthesized using replacement net ananlysis. A total of 120 peptides are synthesized and placed into the wells of two 96-well plates. RAW 264.7 cells in fresh media are added to the 96 well plates and the cells are stimulated with LPS (10 μg/ml) and the level of NF-κB mRNA determined at an appropriate time.
Likewise, other pro inflammatory cytokine levels may be assayed.
In another embodiment, the results are imput into a database. The database is then sorted
Monkey Experiment
Efficacy of NMPF, here a mixture 1:1:1 of LQGV (SEQ ID NO:1), AQGV (SEQ ID NO:2) and VLPALP (SEQ ID NO:3), administered in a gram-negative induced rhesus monkey sepsis model for prevention of septic shock.
Overwhelming inflammatory and immune responses are essential features of septic shock and play a central part in the pathogenesis of tissue damage, multiple organ failure, and death induced by sepsis. Cytokines, especially tumor necrosis factor (TNF)-α interleukin (IL)-1β, and macrophage migration inhibitory factor (MIF), have been shown to be critical mediators of septic shock. Yet, traditional anti-TNF and anti-IL-1 therapies have not demonstrated much benefit for patients with severe sepsis. We have designed peptides that block completely LPS induced septic shock in mice, even when treatment with these peptides is started up to 24 hours after LPS injection. These peptides are also able to inhibit the production of MIF. This finding provides the possibility of therapeutic use of these peptides for the treatment of patients suffering from septic shock. Since primates are evolutionary more closer to humans, we tested these peptides for their safety and effectiveness in a primate system.
Only naive monkeys were used in this preclinical study to exclude any interaction with previous treatments. The animals were sedated with ketamine hydrochloride. Animals were intubated orally and allowed to breathe freely. The animals were kept anesthetized with O2/N2O/isoflurane. The animals received atropin as pre-medication for O2/N2O/isoflurane anesthesia. A level of surgical anesthesia was maintained during the 2 h infusion of E. coli and for 6 h following E. coli challenge, after which the endothracheal tubes were removed and the animals were euthanized. Before bacteria were induced, a 1 hour pre-infusion monitoring of heart-rate and blood pressure was performed.
Two rhesus monkeys were infused with a 1010 CFU per kg of the Gram negative bacterium E. coli to induce a fatal septic shock. One monkey received placebo-treatment and was sacrificed within 7 hours after infusion of the bacteria without recovery from the anesthesia. The second monkey received treatment with test compound and was sacrificed at the same time point.
In a limited dose-titration experiment performed with the same bacterium strain in 1991, the used dose proved to induce fatal shock within 8 hours. In recent experiments, a 3-fold lower dose was used inducing clear clinical and pathomorphological signs of septic shock without fatal outcome.
The monkeys were kept anesthetized throughout the observation period and sacrificed 7 hours after the start of the bacterium infusion for pathological examination. The animals underwent a gross necropsy in which the abdominal and thorax cavities were opened and internal organs examined in situ.
Full Description of the Experiment with Three Rhesus Monkeys
The study was conducted in rhesus monkeys (Maccaca mulatta). Only experimentally naive monkeys were used in the study to exclude any interaction with previous treatments. Prior to the experiment, the state of health of the animals was assessed physically by a veterinarian. All animals had been declared to be in good health and were free of pathogenic ecto- and endoparasites and common bacteriological infections: Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Shigella, Aeromonas hydrophilia, pathogenic Campylobacter species and Salmonella.
Reagents. The Escherichia coli strain was purchased from ATCC (E. coli; 086a: K61 serotype, ATCC 33985). In a control experiment, the strain proved equally susceptible to bactericidal factors in human and rhesus monkey serum. Prior to the experiment, a fresh culture was set-up; the E. coli strain was cultured for one day, harvested and washed thoroughly to remove free endotoxine. Prior to infusion into the animal, the number and viability of the bacteria were assessed. Serial dilutions of the E. coli stock were plated on BHI agar and cultured overnight at 37° C. The colonies on each plate were counted and the number of colony-forming units per ml was calculated. The body weight measurement of the day of the experiment was used to calculate the E. coli dose and E. coli stock was suspended in isotonic saline (N.P.B.I., Emmer-Compascuum, NL) at the concentration needed for infusion (total dose volume for infusion approximately 10 ml/kg. The E. coli suspension was kept on ice until infusion.
Antibiotic was used to synchronize the shock induction in the monkeys. Baytril (Baytril 2.5%, Bayer, Del.) was used instead of gentamycin, as the strain proved only marginally susceptible to the latter antibiotic. Individual animals were identified by a number or letter combination tattooed on the chest.
Experimental design.
Anesthesia. All animals were fasted overnight prior to the experiment. On the morning of the experiment, the animals were sedated with ketamine hydrochloride (Tesink, NL) and transported to the surgery. The animal was placed on its side on a temperature-controlled heating pad to support body temperature. Rectal temperature was monitored using a Vet-OX 5700. The animals were intubated orally and were allowed to breathe freely. The animals were kept anesthesized using O2/N2O/isoflurane inhalation anesthesia during the E. coli infusion and the 7 hour observation period following E. coli challenge, after which the endothracheal tubes were removed and the animals were euthanized or allowed to recover from anesthesia. The femoral or the cephalic vein was cannulated and used for infusing isotonic saline, live E. coli and antibiotic administration. Insensible fluid loss was compensated for by infusing isotonic saline containing 2.5% glucose (Fresenius, 's Hertogenbosch, NL) at a rate of 3.3 ml/kg/hr.
Preparative actions. During anesthesia the animals were instrumented for measurement of blood pressure (with an automatic cuff), heart rate and body temperature. Isotonic saline was infused at 3.3 ml/kg/hr to compensate for fluid loss. Femoral vessels were cannulated for infusion of E. coli and antibiotics. Temperature-controlled heating pads were used to support body temperature. The monkeys were continuously monitored during the E. coli challenge and for the 6 hr period following E. coli administration. After 7 hrs, 2 animals (the control animal and one treated with NMPF) were sacrificed to compare the direct effect of the compound at the level of histology. The 3rd animal, treated with NMPF, was allowed to recover from anesthesia and was intensively observed during the first 12 hours after recovery followed by frequent daily observation. The decision to allow the 3rd animal to recover was made after consulting with the veterinarian.
Induction of septic shock. Before the infusion of E. coli, a 1 hr pre-infusion monitoring of heart-rate and blood pressure was performed. All three animals received an i.v. injection of E. coli 086 (k61 serotype; ATCC 33985) at a lethal dose of 10×109 CFU/kg body weight. In a dose titration study with this batch performed in 1991, this bacterial dose induced lethal shock within 8 hrs after the start of the infusion. The infusion period was 2 hrs.
Antibiotics. Baytril was administered intravenously immediately after completion of the 2 h.E. coli infusion (i.v.; dose 9 mg/kg).
Treatment with NMPF. 30 minutes post-onset of E. coli infusion, the animals were administered a single intravenous bolus injection of a mixer of NMPF peptides. The peptide mixture contained the following NMPF peptides: LQGV (SEQ ID NO:1) (5 mg/kg), AQGV (SEQ ID NO:2) (5 mg/kg) and VLPALP (SEQ ID NO:3) (5 mg/kg). These NMPF peptides were dissolved in 0.9% sodium chloride for injection (N.P.B.I., Emmer Compascuum, NL).
RESULTS
Preliminary Monkey Results
An anti-shock effect of the test compound on sepsis in the monkey treated with the oligopeptide mixture, namely the inhibition of the effect of the sepsis in this early 7-hour trajectory of this primate model, was observed. Immunomodulatory effects with these peptides have been observed in vitro/ex vivo such as in T-cell assays, the inhibition of pathological Th1 immune responses, suppression of inflammatory cytokines (MIF), increase in production of anti-inflammatory cytokines (IL-10, TGF-beta) and immunomodulatory effects on antigen-presenting cells (APC) like dendritic cells and macrophages.
The following organs were weighed and a bacterial count was performed: kidneys, liver, lungs, lymph nodes, and gross lesions.
Tissues of all organs were preserved in neutral aqueous phosphate buffered 4% solution of formaldehyde. Lymphoid organs were cryopreserved. All tissues will be processed for histopathological examination.
Further Results Obtained in the Three-Monkey Experiment
Monkey 429(control). Female monkey (5.66 kg) received an i.v. injection of E. coli 086 (10E10 CFU/kg). In a dose titration study with this batch performed in 1991, this bacterial dose induced lethal shock within 8 hrs after the start of the infusion. The infusion period was 2 hrs. Baytril was administered intravenously immediately after completion of the 2 h. E. coli infusion (i.v.; dose 9 mg/kg). After the E. coli injection, the monkey was observed by the authorized veterinarian without knowing which of the monkeys received NMPF treatment. The clinical observations were as follows: vomiting, undetectable pulse, heart arythmia, abnormalities in ECG: signs of ventricle dilatation/heart decompensation (prolonged QRS complex, extra systoles), decreased blood clotting and forced respiration. In addition, there was big fluctuation in heart rate (30-150 beats per minute), collapse of both systolic and diastolic blood pressure (35/20 mmHg) and decrease in blood oxygen concentration (80-70%). Seven hours after the start of the E. coli infusion, monkey began to vomit blood and feces, and have convulsions. After final examination, the veterinarian did not give permission to let this monkey awake. At this time point, the control monkey was euthanized. Hereafter, post-mortem examination was conducted and internal organs were examined in situ. A number of internal bleedings were found by the pathologist.
Monkey 459(NMPF). Female monkey (5.44 kg) received an i.v. injection of E. coli 086 (10E10 CFU/kg). In a dose titration study with this batch performed in 1991, this bacterial dose induced lethal shock within 8 hrs after the start of the infusion. The infusion period was 2 hrs. Thirty minutes after the initiation of E. coli infusion, NMPF was i.v. injected in a single bolus injection. Baytril was administered intravenously immediately after completion of the 2 h. E. coli infusion (i.v.; dose 9 mg/kg). After the E. coli injection, this monkey was also observed by the authorized veterinarian without knowing which of the monkeys received NMPF treatment. The clinical observations were as follows: normal pulse, heart sounds normal, normal ECG, higher heart-rate but otherwise stable (180 beats per minute), no hypotension (75/30 mmHg), normal blood oxygen concentration (95-85%), lungs sound normal, normal turgor. Seven hours after the start of the E. coli infusion, the clinical condition of the monkey was stable. After final examination, the veterinarian did give permission to let this monkey awake due to her stable condition. In order to compare the hematological and immunological parameters between the control and NMPF-treated monkey, at this time point the NMPF-treated monkey 459 was euthanized. Hereafter, post-mortem examination was conducted and internal organs were examined in situ. No macroscopic internal bleedings were found by the pathologist.
Monkey 427(NMPF). Female monkey (4.84 kg) received an i.v. injection of E. coli 086 (10E10 CFU/kg). In a dose titration study with this batch performed in 1991, this bacterial dose induced lethal shock within 8 hrs after the start of the infusion. The infusion period was 2 hrs. Thirty minutes after the initiation of E. coli infusion, NMPF was i.v. injected. Baytril was administered intravenously immediately after completion of the 2 h. E. coli infusion (i.v.; dose 9 mg/kg). After the E. coli injection, this monkey was also observed by the authorized veterinarian doctor without knowing which of the monkeys received NMPF treatment. The clinical observations were as follows: normal pulse, heart sounds normal, normal ECG, moderately higher heart-rate but otherwise stable (160 beats per minute), no hypotension (70/30 mmHg), normal blood oxygen concentration (95-90%), lungs sound normal, normal turgor. Seven hours after the start of the E. coli infusion, the clinical condition of the monkey was stable. After final examination, the veterinarian did give permission to let this monkey wake up due to her stable condition. The monkey woke up quickly, she was alert and there was a slow disappearance of oedema.
Cells, for example, 3T3 cells, are transfected with an expression cassette that expresses the peptide of SEQ ID NO:1 or SEQ ID NO:34, which is found to increase production of NF-κB. Production of NF-κB may be assayed by electrophoretic mobility shift assays. For example, an oligonucleotide representing NF-κB binding sequence such as (5′-AGC TCA GAG GGG GAC TTT CCG AGA G-3′) (SEQ ID NO: 51) are synthesized. Hundred pico mol sense and antisense oligo are annealed and labeled with α-32P-dATP using T4 polynucleotide kinase according to manufacture's instructions (Promega, Madison, Wis.). Cytosolic extract or nuclear extract (5-7.5 μg) from cells expressing the regulatory peptide or from untreated cells (e.g., as a control) is incubated for 30 minutes with 75000 cpm probe in binding reaction mixture (20 μl) containing 0.5 μg poly dI-dC (Amersham Pharmacia Biotech) and binding buffer BSB (25 mM MgC12, 5 mM CaCl2, 5 mM DTT and 20% Ficoll) at room temperature. Or cytosolic and nuclear extract from untreated cells or from cells treated with stimuli could also be incubated with probe in binding reaction mixture and binding buffer. The DNA-protein complexes are resolved from free oligonucleotide by electrophoresis in a 4-6% polyacrylamide gel (150 V, 2-4 hours). The gel is then dried and exposed to x-ray film.
Increased Expression
Candidate Peptides can also be biotinylated and incubated with cells. Cells are then washed with phosphate-buffered saline, harvested in the absence or presence of certain stimulus (LPS, PHA, TPA, anti-CD3, VEGF, TSST-1, VIP or know drugs etc.). After culturing, cells are lysed and cells lysates (whole lysate, cytosolic fraction or nuclear fraction) containing 200 micro gram of protein are incubated with 50 miroliters of Neutr-Avidin-plus beads for 1 h at 4° C. with constant shaking. Beads are washed five times with lysis buffer by centrifugation at 6000 rpm for 1 min. Proteins are eluted by incubating the beads in 0.05 N NaOH for 1 min at room temperature to hydrolyze the protein-peptide linkage and analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoprecipitated with agarose-conjugated anti-NF-κB subunits antibody or immunoprecipitated with antibody against the target, where that target is other than NF-κB. After hydrolyzing the protein-peptide linkage, the sample may be analyzed on HPLS and mass-spectrometry. Purified NF-κB subunits or cell lysate interaction with biotinylated regulatory peptide can be analyzed using biosensor technology. Peptides can be labeled with FITC and incubated with cells in the absence or presence of different stimulus. After culturing, cells can be analyzed with fluorescent microscopy, confocal microscopy, flow cytometry (cell membrane staining and/or intracellular staining) or cells lysates are made and analyzed on HPLC and mass-spectrometry. NF-κB transfected (reporter gene assay) cells and gene array technology can be used to determine the regulatory effects of peptides.
An exemplary embodiment of the invention provides an improvement in a method of screening a candidate compound for biological activity by screening the compound in a cell line, the improvement comprising: screening said candidate compound in a cell line wherein said cell line has been contacted with at least one exogenously added biologically active peptide having gene regulatory activity.
RNA Extraction and DNA Microarray Procedures
RNA was isolated using RNeasy columns as described by the manufacturer (Qiagen, Hilden, Germany). The integrity of the RNA was tested on 1% formaldehyde containing agarose gels. A total of 5 μg of RNA was used to generate ds cDNA using superscript reverse transcriptase and a T7-oligodT primer. The resulting cDNA was used in an in vitro cRNA reaction using T7 RNA polymerase and biotinylated ribonucleotides employing an ENZO kit (ENZO, Farmingdale, N.Y., USA). The biotinylated cRNA was cleaned-up using RNeasy spin columns (Qiagen) and quantified by spectrophotometric methods. An adjusted cRNA yield was calculated to reflect carryover of unlabeled total RNA. Fragmentation of 20 μg cRNA was performed at 95° C. for 35 min. Fragmented cRNA (10 μg) was subsequently hybridized for 16 h to U95A microarrays (Affymetrix) at 45° C. After washing and staining with PE-conjugated streptavidin, the arrays were scanned in an HPAffymetrix scanner at 570 nm using a kryptonargon laser.
The scanned images were analyzed using Affymetrix Microarray suite 4.2 software, using either LPS, PHA or only PBS treated sample as baseline.
Ratios between the 5′ oligonucleotides and the 3′ oligonucleotides of GAPDH transcripts were <1.5 (usually 0.91.1.), indicating that the amount of labeling was equally distributed over the RNA molecules. This implies that no major degradation of RNA occurred. In comparison experiments, care was taken that the scaling factor, noise, and presence calls were all comparable.
Analysis of the result was performed as follows: for example two arrays were compared, LPS and PBS treated sample, using Affymetrix Microarray suite 4.2. PBS array was used as baseline. Genes absent in both arrays and genes not changed were deleted. The ten most decreased and increased genes were collected and are described in tables. Affymetrix probeset descriptions are collected from either the internet site of Affymetrix (affymetrix.com/index.affx) or from EASE version 2.0 software. A description of the gene accession numbers are also shown at nucleotide database of internet site (ncbi.nlm.nih.gov/entrez).
The peptides that are used in the present experimental design are usually of synthetic origin and produced by chemical synthesis. Nevertheless, where desirable and appropriate, the peptides may also be isolated by fractionating a biological sample or produced by recombinant techniques known in the art. In addition, the peptides, whether chemically synthesized or produced by recombinant techniques, may contain modifications known in the art.
Based on the disclosure herein, a person of ordinary skill in the art will recognize that the peptides tested, may be of any length, particularly desirable are trimers and tetramers, but any peptide fragment may be tested using the methodology disclosed herein. Furthermore, the results of the test may beneficially be compiled into a database, which may then be searched and/or used to identify peptides with desirable activities.
Results:
Analysis of different peptides, which may optionally be in a database or use an exisiting database, may be conducted using: proteomics tools and/or sequence alignment tools, such as BLAST database (ExPasy, NCBI), SMART (EMBL), and PATTINPROT (PBIL); Post-translational modification prediction tools, for example, SignalP (CBS), Primary structure analysis; HLA Peptide Binding Predictions (e.g., BIMAS); Prediction of MHC type I and II peptide binding (e.g., SYFPEITHI); Amino acid scale representation (which may measure hydrophobicity, and other conformational parameters) (e.g., PROTSCALE); and Representations of a protein fragment as a helical wheel (e.g., HelixWheel/HelixDraw).
All references, including database accession numbers, publications, patents, and patent applications, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 00201139.3 | Mar 2000 | EP | regional |
| 01203748.7 | Oct 2001 | EP | regional |
This application is a continuation in part of co-pending application, U.S. Ser. No. 10/753,510, filed Jan. 7, 2004, the contents of the entirety of which is incorporated by this reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10262522 | Sep 2002 | US |
| Child | 10753510 | Jan 2004 | US |
| Parent | PCT/NL01/00259 | Mar 2001 | US |
| Child | 10262522 | Sep 2002 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10753510 | Jan 2004 | US |
| Child | 10821240 | Apr 2004 | US |
| Parent | 10028075 | Dec 2001 | US |
| Child | 10753510 | Jan 2004 | US |
| Parent | PCT/NL02/00639 | Apr 2003 | US |
| Child | 10753510 | Jan 2004 | US |
