Patent Grant
7862826
The invention relates to combinations of neurochemically active agents for treating a nervous system and the methods of treating a nervous system with the combinatorial treatments.
The contents of the electronic submission of text file Sequence Listing is incorporated herein by reference in its entirety.
The nervous system is comprised of two divisions: the central nervous system (CNS) and the peripheral nervous system (PNS). The CNS includes the brain and the spinal cord and controls most functions of the body and mind. The remainder of the nervous system is the PNS. Nerves of the PNS connect the CNS to sensory organs (such as the eyes and ears), other organs of the body, muscles, blood vessels, and glands. The peripheral nerves include the cranial nerves, the spinal nerves, and roots.
The CNS controls all voluntary movement, such as movement of the legs during walking, and all involuntary movement, such as beating of the heart. The spinal cord connects the body and the brain by transmitting information to and from the body and the brain.
The nervous system can be injured in numerous ways, and injuries can be traumatic. For instance, sudden physical assault on a portion of the nervous system results in a traumatic injury. In the case of a traumatic brain injury, the injury can be focal, i.e., confined to a specific area of the brain, or diffuse, i.e., involving more than one area of the brain.
Injuries to the nervous system include contusions, which are bruises of the nervous system, and blood clots. Blood clots can form in or around the nervous system. For example, when bleeding occurs between the skull and the brain, the blood forms a clot. This puts pressure on the brain, which can lead to changes in brain function.
Spinal cord injuries (SCI) are a particular type of injury to the nervous system. As of the year 2000, approximately 450,000 people in the United States have sustained SCI, with more than 10,000 new cases reported in the United States every year. Motor vehicle accidents are the leading cause of SCI (44 percent), followed by acts of violence (24 percent), falls (22 percent), sports injuries (8 percent), and other causes (2 percent). Of the 10,000 new cases of SCI in the United States each year, 51.7% have tetraplegia, i.e., injuries to one of the eight cervical segments of the spinal cord, and 56.7% have paraplegia, i.e., lesions in the thoracic, lumbar, or sacral regions of the spinal cord. Since 1990, the most frequent neurologic category is incomplete tetraplegia (29.5%), followed by complete paraplegia (27.9%), incomplete paraplegia (21.3%), and complete tetraplegia (18.5%).
With spinal cord injuries in the neck, significant impairment of breathing may result. The most frequent site of spinal injury is the neck or cervical region and, of these, the major cause of death arises from respiratory complications. For patients that survive a major spinal cord injury in the neck, they may spend the rest of their lives depending on an artificial ventilator or phrenic nerve pacemaker to sustain their lives. For others with less severe respiratory impairment, they may be able to breathe normally, but are unable to sigh or breathe deeply and maintain the integrity of the lung. As a consequence, regions of the lung will collapse in these patients, causing pneumonia and allowing other respiratory infections to become established. Clearly, restoration of normal breathing ability, including deep breaths and sighs, is a major goal in the treatment of spinal cord injury patients.
Injury to the spinal cord and other parts of the nervous system may be particularly devastating to life and the quality of life. In addition, injury to the nervous system can engender serious economic losses to the individual and to society. Currently, there are few effective treatment options available for patients with spinal cord injuries, although there are a few promising indications that physical therapy or chronic intermittent hypoxia (CIH), may have beneficial effects. Exposure to intermittent hypoxic episodes has been shown to initiate spinal protein synthesis. However, studies have also shown that chronic intermittent hypoxia has other drawbacks as a treatment for spinal cord injuries. For example, certain CIH treatment methods can cause systemic hypertension, altered sympathetic chemoreflexes, and hippocampal cell death by the process of apoptosis.
Physical training and preconditioning have been used to treat SCI. Almost all patients with spinal cord injuries can now achieve a partial return of function with proper physical therapy that maintains flexibility and function of the muscles and joints, and strengthens the neural pathways that underlie movement. Physical therapy can also help reduce the risk of blood clots and boost the patient's morale. Physical training currently being investigated includes body weight-supported treadmill training, in which patients with partial spinal cord injury “walk” on a treadmill while they are partially supported through the use of a specially designed harness attached to an overhead lift. Unfortunately, this type of therapy is very expensive, and efficacy is far from complete.
The invention, which is defined by the claims set out at the end of this disclosure, is intended to solve at least some of the problems noted above. A composition is provided that includes an effective amount of at least one of an antimicrobial peptide and a substance having an antimicrobial peptide effect. The composition also includes an effective amount of a neurotrophin.
In another embodiment, the composition also includes an effective amount of at least one of a growth factor and a neuropeptide.
Also provided is a method of treating an injury to a nervous system of an animal. In one embodiment, the method includes the steps of identifying the injury to the nervous system and applying to the injury an effective amount of at least one of antimicrobial peptide and a substance having an antimicrobial peptide effect.
In another embodiment, an injury to the nervous system is identified. An effective amount of at least one of an antimicrobial peptide and a substance having an antimicrobial peptide effect is combined with an effective amount of one or more trophic factors selected from the group consisting of a growth factor, a neurotrophin, and a neuropeptide. The combination is applied to the injury.
A kit is also provided. In an embodiment, the kit includes at least one of an antimicrobial peptide and a substance having an antimicrobial peptide effect. The kit also includes a neurotrophin. In another embodiment, the kit also includes a viscous substance. In some embodiments, the kit also includes at least one of a growth factor and a neuropeptide.
Preferred exemplary embodiments of the invention are illustrated in the accompanying drawings in which:
Before explaining embodiments of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
To facilitate understanding of the invention, a number of terms are defined below.
As used herein, the term “antimicrobial polypeptide” refers to polypeptides that inhibit the growth of microbes (e.g., bacteria). Examples of antimicrobial polypeptides include, but are not limited to, the polypeptides described in Tables 1 and 2 below. Antimicrobial polypeptides include peptides synthesized from both L-amino and D-amino acids.
As used herein, the term “pore forming agent” refers to any agent (e.g., peptide or other organic compound) that forms pores in a biological membrane. When the pore forming agent is a peptide, the peptide can be synthesized from both L-amino and D-amino acids.
As used herein, the term “growth factor” refers to any compound that is involved in cell differentiation and growth. Growth factors can be proteins (e.g., IGF-1 (insulin-like growth factor 1), IGF-2 (insulin-like growth factor 2), NGF-β (nerve growth factor-β), EGF (epidermal growth factor), CSGF (colony-stimulating growth factor), FGF (fibroblast growth factor), PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor), TGF-β (transforming growth factor β, and bone morphogenetic proteins)), either purified from natural sources or genetically engineered, as well as fragments, mimetics, and derivatives or modifications thereof. Further examples of growth factors are provided in U.S. Pat. Nos. 5,183,805; 5,218,093; 5,130,298; 5,639,664; 5,457,034; 5,210,185; 5,470,828; 5,650,496; 5,998,376; and 5,410,019; all of which are incorporated herein by reference.
The term “trophic factor” as used herein refers to a substance that stimulates growth and development or stimulates increased activity.
The term “hyaluronic acid” includes hyaluronic acid and its derivatives, for instance, esters, salts such as the sodium, potassium, magnesium, calcium, alkaline, alkaline earth metals, and the like, and derivatives such as sulphated or polysulphated hyaluronates, or hyaluronates that have been otherwise modified in a manner way such that the function of hyaluronic acid is retained.
The term “recombinant protein” or “recombinant polypeptide” as used herein refers to a protein molecule expressed from a recombinant DNA molecule. In contrast, the term “native protein” or “native polypeptide” is used herein to indicate a protein isolated from a naturally occurring (i.e., a nonrecombinant) source. Molecular biological techniques may be used to produce a recombinant form of a protein or polypeptide with similar or identical properties as compared to the native form of the protein.
Where an amino acid sequence is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, amino acid sequence and like terms, such as polypeptide or protein are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
As used herein in reference to an amino acid sequence or a protein, the term “portion” (as in “a portion of an amino acid sequence”) refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid (e.g., 5, 6, 7, 8, . . . x−1).
As used herein, the term “variant,” when used in reference to a protein, refers to a protein encoded by partially homologous nucleic acids so that the amino acid sequence of the protein varies. As used herein, the term “variant” encompasses proteins encoded by homologous genes having both conservative and nonconservative amino acid substitutions that do not result in a change in protein function, as well as proteins encoded by homologous genes having amino acid substitutions that cause decreased protein function or increased protein function.
As used herein, the term “fusion protein” refers to a chimeric protein containing the protein of interest (e.g., defensins and fragments thereof) joined to a heterologous protein fragment (e.g., the fusion partner which consists of a non-defensin protein). The fusion partner may enhance the solubility of a defensin as expressed in a host cell, may provide an affinity tag to allow purification of the recombinant fusion protein from the host cell or culture supernatant, or both. If desired, the fusion protein may be removed from the protein of interest (e.g., defensin or fragments thereof) by a variety of enzymatic or chemical processes known to the art.
As used herein, the term “purified” refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated, or separated. The percent of a purified component is thereby increased in the sample. For example, an isolated defensin is therefore a purified defensin. Substantially purified molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are naturally associated.
The term “gene” as used herein, refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or protein precursor. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence, as long as the desired protein activity is retained.
The term “homology” refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A “partially complementary sequence” is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid. This situation is referred to using the functional term “substantially homologous.” The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (e.g., Southern or Northern blot, solution hybridization, and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence or probe to a target under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target that lacks even a partial degree of complementarity (e.g., less than about 30% identity). In this case, in the absence of non-specific binding, the probe will not hybridize to the second non-complementary target.
When used in reference to a double-stranded nucleic acid sequence such as a cDNA or a genomic clone, the term “substantially homologous” refers to any probe which can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described herein.
As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acid strands. Hybridization and the strength of hybridization (i.e., the strength of the association between nucleic acid strands) is impacted by many factors well known in the art including the degree of complementarity between the nucleic acids, stringency of the conditions involved affected by such conditions as the concentration of salts, the Tm (melting temperature) of the formed hybrid, the presence of other components (e.g., the presence or absence of polyethylene glycol), the molarity of the hybridizing strands, and the G:C content of the nucleic acid strands.
As used herein, the term “stringency” is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds, under which nucleic acid hybridizations are conducted. With high stringency conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of medium or low stringency are often required when it is desired that nucleic acids that are not completely complementary to one another be hybridized or annealed together. It is well known in the art that numerous equivalent conditions can be employed to comprise medium or low stringency conditions. The choice of hybridization conditions is generally evident to one skilled in the art and is normally guided by the purpose of the hybridization, the type of hybridization (DNA-DNA or DNA-RNA), and the level of desired relatedness between the sequences (e.g., Sambrook et al., 1989, Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington D.C., 1985, for a general discussion of the state of the art).
The stability of nucleic acid duplexes is known to decrease with an increased number of mismatched bases, and further to be decreased to a greater or lesser degree depending on the relative positions of mismatches in the hybrid duplexes. Thus, the stringency of hybridization can be used to maximize or minimize stability of such duplexes. Hybridization stringency can be altered, for example, by adjusting the temperature of hybridization; adjusting the percentage of helix destabilizing agents, such as formamide, in the hybridization mix; and adjusting the temperature and/or salt concentration of the wash solutions. For filter hybridizations, the final stringency of hybridizations can be determined by the salt concentration and/or temperature used for the post-hybridization washes.
“High stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4.H2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when a probe of about 500 nucleotides in length is employed.
“Medium stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4.H2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when a probe of about 500 nucleotides in length is employed.
“Low stringency conditions” comprise conditions equivalent to binding or hybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4.H2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500 nucleotides in length is employed.
As used herein, the term “Tm” is used in reference to the melting temperature, which is the temperature at which 50% of a population of double-stranded nucleic acid molecules becomes dissociated into single strands. The equation for calculating the Tm of nucleic acids is well known in the art. The Tm of a hybrid nucleic acid can be estimated using a formula adopted from hybridization assays in 1 M salt, and commonly used for calculating Tm for PCR primers: [(number of A+T)×2° C.+(number of G+C)×4° C.]. (C. R. Newton et al., PCR, 2nd Ed., Springer-Verlag (New York, 1997), p. 24). This formula was found to be inaccurate for primers longer than 20 nucleotides. (Id.) Another simple estimate of the Tm value can be calculated by the equation: Tm=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl. (e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985). Other more sophisticated computations exist in the art which take structural as well as sequence characteristics into account for the calculation of Tm. A calculated Tm is merely an estimate; the optimum temperature is commonly determined empirically.
As used herein, the term “vector” is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another and capable of replication in a cell. Vectors may include plasmids, bacteriophages, viruses, cosmids, and the like.
The terms “recombinant vector” and “expression vector” as used herein refer to DNA or RNA sequences containing a desired coding sequence and appropriate DNA or RNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Prokaryotic expression vectors include a promoter, a ribosome binding site, an origin of replication for autonomous replication in host cells and can also include other sequences, e.g., an optional operator sequence. A “promoter” is defined as a DNA sequence that directs RNA polymerase to bind to DNA and to initiate RNA synthesis. Eukaryotic expression vectors include a promoter, polyadenlyation signal and optionally an enhancer sequence.
As used herein the term “coding region” when used in reference to structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. Typically, the coding region is bounded on the 5′ side by the nucleotide triplet ATG, which encodes the initiator methionine, and on the 3′ side by a stop codon (e.g., TAA, TAG, TGA). In some cases, the coding region is also known to initiate by a nucleotide triplet TTG.
The terms “buffer” or “buffering agents” refer to materials that when added to a solution, cause the solution to resist changes in pH.
The term “monovalent salt” refers to any salt in which the metal (e.g., Na, K, or Li) has a net 1+ charge in solution (i.e., one more proton than electron).
The term “divalent salt” refers to any salt in which a metal (e.g., Mg, Ca, or Sr) has a net 2+ charge in solution.
The term “solution” refers to an aqueous mixture.
The term “buffering solution” refers to a solution containing a buffering reagent.
The present invention relates to neurochemically active agents and combinations thereof. Neurochemically active agents include one or more antimicrobial peptide and/or a substance having an antimicrobial peptide effect. Antimicrobial peptides themselves are known to have trophic effects. As such, an antimicrobial peptide and/or a substance having an antimicrobial peptide effect can be used by itself in the methods of the invention. Neurochemically active agents also include one or more growth factor, neurotrophin, and neuropeptide. Combinations of neurochemically active agents are referred to herein as “trophic factor combinations.”
According to the invention, neurochemically active agents can be used alone or in combination to treat injuries to the nervous system, i.e., the central nervous system and the peripheral nervous system. The one or more neurochemically active agents can be used to treat nervous system injuries, including trauma induced injuries, degenerative induced injuries, age induced injuries, and infection induced injuries. Injuries that can be treated include, but are not limited to, spinal cord injury, including severed spinal cords; peripheral nerve damage, brain injuries, e.g., blood clots, tumors, strokes, and ischemis and perfusion; and Parkinson's disease, Alzheimer disease, muscular dystrophy, amyotrophic lateral sclerosis, multiple sclerosis, Pick's disease, prion diseases, Huntington disease, and related disorders.
When applied to a the nervous system, trophic factor combinations of the invention result in at least one of the following: lower loss in body weight after the injury when compared to controls not receiving the trophic factor combinations, strengthened motor recovery in injured animals treated with the trophic factor combination when compared to animals not treated with the trophic factor combination, larger evoked potentials in nerves when compared to controls not receiving the trophic factor combination, and a lower current required to evoke a response (threshold current) when compared to controls not receiving the trophic factor combination.
It is contemplated that the trophic factor combinations of the present invention used to treat injuries of the nervous system result in reduced inflammation, growth of new cells, increased plasticity, among other beneficial effects.
I. Trophic Factor Combinations
The present invention contemplates the use of trophic factor combinations and their individual components for treatment of injuries to the nervous system. Trophic factor combinations according to the invention can include one or more of the following elements: antimicrobial polypeptides (e.g., defensins), a substance having an effect of an antimicrobial peptide, a growth factor, a neurotrophin, and a neuropeptide. Additional components can also be included and are discussed below.
A. Antimicrobial Peptides
In some embodiments, one or more antimicrobial polypeptides and/or one or more substances having an antimicrobial peptide effect are used as a trophic factor to treat an injury to a nervous system. For additional information on antimicrobial peptides, see, for example, Antimicrobial Peptide Protocols, ed. W. M. Shafer, Humana Press, Totowa, N.J., 1997; and databases including http://aps.unmc.edu/AP/main.php (discussed in Wang Z, Wang G., APD: the Antimicrobial Peptide Database, Nucleic Acids Res. 2004 Jan. 1; 32(Database issue):D590-2), http://sdmc.lit.org.sg/Templar/DB/Antimic/, and http://www.bbcm.units.it/˜zelezetsk/hdpdb.html (database of defense peptides) and Table 1 below.
In some embodiments, the antimicrobial peptide is a compound or peptide selected from the following: bovine defensin peptide (BNP-1, Romeo et al., J. Biol. Chem. 263(15):9573-9575 [1988]), magainin (e.g., magainin I, magainin II, xenopsin, xenopsin precursor fragment, caerulein precursor fragment), magainin I and II analogs (PGLa, magainin A, magainin G, pexiganin, Z-12, pexigainin acetate, D35, MSI-78A, MG0 [K10E, K11E, F12W-magainin 2], MG2+ [KIOE, F12W-magainin-2], MG4+ [F12W-magainin 2], MG6+ [f12W, E19Q-magainin 2 amide], MSI-238, reversed magainin II analogs [e.g., 53D, 87-ISM, and A87-ISM], Ala-magainin II amide, magainin II amide), cecropin P1, cecropin A, cecropin B, indolicidin, nisin, ranalexin, lactoferricin B, poly-L-lysine, cecropin A (1-8)-magainin II (1-12), cecropin A (1-8)-melittin (1-12), CA(1-13)-MA(1-13), CA(1-13)-ME(1-13), gramicidin, gramicidin A, gramicidin D, gramicidin S, alamethicin, protegrin, histatin, dermaseptin, lentivirus amphipathic peptide or analog, parasin I, lycotoxin I or II, globomycin, gramicidin S, surfactin, ralinomycin, valinomycin, polymyxin B, PM2 [(+/−) 1-(4-aminobutyl)-6-benzylindane], PM2c [(+/−)-6-benzyl-1-(3-carboxypropyl)indane], PM3 [(+/−) 1-benzyl-6-(4-aminobutyl)indane], tachyplesin, buforin I or II, misgurin, melittin, PR-39, PR-26, 9-phenylnonylamine, (KLAKKLA)n, (KLAKLAK)n, where n=1, 2, or 3, (KALKALK)3, KLGKKLG)n, and KAAKKAA)n, wherein N=1, 2, or 3, paradaxin, Bac 5, Bac 7, ceratoxin, mdelin 1 and 5, bombin-like peptides, PGQ, cathelicidin, HD-5, Oabac5alpha, ChBac5, SMAP-29, Bac7.5, lactoferrin, granulysin, thionin, hevein and knottin-like peptides, MPG1, 1bAMP, snakin, lipid transfer proteins, and plant defensins. Exemplary sequences for the above listed compounds are provided in Table 1. In some embodiments, the antimicrobial peptides or substances having an antimicrobial peptide effect (where they are peptides) are synthesized from L-amino acids, while in other embodiments, the peptides are synthesized from or comprise D-amino acids.
The compounds listed above can be isolated and purified from natural sources as appropriate. The compounds can also be produced recombinantly or synthetically, as described below.
In preferred embodiments, the trophic factor combinations of the present invention comprise one or more antimicrobial polypeptides and/or one or more substance having an antimicrobial peptide effect at a concentration of about 0.01 to about 1000 mg/L. In preferred embodiments, the trophic factor combinations comprise a solution comprising one or more antimicrobial polypeptides at a concentration of about 0.1 to about 5 mg/L.
In some embodiments of the present invention, the antimicrobial polypeptide is a defensin. In preferred embodiments, the trophic factor combinations of the present invention comprise one or more defensins. In further preferred embodiments, the trophic factor combination comprises a solution comprising purified defensins at a concentration of about 0.01 to 1000 mg/L. In particularly preferred embodiments, the trophic factor combinations comprise a solution comprising defensins at a concentration of about 0.1 to 5 mg/L. In still further preferred embodiments, the antimicrobial polypeptide is BNP1 (also known as bactanecin and bovine dodecapeptide). In certain embodiments, the defensin comprises the following consensus sequence: X1CN1CRN2CN3ERN4CN5GN6CCX2, wherein N and X represent conservatively or nonconservatively substituted amino acids and N1=1, N2=3 or 4, N3=3 or 4, N4=1, 2, or 3, N6=5-9, X1 and X2 may be present, absent, or equal from 1-2.
The present invention is not limited to any particular defensin. Indeed, trophic factor combinations comprising a variety of defensins are contemplated. Representative defensins are provided in Tables 1 and 2 below. In general, defensins are a family of highly cross-linked, structurally homologous antimicrobial peptides that can be found in the azurophil granules of polymorphonuclear leukocytes (PMNs) with homologous peptides being present in macrophages (e.g., Selsted et al., Infect. Immun. 45:150-154 [1984]). Originally described as “Lysosomal Cationic Peptides” in rabbit and guinea pig PMN (Zeya et al., Science 154:1049-1051 [1966]; Zeya et al., J. Exp. Med. 127:927-941 [1968]; Zeya et al., Lab. Invest. 24:229-236 [1971]; Selsted et al., [1984], supra.), this mixture was found to account for most of the microbicidal activity of the crude rabbit PMN extract against various microorganisms (Zeya et al., [1966], supra; Lehrer et al., J. Infect. Dis. 136:96-99 [1977]; Lehrer et al., Infect. Immun. 11:1226-1234 [1975]). Six rabbit neutrophil defensins have been individually purified and are designated NP-1, NP-2, NP-3A, NP-3B, NP-4, and NP-5. Their amino acid sequences were determined, and their broad spectra of activity were demonstrated against a number of bacteria (Selsted et al., Infect. Immun. 45:150-154 [1984]), viruses (Lehrer et al., J. Virol. 54:467 [1985]), and fungi (Selsted et al., Infect. Immun. 49:202-206 [1985]; Segal et al., 151:890-894 [1985]). Defensins have also been shown to possess mitogenic activity (e.g., Murphy et al., J. Cell. Physiol. 155:408-13 [1993]).
Four peptides of the defensin family have been isolated from human PMN's and are designated HNP-1, HNP-2, HNP-3, and LNP-4 (Ganz et al., J. Clin. Invest. 76:1427-1435 [1985]; Wilde et al., J. Biol. Chem. 264:11200-11203 [1989]). The amino acid sequences of HNP-1, HNP-2, and HNP-3 differ from each other only in their amino terminal residues, while each of the human defensins are identical to the six rabbit peptides in 10 or 11 of their 29 to 30 residues. These are the same 10 or 11 residues that are shared by all six rabbit peptides. Human defensin peptides have been shown to share with the rabbit defensins a broad spectrum of antimicrobial activity against bacteria, fungi, and enveloped viruses (Ganz et al., [1985], supra).
Three defensins designated RatNP-1, RatNP-2, and RatNP-4, have been isolated from rat (Eisenhauer et al., Infection and Immunity 57:2021-2027 [1989]). A guinea pig defensin (GPNP) has also been isolated, purified, sequenced and its broad spectrum antimicrobial properties verified (Selsted et al., Infect. Immun. 55:2281-2286 [1987]). Eight of its 31 residues were among those invariant in six rabbit and three human defensin peptides. The sequence of GPNP also included three nonconservative substitutions in positions otherwise invariant in the human and rabbit peptides. Of the defensins tested in a quantitative assay HNP-1, RatNP-1, and rabbit NP-1 possess the most potent antimicrobial properties, while NP-5 possesses the least amount of antimicrobial activity when tested against a panel of organisms in stationary growth phase (Selsted et al., Infect. Immun. 45:150-154 [1984]; Ganz et al., J. Clin. Invest. 76:1427-1435 [1985]). Defensin peptides are further described in U.S. Pat. Nos. 4,543,252; 4,659,692; and 4,705,777 (each of which is incorporated herein by reference).
Defensin peptides suitable for use alone in the methods and/or in trophic factor combinations of the present invention include natural defensin peptides isolated from known cellular sources, synthetic peptides produced by solid phase or recombinant DNA techniques, and defensin analogs which may be smaller peptides or other molecules having similar binding and biological activity as the natural defensin peptides (e.g., peptide mimetics). Methods for the purification of defensin peptides are described in U.S. Pat. Nos. 4,543,252; 4,659,692; and 4,705,777, the disclosures of which are incorporated herein by reference.
In preferred embodiments, suitable synthetic peptides will comprise all or part of the amino acid sequence of a known peptide, more preferably incorporating at least some of the conserved regions identified in Table 2. In particularly preferred embodiments, the synthetic peptides incorporate at least one of the conserved regions, more typically incorporating two of the conserved regions, preferably conserving at least three of the conserved regions, and more preferably conserving four or more of the conserved regions. In preferred embodiments, the synthetic peptides comprise fifty amino acids or fewer, although there may be advantages in increasing the size of the peptide above that of the natural peptides in certain instances. In certain embodiments, the peptides have a length in the range from about 10 to 50 amino acids, preferably being in the range from about 10 to 40 amino acids, and most preferably being in the range from about 30 to 35 amino acids which corresponds generally to the length of the natural defensin peptides.
In some cases, it may be desirable to incorporate one or more non-natural amino acids in the synthetic defensin peptides of the present invention. In preferred embodiments, non-natural amino acids comprise at least an N-terminus and a C-terminus of the peptide and have side chains that are either identical to or chemically modified or substituted from a natural amino acid counterpart. An example of a non-natural amino acid is an optical isomer of a naturally-occurring L-amino acid, such as a peptide containing all D-amino acids. Examples of the synthesis of peptides containing all D-amino acids include Merrifield et al., Ciba Found Symp. 186:5-26 (1994); Wade et al., Proc. Natl. Acad. Sci. USA 87(12):4761-5 (1990); and U.S. Pat. No. 5,792,831, which is herein incorporated by reference. Examples of chemical modifications or substitutions include hydroxylation or fluorination of C—H bonds within natural amino acids. Such techniques are used in the manufacture of drug analogs of biological compounds and are known to one of ordinary skill in the art.
Synthetic peptides having biological and binding activity the same or similar to that of natural defensin peptides can be produced by either of two exemplary approaches. First, the polypeptides can be produced by the well-known Merrifield solid-phase chemical synthesis method wherein amino acids are sequentially added to a growing chain (Merrifield, J. Am. Chem. Soc. 85:2149-2156 [1963]). Automatic peptide synthesis equipment is available from several commercial suppliers, including PE Biosystems, Inc., Foster City, Calif.; Beckman Instruments, Inc., Waldwick, N.J.; and Biosearch, Inc., San Raphael, Calif. Using such automatic synthesizers according to manufacturer's instructions, peptides can be produced in gram quantities for use in the present invention.
Second, the synthetic defensin peptides of the present invention can be synthesized by recombinant techniques involving the expression in cultured cells of recombinant DNA molecules encoding a gene for a desired portion of a natural or analog defensin molecule. The gene encoding the defensin peptide can itself be natural or synthetic. Conveniently, polynucleotides can be synthesized by well-known techniques based on the desired amino acid sequence. For example, short single-stranded DNA fragments can be prepared by the phosphoramidite method (Beaucage et al., Tetra. Lett. 22:1859-1862 [1981]). A double-stranded fragment can then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence. The natural or synthetic DNA fragments coding for the desired defensin peptide can then be incorporated in a suitable DNA construct capable of introduction to and expression in an in vitro cell culture. The DNA fragments can be portions or variants of wild-type nucleic acids encoding defensins. Suitable variants include those both with conservative and nonconservative amino acid substitutions.
The methods, compositions, and trophic factor combinations of the present invention can also employ synthetic non-peptide compositions that have biological activity functionally comparable to that of known defensin peptides. By functionally comparable, it is meant that the shape, size, flexibility, and electronic configuration of the non-peptide molecule is such that the biological activity of the molecule is similar to defensin peptides. In particular, the non-peptide molecules should display comparable mitogenic activity and/or antimicrobial activity or pore forming ability, preferably possessing both activities. Such non-peptide molecules will typically be small molecules having a molecular weight in the range from about 100 to about 1000 daltons. The use of such small molecules is frequently advantageous in the preparation of trophic factor combinations. Candidate mimetics can be screened in large numbers to identify those having the desired activity.
The identification of such nonpeptide analog molecules can be performed using techniques known in the art of drug design. Such techniques include, but are not limited to, self-consistent field (SCF) analysis, configuration interaction (CI) analysis, and normal mode dynamics computer analysis, all of which are well described in the scientific literature (e.g., Rein et al., Computer-Assisted Modeling of Receptor-Ligand Interactions, Alan Liss, N.Y., [1989]). Preparation of the identified compounds will depend on the desired characteristics of the compounds and will involve standard chemical synthetic techniques (e.g., Cary et al., Advanced Organic Chemistry, part B, Plenum Press, New York [1983]).
In some embodiments of the present invention, one or more substances having an effect that an antimicrobial peptide has can be used. Effects that antimicrobial peptides have include, but are not limited to, the following: form pores on the cell membrane; enter cells without membrane lysis and, once in the cytoplasm, bind to, and inhibit the activity of specific molecular targets essential to bacterial growth, thereby causing cell death; induce expression of syndecan, an integral membrane proteoglycan associated largely with epithelial cells, in mesenchymal cells and inhibit the NADPH oxidase activity of neutrophils, suggesting a role of this peptide in wound repair and inflammation; exert a protective effect in various animal models of ischemia-reperfusion injury, preventing the post-ischemic oxidant production; induce angiogenesis both in vitro and in vivo; inhibit membrane protein synthesis; inhibit DNA synthesis; antitumor effect; stimulate cell proliferation; interfere with signal pathways; chemoattractant for immune cells; stimulate cytokine expression; stimulate adhesion molecule expression; angiogenesis; and chloride secretion.
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Carcinus
maenas
Cyprinus
carpio
Androctonus
australis
Leiurus
quinquestriatus
Ovis aries
Bombus
pascuorum
Apis
mellifera
Acalolepta
luxuriosa
Achatina
fulica
Sus scrofa
Phyllomedusa
bicolor
Macaca
mulatta
Macaca
mulatta
Macaca
mulatta
Bos taurus
Androctonus
australis
Drosophila
mauritiana
Drosophila
melanogaster
Drosophila
orena
Drosophila
sechellia
Drosophila
simulans
Drosophila
teissieri
Drosophila
yakuba
Bombina
maxima
Carcinus
maenas
Bos taurus
Bos taurus
Bos taurus
Bombyx
mori
Sus scrofa
Sus scrofa
Sus scrofa
Carcinus
maenas
Sus scrofa
Sus scrofa
Cavia
porcellus
Galleria
mellonella
Sarcophaga
peregrina
Xenopus
laevis
Acrocinus
longimanus
Acrocinus
longimanus
Acrocinus
longimanus
Glossina
morsitans
Manduca
sexta
Gallus
gallus
Gallus
gallus
Glossina
morsitans
Glossina
morsitans
Equus
caballus
Equus
caballus
Manduca
sexta
Sus scrofa
Lumbricus
rubellus
Mytilus
galloprovincialis
Manduca
sexta
Equus
caballus
Xenopus
laevis
Meleagris
gallopavo
Meleagris
gallopavo
Meleagris
gallopavo
Manduca
sexta
Oryctolagus
cuniculus
Pheretima
tschiliensis
Bombus
pascuorum
Apis
mellifera
Apis
mellifera
Apis
mellifera
Apis
mellifera
Bos taurus
Ascaris
suum
Ascaris
suum
Ascaris
suum
Ascaris
suum
Drosophila
melanogaster
Trichoplusia
ni
Drosophila
melanogaster
Hyalophora
cecropia
Hyalophora
cecropia
Bombyx
mori
Drosophila
melanogaster
Drosophila
melanogaster
Sus scrofa
Bos taurus
Ovis aries
Bos taurus
Capra
hircus
Bos taurus
Ovis aries
Bos taurus
Mus
musculus
Mus
musculus
Capra
hircus
Bos taurus
Capra
hircus
Sus scrofa
Pan
troglodytes
Mus
musculus
Rattus
norvegicus
Macaca
mulatta
Ovis aries
Bos taurus
Bos taurus
Macaca
mulatta
Bos taurus
Macaca
fascicularis
Bos taurus
Macaca
mulatta
Bos taurus
Mus
musculus
Rattus
norvegicus
Ovis aries
Mus
musculus
Bos taurus
Mus
musculus
Bos taurus
Mus
musculus
Bos taurus
Mus
musculus
Bos taurus
Mus
musculus
Bos taurus
Mus
musculus
Mus
musculus
Mus
musculus
Bos taurus
Bos taurus
Bos taurus
Gallus
gallus
Meleagris
gallopavo
Equus
caballus
Mus
musculus
Pan
troglodytes
Bos taurus
Pan
troglodytes
Bos taurus
Bos taurus
Bos taurus
Canis
familiaris
Canis
familiaris
Canis
familiaris
Tachypleus
tridentatus
Bombina
variegata
Bombina
variegata
Bombina
variegata
Bombina
variegata
Bombina
orientalis
Bombina
orientalis
Bombina
orientalis
Bombina
orientalis
Bombina
variegata
Bombina
variegata
Bombina
orientalis
Bos taurus
Rana
brevipoda
Rana
berlandieri
Rana
berlandieri
Rana
berlandieri
Rana
berlandieri
Rana
berlandieri
Rana
berlandieri
Rana
esculenta
Rana
esculenta
Rana
esculenta
Rana
esculenta
Rana
luteiventris
Rana
luteiventris
Rana
pipiens
Rana
pipiens
Rana
pipiens
Rana
pipiens
Rana
pipiens
Rana
sphenocephala
Rana
sphenocephala
Rana
sphenocephala
Rana
sylvatica
Rana
temporaria
Rana
temporaria
Rana
brevipoda
Rana
esculenta
Rana
esculenta
Rana
esculenta
Rana
esculenta
Rana
esculenta
Rana
esculenta
Rana
esculenta
Rana
temporaria
Rana
temporaria
Rana
temporaria
Rana
temporaria
Bufo
gargarizans
Androctonus
australis
Litoria
chloris
Litoria
xanthomera
Litoria
chloris
Litoria
xanthomera
Litoria
caerulea
Litoria
splendida
Litoria
chloris
Litoria
xanthomera
Litoria
chloris
Litoria
xanthomera
Litoria
xanthomera
Mus
musculus
Mus
musculus
Ovis aries
Ovis aries
Ovis aries
Ovis aries
Bombyx
mori
Ceratitis
capitata
Drosophila
virilis
Ceratitis
capitata
Drosophila
virilis
Drosophila
virilis
Aedes
aegypti
Bombyx
mori
Trichoplusia
ni
Hyalophora
cecropia
Spodoptera
litura
Drosophila
melanogaster
Antheraea
pernyi
Drosophila
melanogaster
Spodoptera
litura
Hyalophora
cecropia
Bombyx
mori
Drosophila
erecta
Drosophila
mauritiana
Bombyx
mori
Hyalophora
cecropia
Sus scrofa
Ceratitis
capitata
Ceratitis
capitata
Ceratitis
capitata
Ceratitis
capitata
Ceratitis
capitata
Ceratitis
capitata
Chlamys
islandica
Bos taurus
Bos taurus
Pagrus
major
Pagrus
major
Pagrus
major
Cicada
flammata
Litoria
citropa
Litoria
citropa
Litoria
citropa
Litoria
citropa
Litoria
citropa
Litoria
citropa
Litoria
citropa
Litoria
citropa
Litoria
citropa
Styela
clava
Styela
clava
Styela
clava
Styela
clava
Styela
clava
Zophobas
atratus
Oryctolagus
cuniculus
Oryctolagus
cuniculus
Oryctolagus
cuniculus
Oryctolagus
cuniculus
Oryctolagus
cuniculus
Oryctolagus
cuniculus
Vespa
crabro
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Bos taurus
Ovis aries
Cercopithecus
aethiops
Cercopithecus
erythrogaster
Gorilla
gorilla
Hylobates
concolor
Pan
troglodytes
Presbytis
obscura
Saguinus
oedipus
Mus
musculus
Aeshna
cyanea
Allomyrina
dichotoma
Anopheles
gambiae
Anopheles
gambiae
Bombus
pascuorum
Branchiostoma
belcheri
Drosophila
melanogaster
Drosophila
melanogaster
Drosophila
melanogaster
Drosophila
melanogaster
Drosophila
simulans
Mamestra
brassicae
Musca
domestica
Ornithodoros
moubata
Ornithodoros
moubata
Palomena
prasina
Phlebotomus
duboscqi
Pyrocoelia
rufa
Pyrrhocoris
apterus
Aedes
albopictus
Apis
mellifera
Stomoxys
calcitrans
Acalolepta
luxuriosa
Stomoxys
calcitrans
Stomoxys
calcitrans
Stomoxys
calcitrans
Stomoxys
calcitrans
Rattus
norvegicus
Aedes
aegypti
Mytilus
edulis
Ornithodoros
moubata
Rhodnius
prolixus
Aedes
aegypti
Aedes
aegypti
Aedes
aegypti
Aedes
aegypti
Aedes
aegypti
Macaca
mulatta
Macaca
mulatta
Macaca
mulatta
Macaca
mulatta
Macaca
mulatta
Macaca
mulatta
Aedes
aegypti
Ornithodoros
moubata
Rhodnius
prolixus
Mytilus
edulis
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Rhodnius
prolixus
Zophobas
atratus
Aedes
aegypti
Aedes
albopictus
Heliothis
virescens
Heliothis
virescens
Aedes
aegypti
Aedes
aegypti
Aedes
aegypti
Aedes
aegypti
Mytilus
galloprovincialis
Mytilus
galloprovincialis
Mytilus
galloprovincialis
Rattus
norvegicus
Rattus
norvegicus
Anopheles
gambiae
Drosophila
melanogaster
Oryctes
rhinoceros
Spodoptera
frugiperda
Culex
pipiens
Rattus
norvegicus
Rattus
norvegicus
Mus
musculus
Mus
musculus
Mus
musculus
Zophobas
atratus
Macaca
mulatta
Macaca
mulatta
Mus
musculus
Ornithorhynchus
anatinus
Ornithorhynchus
anatinus
Ornithorhynchus
anatinus
Mesobuthus
martensii
Macaca
mulatta
Macaca
mulatta
Phyllomedusa
sauvagei
Phyllomedusa
bicolor
Phyllomedusa
bicolor
Phyllomedusa
bicolor
Protophormia
terraenovae
Protophormia
terraenovae
Protophormia
terraenovae
Protophormia
terraenovae
Dolabella
auricularia
Drosophila
melanogaster
Drosophila
melanogaster
Drosophila
melanogaster
Drosophila
melanogaster
Drosophila
melanogaster
Drosophila
melanogaster
Bombyx
mori
Sus scrofa
Bubalus
bubalis
Bos taurus
Cavia
porcellus
Mus
musculus
Rana
esculenta
Rana
esculenta
Rana
esculenta
Rana
esculenta
Rana
berlandieri
Rana
esculenta
Rana
luteiventris
Rana
pipiens
Myrmecia
gulosa
Myrmecia
gulosa
Rana
rugosa
Rana
rugosa
Rana
rugosa
Rana
rugosa
Rana
rugosa
Rana
rugosa
Gallus
gallus
Gallus
gallus
Gallus
gallus
Gallus
gallus
Gallus
gallus
Gallus
gallus
Sus scrofa
Hyalophora
cecropia
Acanthoscurria
gomesiana
Cavia
Hadrurus
aztecus
Pyrrhocoris
apterus
Mus
musculus
Danio rerio
Morone
chrysops ×
Morone
saxatilis
Mus
musculus
Rattus
norvegicus
Oncorhynchus
mykiss
Bufo
gargarizans
Hippoglossus
hippoglossus
Ictalurus
punctatus
Ictalurus
punctatus
Holotrichia
diomphalia
Holotrichia
diomphalia
Holotrichia
diomphalia
Holotrichia
diomphalia
Apis
mellifera
Bos taurus
Bos taurus
Protophormia
terraenovae
Mus sp.
Rana
japonica
Rana
japonica
Bos taurus
Sus scrofa
Bos taurus
Bombyx
mori
Bombyx
mori
Limulus
polyphemus
Tachypleus
tridentatus
Bos taurus
Bos taurus
Macaca
mulatta
Mus
musculus
Sus scrofa
Cavia
porcellus
Sus scrofa
Heliothis
virescens
Alopochen
aegyptiacus
Chrysolophus
pictus
Lophophorus
impejanus
Manduca
sexta
Ovis aries
Rattus
norvegicus
Bos taurus
Cervus axis
Ovis aries
Ovis aries
Bos taurus
Drosophila
melanogaster
Papio sp.
Numida
meleagris
Coturnix
japonica
Hyalophora
cecropia
Phasianus
colchicus
Drosophila
melanogaster
Rhea
americana
Casuarius
casuarius
Drosophila
melanogaster
Opisthocomus
hoazin
Drosophila
melanogaster
Drosophila
melanogaster
Drosophila
melanogaster
Callipepla
californica
Colinus
virginianus
Columba
livia
Equus
asinus
Ortalis
vetula
Oryctolagus
cuniculus
Syrmaticus
soemmerringii
Felis catus
Pseudocheirus
peregrinus
Lophura
leucomelanos
Pavo
cristatus
Phasianus
versicolor
Syrmaticus
reevesi
Cervus axis
Bos taurus
Bos taurus
Bos taurus
Tachyglossus
aculeatus
Oncorhynchus
mykiss
Coturnix
japonica
Meleagris
gallopavo
Presbytis
entellus
Gallus
gallus
Rattus
norvegicus
Mus
musculus
Mus
musculus
Anas
platyrhynchos
Anas
platyrhynchos
Anser
anser
Cygnus
atratus
Struthio
camelus
Gallus
gallus
Drosophila
melanogaster
Chlamys
islandica
Bombyx
mori
Hyalophora
cecropia
Litoria
genimaculata
Litoria
genimaculata
Litoria
genimaculata
Litoria
genimaculata
Xenopus
laevis
Rana
temporaria
Drosophila
melanogaster
Drosophila
melanogaster
Mytilus
galloprovin
cialis
Misgurnus
anguillicau
datus
Bombyx
mori
Bombyx
mori
Ovis aries
Ovis aries
Mytilus
galloprovincialis
Mytilus
galloprovincialis
Mytilus
edulis
Mytilus
edulis
Mytilus
galloprovincialis
Mytilus
edulis
Rattus
norvegicus
Rattus
norvegicus
Rattus
norvegicus
Rattus
norvegicus
Oryctolagus
cuniculus
Rattus
norvegicus
Oryctolagus
cuniculus
Bos taurus
Bos taurus
Cavia
porcellus
Cavia
porcellus
Cavia
porcellus
Cavia
porcellus
Mesocricetus
auratus
Mesocricetus
auratus
Macaca
mulatta
Mesocricetus
auratus
Mesocricetus
auratus
Macaca
mulatta
Macaca
mulatta
Macaca
mulatta
Sus scrofa
Oncorhynchus
mykiss
Bos taurus
Opistophthalmus
carinatus
Opistophthalmus
carinatus
Pandinus
imperator
Pandinus
imperator
Parabuthus
schlechteri
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
setiferus
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
setiferus
Litopenaeus
setiferus
Litopenaeus
setiferus
Litopenaeus
setiferus
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
setiferus
Protophormia
terraenovae
Phyllomedusa
bicolor
Pseudople
uronectes
americanus
Pseudople
uronectes
americanus
Pseudople
uronectes
americanus
Pseudople
uronectes
americanus
Pseudople
uronectes
americanus
Pseudople
uronectes
americanus
Pseudople
uronectes
americanus
Pseudople
uronectes
americanus
Pseudople
uronectes
americanus
Limulus
polyphemus
Limulus
polyphemus
Pachycondyla
goeldii
Pachycondyla
goeldii
Pachycondyla
goeldii
Pachycondyla
goeldii
Pachycondyla
goeldii
Pachycondyla
goeldii
Pachycondyla
goeldii
Pachycondyla
goeldii
Pachycondyla
goeldii
Pachycondyla
goeldii
Pachycondyla
goeldii
Pachycondyla
goeldii
Pachycondyla
goeldii
Pachycondyla
goeldii
Pachycondyla
goeldii
Boophilus
microplus
Ixodes
ricinus
Riptortus
clavatus
Riptortus
clavatus
Bos taurus
Sus scrofa
Sus scrofa
Sus scrofa
Sus scrofa
Sus scrofa
Sus scrofa
Sus scrofa
Sus scrofa
Pseudis
paradoxa
Pseudis
paradoxa
Pseudis
paradoxa
Pseudis
paradoxa
Litopenaeus
setiferus
Litopenaeus
setiferus
Litopenaeus
setiferus
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
vannamei
Litopenaeus
vannamei
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mesobuthus
martensii
Xenopus
laevis
Pyrrhocoris
apterus
Rana
catesbeiana
Rana
clamitans
Rana
clamitans
Rana
clamitans
Rana
berlandieri
Rana
clamitans
Rana
clamitans
Rana
luteiventris
Rana
pipiens
Rana
luteiventris
Oryctes
rhinoceros
Apis
mellifera
Rana
rugosa
Rana
rugosa
Rana
rugosa
Sarcophaga
peregrina
Sarcophaga
peregrina
Sarcophaga
peregrina
Sarcophaga
peregrina
Sarcophaga
peregrina
Sarcophaga
peregrina
Sarcophaga
peregrina
Sarcophaga
peregrina
Sarcophaga
peregrina
Sarcophaga
peregrina
Sarcophaga
peregrina
Pandinus
imperator
Bos taurus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Pseudacan
thotermes
spiniger
Styela
clava
Styela
clava
Styela
clava
Styela
clava
Styela
clava
Caenorhabditis
elegans
Tachypleus
tridentatus
Tachypleus
tridentatus
Tachypleus
tridentatus
Tachypleus
tridentatus
Rana
temporaria
Rana
temporaria
Rana
temporaria
Rana
temporaria
Rana
temporaria
Rana
temporaria
Rana
temporaria
Rana
temporaria
Rana
temporaria
Rana
temporaria
Rana
clamitans
Rana
clamitans
Rana
clamitans
Rana
clamitans
Rana
clamitans
Rana
japonica
Rana
luteiventris
Rana
luteiventris
Rana
luteiventris
Rana
pipiens
Tenebrio
molitor
Tenebrio
molitor
Pseudacanthotermes
spiniger
Mus
musculus
Mus
musculus
Mus
musculus
Podisus
maculiventris
Macaca
mulatta
Macaca
mulatta
Macaca
mulatta
Macaca
mulatta
Hoplobatrachus
tigerinus
Hoplobatrachus
tigerinus
Hoplobatrachus
tigerinus
Hoplobatrachus
tigerinus
Bos taurus
Bos taurus
Xenopus
laevis
Zea mays
Amaranthus
caudatus
Zea mays
Basella
alba
Linum
usitatissimum
Diospyros
texana
Raphanus
sativus
Raphanus
sativus
Phytolacca
americana
Eucommia
ulmoides
Eucommia
ulmoides
Gastrodia
elata
Gastrodia
elata
Ipomoea nil
Capsicum
annuum
Ipomoea nil
Sinapis
alba
Brassica
napus
Arabidopsis
thaliana
Raphanus
sativus
Malva
parviflora
Phytolacca
americana
Malva
parviflora
Sinapis
alba
Malva
parviflora
Malva
parviflora
Malva
parviflora
Malva
parviflora
Beta
vulgaris
Beta
vulgaris
Gastrodia
elata
Medicago
sativa
Gastrodia
elata
Hordeum
vulgare
Hordeum
vulgare
Arabidopsis
thaliana
Pisum
sativum
Macadamia
integrifolia
Mesembryanthemum
crystallinum
Macadamia
integrifolia
Mirabilis
jalapa
Amaranthus
caudatus
Mirabilis
jalapa
Spinacia
oleracea
Spinacia
oleracea
Spinacia
oleracea
Spinacia
oleracea
Spinacia
oleracea
Spinacia
oleracea
Spinacia
oleracea
Zea mays
Capsella
bursa-
pastoris
Impatiens
balsamina
Allium cepa
Ipomoea nil
Amaranthus
hypochondriacus
Phytolacca
americana
Avena
sativa
Zea mays
Zea mays
Zea mays
Zea mays
Basella
alba
Nicotiana
tabacum
Nicotiana
tabacum
Nicotiana
tabacum
Zea mays
Zea mays
Nicotiana
tabacum
Hydrangea
macrophylla
Hordeum
vulgare
Chassalia
parviflora
Chassalia
parviflora
Psychotria
longipes
Brassica
rapa
Sinapis
alba
Raphanus
sativus
Arabidopsis
thaliana
Brassica
napus
Brassica
rapa
Raphanus
sativus
Sinapis
alba
Sinapis
alba
Brassica
napus
Raphanus
sativus
Raphanus
sativus
Pisum
sativum
Pisum
sativum
Capsicum
annuum
Brassica
rapa
Helianthus
annuus
Helianthus
annuus
Triticum
aestivum
Heuchera
sanguinea
Dahlia
merckii
Aesculus
hippocastanum
Clitoria
ternatea
Dahlia
merckii
Helianthus
annuus
Elaeis
guineensis
Capsicum
annuum
Capsicum
annuum
Brassica
oleracea
Prunus
persica
Zea mays
Zea mays
Vicia faba
Vicia faba
Petunia ×
hybrida
Petunia ×
hybrida
Helianthus
annuus
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Eutrema
wasabi
Lycopersicon
esculentum
Gastrodia
elata
Gastrodia
elata
Gastrodia
elata
Gastrodia
elata
Arabidopsis
thaliana
Ginkgo
biloba
Hevea
brasiliensis
Euonymus
europaeus
Euonymus
europaeus
Hordeum
vulgare
Gastrodia
elata
Nicotiana Alata
Nicotiana
tabacum
Nicotiana
tabacum
Nicotiana
tabacum
Lycopersicon
esculentum
Lycopersicon
esculentum
Arabidopsis
thaliana
Arabidopsis
thaliana
Pyrus
pyrifolia
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Pyrus
pyrifolia
Pyrus
pyrifolia
Capsicum
annuum
Arabidopsis
thaliana
Picea abies
Sorghum
bicolor
Lycopersicon
esculentum
Lycopersicon
esculentum
Lycopersicon
esculentum
Triticum
aestivum
Macadamia
integrifolia
Macadamia
integrifolia
Macadamia
integrifolia
Triticum
aestivum
Triticum
aestivum
Zea mays
Zea mays
C
YCRIPACIAGERRYGTCIYQGRLWAFCC
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B. Growth Factors
In some embodiments of the present invention, trophic factor combinations for treating injured nervous systems comprise one or more growth factors. Growth factors useful in the present invention include, but are not limited to, the following broad classes of cytoactive compounds: Insulin, Insulin like growth factors such as IGF-I, IGF-IB, IGF-II, and IGF-BP; Heparin-binding growth factors such as Pleiotrophin (NEGF1) and Midkine (NEGF2); PC-cell derived growth factors (PCDGF); Epidermal Growth Factors such as α-EGF and β-EGF; EGF-like molecules such as Keratinocyte-derived growth factor (which is identical to KAF, KDGF, and amphiregulin) and vaccinia virus growth factor (VVGF); Fibroblast Growth Factors such as FGF-1 (Basic FGF Protein), FGF-2 (Acidic FGF Protein), FGF-3 (Int-2), FGF-4 (Hst-1), FGF-5, FGF-6, and FGF-7 (identical to KGF); FGF-Related Growth Factors such as Endothelial Cell Growth Factors (e.g., ECGF-α and ECGF-β); FGF- and ECGF-Related Growth Factors such as Endothelial cell stimulating angiogenesis factor and Tumor angiogenesis factor, Retina-Derived Growth Factor (RDGF), Vascular endothelium growth factors (VEGF, VEGF-B, VEGF-C, and VEGF-D), Brain-Derived Growth Factor (BDGF A- and -B), Astroglial Growth Factors (AGF 1 and 2), Omentum-derived factor (ODF), Fibroblast-Stimulating factor (FSF), and Embryonal Carcinoma-Derived Growth Factor; Neurotrophic Growth Factors such as α-NGF, β-NGF, γ-NGF, Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3, Neurotrophin-4, and Ciliary Nuerotrophic Factor (CNTF); Glial Growth Factors such as GGF-I, GGF-II, GGF-III, Glia Maturation Factor (GMF), and Glial-Derived Nuerotrophic Factor (GDNF); Organ-Specific Growth Factors such as Liver Growth Factors (e.g., Hepatopoietin A, Hepatopoietin B, and Hepatocyte Growth Factors (HCGF or HGF), Prostate Growth Factors (e.g., Prostate-Derived Growth Factors [PGF] and Bone Marrow-Derived Prostate Growth Factor), Mammary Growth Factors (e.g., Mammary-Derived Growth Factor 1 [MDGF-1] and Mammary Tumor-Derived Factor [MTGF]), and Heart Growth Factors (e.g., Nonmyocyte-Derived Growth Factor [NMDGF]); Cell-Specific Growth Factors such as Melanocyte Growth Factors (e.g., Melanocyte-Stimulating Hormone [α-, β-, and γ-MSH] and Melanoma Growth-Stimulating Activity [MGSA]), Angiogenic Factors (e.g., Angiogenin, Angiotropin, Platelet-Derived ECGF, VEGF, and Pleiotrophin), Transforming Growth Factors (e.g., TGF-α, TGF-β, and TGF-like Growth Factors such as TGF-β2, TGF-β3, TGF-e, GDF-1, GDF-9, CDGF and Tumor-Derived TGF-β-like Factors), ND-TGF, and Human epithelial transforming factor [h-TGFe]); Regulatory Peptides with Growth Factor-like Properties such as Bombesin and Bombesin-like peptides (e.g., Ranatensin, and Litorin], Angiotensin, Endothelin, Atrial Natriuretic Factor, Vasoactive Intestinal Peptide, and Bradykinin; Cytokines such as connective tissue growth factor (CTGF), the interleukins IL-1 (e.g., Osteoclast-activating factor (OAF), Lymphocyte-activating factor (LAF), Hepatocyte-stimulating factor (HSF), Fibroblast-activating factor (FAF), B-cell-activating factor (BAF), Tumor inhibitory factor 2 (TIF-2), Keratinocyte-derived T-cell growth factor (KD-TCGF)), IL-2 (T-cell growth factor (TCGF), T-cell mitogenic factor (TCMF)), IL-3 (e.g., Hematopoietin, Multipotential colony-stimulating factor (multi-CSF), Multilineage colony-stimulating activity (multi-CSA), Mast cell growth factor (MCGF), Erythroid burst-promoting activity (BPA-E), IL-4 (e.g., B-cell growth factor I (BCGF-I), B-cell stimulatory factor 1 (BSF-1)), IL-5 (e.g., B-cell growth factor II (BCGF-II), Eosinophil colony-stimulating factor (Eo-CSF), Immunoglobulin A-enhancing factor (IgA-EF), T-cell replacing factor (TCRF)), IL-6 (B-cell stimulatory factor 2 (BSF-2), B-cell hybridoma growth factor (BCHGF), Interferon β2 (IFN-B), T-cell activating factor (TAF), IL-7 (e.g., Lymphopoietin 1 (LP-1), Pre-B-cell growth factor (pre-BCGF)), IL-8 (Monocyte-derived neutrophil chemotactic factor (MDNCF), Granulocyte chemotatic factor (GCF), Neutrophil-activating peptide 1 (NAP-1), Leukocyte adhesion inhibitor (LAI), T-lymphocyte chemotactic factor (TLCF)), IL-9 (e.g., T-cell growth factor III (TCGF-III), Factor P40, MegaKaryoblast growth factor (MKBGF), Mast cell growth enhancing activity (MEA or MCGEA)), IL-10 (e.g., Cytokine synthesis inhibitory factor (CSIF)), IL-11 (e.g., Stromal cell-derived cytokine (SCDC)), IL-12 (e.g., Natural killer cell stimulating factor (NKCSF or NKSF), Cytotoxic lymphocyte maturation factor (CLMF)), TNF-α (Cachectin), TNF-β (Lymphotoxin), LIF (Differentiation-inducing factor (DIF), Differentiation-inducing activity (DIA), D factor, Human interleukin for DA cells (HILDA), Hepatocyte stimulating factor III (HSF-III), Cholinergic neuronal differentiation factor (CNDF), CSF-1 (Macrophage colony-stimulating factor (M-CSF)), CSF-2 (Granulocyte-macrophage colony-stimulating factor (GM-CSF)), CSF-3 (Granulocyte colony-stimulating factor (G-CSF)), and erythropoietin; Platelet-derived growth factors (e.g., Placental growth factor (PlGF), PDGF-A, PDGF-B, PDGF-AB, p28-sis, and p26-cis), and Bone Morphogenetic proteins (e.g., BMP and BMP-15), neuropeptides (e.g., Substance P, calcitonin gene-regulated peptide, and neuropeptide Y), and neurotransmitters (e.g., norepinephrine and acetylcholine).
Suitable growth factors may be obtained from commercial sources, purified from natural sources, or be produced by recombinant methods. Recombinant growth factors can be produced from wild-type coding sequences or from variant sequences that encode functional growth factors. Suitable growth factors also include analogs that may be smaller peptides or other molecules having similar binding and biological activity as the natural growth factors. Methods for producing growth factors are described in U.S. Pat. Nos. 5,183,805; 5,218,093; 5,130,298; 5,639,664; 5,457,034; 5,210,185; 5,470,828; 5,650,496; 5,998,376; and 5,410,019; all of which are incorporated herein by reference.
C. Neurotrophins
The trophic factor combinations provided herein also can include one or more neurotrophic growth factors such as Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3, Neurotrophin-4, and Ciliary Nuerotrophic Factor (CNTF).
Nerve growth factors, such as α-NGF, β-NGF, γ-NGF, and the like, are neurotrophins. In an embodiment, the trophic factor combination does not include a nerve growth factor, which results in lessened pain.
D. Neuropeptides
The trophic factor combinations provided herein also can include one or more neuropeptides, e.g., PBAN-type neuropeptides (e.g., Diapause hormone homolog (DH); Alpha-SG neuropeptide (MAB-alpha-NP); Beta-SG neuropeptide (MAB-beta-NP)); Pheromone biosynthesis activating neuropeptide (M); PBAN-type neuropeptides (e.g., Diapause hormone (DH); Alpha-SG neuropeptide (Alpha-SGNP); Beta-SG neuropeptide (Beta-SGNP); Pheromone biosynthesis activating neuropeptide I (PBAN-I) (BoM)); Neuropeptides B/W receptor type 2 (G protein-coupled receptor 8); Neuropeptides B/W receptor type 1 (G protein-coupled receptor 7); Neuropeptides B/W receptor type 1 (G protein-coupled receptor 7) (Fragment); neuropeptides [similarity]; Glucagon-family neuropeptides (e.g., Growth hormone-releasing factor (GRF) (Growth hormone-releasing hormone) (GHRH); Pituitary adenylate cyclase activating polypeptide (PACAP)); Pol-RFamide neuropeptides; Antho-RFamide neuropeptides type 1; LWamide neuropeptides (e.g., LWamide I; Metamorphosin A (LWamide II) (MMA); LWamide III; LWamide IV; LWamide V; LWamide VI; LWamide VII; LWamide VIII; LWamide IX); Antho-RFamide neuropeptides type 2; Glucagon-family neuropeptides (e.g., Growth hormone-releasing factor (GRF) (Growth hormone-releasing hormone) (GHRH); Pituitary adenylate cyclase activating polypeptide-27 (PACAP-27) (P)); Glucagon-family neuropeptides (e.g., Growth hormone-releasing factor (GRF) (Growth hormone-releasing hormone) (GHRH); Pituitary adenylate cyclase activating polypeptide-27 (PACAP-27) (P)); LWamide neuropeptides (e.g., LWamide I; LWamide II; LWS); Glucagon-family neuropeptides (e.g., Growth hormone-releasing factor (GRF) (Growth hormone-releasing hormone) (GHRH); Pituitary adenylate cyclase activating polypeptide (PACAP)]; FMRFamide-like neuropeptides); PBAN-type neuropeptides (e.g., Diapause hormone homolog (DH); Alpha-SG neuropeptide; Beta-SG neuropeptide); Pheromone biosynthesis activating neuropeptide (AgI-PBAN); Gamma-SG neuropeptid; FMRFamide-related neuropeptides; Myomodulin neuropeptides (e.g., GLQMLRL-amide; QIPMLRL-amide; SMSMLRL-amide; SLSMLRL-amide; Myomodulin A (PMSMLRL-amide)); FMRFamide neuropeptides; neuropeptides (e.g., Substance P, calcitonin gene-regulated peptide, and neuropeptide Y); LWamide neuropeptides (e.g., LWamide I; LWamide II; LWamide III; LWamide IV; LWamide V; LWamide VI; Metamorphosin A (MMA); Mwamide) (Fragment); PBAN-type neuropeptides (e.g., Diapause hormone homolog (DH); Alpha-SG neuropeptide; Beta-SG neuropeptide); Pheromone biosynthesis activating neuropeptide (HeA-PBAN); Gamma-SG neuropeptid; Antho-RFamide neuropeptides; Neuropeptides capa receptor (Cap2b receptor); Neuropeptides B/W receptor type 2 (G protein-coupled receptor 8); Neuropeptides B/W receptor type 1 (G protein-coupled receptor 7); FMRFamide-like neuropeptides [e.g., Neuropeptide AF10 (GFGDEMSMPGVLRF-amide); Neuropeptide AF20 (GMPGVLRF-amide); Neuropeptide AF3 (AVPGVLRF-amide); Neuropeptide AF4 (GDVPGVLRF-amide); N PBAN-type neuropeptides [e.g., Diapause hormone homolog (DH); Alpha-SG neuropeptide; Beta-SG neuropeptide; Pheromone biosynthesis activating neuropeptide (HeZ-PBAN); Gamma-SG neuropeptid; FMRFamide neuropeptides type FMRF-1 (Fragment); Abdominal ganglion neuropeptides L5-67 (e.g., Luqin; Luqin-B; Luqin-C; Proline-rich mature peptide (PRMP)); FMRFamide neuropeptides type FMRF-2; FMRFamide neuropeptides type FMRF-4 (Fragment); Myomodulin neuropeptides (e.g., Myomodulin A (MM-A) (PMSMLRL-amide) (Neuron B16 peptide); Myomodulin B (MM-B) (GSYRMMRL-amide); Myomodulin D (MM-D) (GLSMLRL-amide); Myomodulin F (MM-F); LWamide neuropeptides (e.g., LWamide I; LWamide II; Metamorphosin A (MMA); Iwamide) (Fragment) (Substance P, calcitonin gene-regulated peptide, and neuropeptide Y.)
E. Other Components
The trophic factor combinations can be used with various delivery systems. In some embodiments, the trophic factor combination is mixed with a viscous substance to increase the viscosity of the combination. The increased viscosity retains the trophic factor combination at the site of the injury longer than it would be retained in the absence of the viscous substance. The viscous substance can be, for example, a polysaccharide, such as hyaluranic acid.
In another embodiment, the trophic factor combination is delivered in a slow release formula, such as in a matrix, for example, a woundhealing matrix, either with or without a viscous substance. In an embodiment, the matrix is a hydrogel, such as a hydrogel disclosed in U.S. Patent Application No. US 20030083389A1, which describes hydrogels wherein a polymer matrix is modified to contain a bifunctional poly(alkylene glycol) molecule covalently bonded to the polymer matrix. The hydrogels can be cross-linked using, for example, glutaraldehyde. The hydrogels can also be crosslinked via an interpenetrating network of a photopolymerizable acrylates. In one embodiment of the invention, the components of the trophic factor combination are incorporated into the hydrogel, for example, through covalent bonds to poly(alkylene glycol) molecules of the hydrogel or through entertainment within the hydrogel. In other embodiments, the matrix is a collagen gel matrix, which can be impregnated with a trophic factor combination. Other matrices can also be used.
The trophic factor combination can also be delivered in a base solution, such as UW solution (DuPont Critical Care, Waukegan, Ill.), or other base solutions.
The neurochemical combinations can be used in conjunction with cell therapy, where transfected cells are produced to release the ingredients and obtain continual delivery of a trophic factor combination. For example, embryonic or adult stem cells can be modified to express trophic factors, antimicrobial peptides, and other relevant neurochemicals, to deliver the trophic factor combination endogenously to the injured spinal cord. In the case of genetically modified cell transplants, the transfected cells can be tagged with cell surface antigens so that the cells can be controlled. For example, antibodies targeting the specific antigen could be used to kill the implanted cells after therapeutic results have been achieved.
Delivery of the neurochemical combinations can also be achieved by media with spaced supports, such as sponges, gels, or biopolymers.
F. Exemplary Formulations
A trophic factor combination includes one or more antimicrobial peptide and/or one or more substance having an antimicrobial peptide effect, alone or with one or more of the following trophic factors: growth factors, neuropeptides, and neurotrophins. Another trophic factor combination includes a viscous substance, such as hyaluronic acid, among others. Another trophic factor combination includes other cytoactive compounds, such as one or more cytokine and/or one or more chemokine. Non-limiting examples of these trophic factor combinations are provided in Tables 3a-3h below. It will be recognized that the trophic factor combinations can comprise one or more antimicrobial polypeptides (e.g., a defensin such as BNP-1). The trophic factor combinations described below can also comprise one or more trophic factors above. Accordingly, in some preferred embodiments, the trophic factor combination is supplemented with one or more of the following trophic factors: trehalose (Sigma, St. Louis Mo.; e.g., about 15 mM), substance P (Sigma; e.g., about 10 μg/ml), IGF-1 (Collaborative Biologicals; e.g., about 10 ng/ml), EGF (Sigma; e.g., about 10 ng/ml), and BDNF (2 μg/ml). In some preferred embodiments, the trophic factor combination is also supplemented with insulin (1-200 units, preferably 40 units) prior to use. In some embodiments, an antimicrobial polypeptide is not included in the trophic factor combination.
In some exemplary embodiments, EGF and/or IGF-1 are included in the trophic factor combination at a concentration of about 1 ng/ml to about 100 ng/ml, most preferably about 10 ng/ml. In other exemplary embodiments, substance P is included at a concentration of about 0.1 μg/ml to about 100 μg/ml, most preferably about 2.5 μg/ml.
It will be recognized that the Tables below provide formulations that are exemplary and non-limiting. For example, alterations in the specific substances used and the number of those substances are all within the scope of the invention. In some embodiments, the antimicrobial polypeptide and/or substance having an antimicrobial peptide effect and/or one or more trophic factor, are provided in stable form that can be reconstituted. Methods for stabilization include, for example, lyophilization. In embodiments where the antimicrobial polypeptide and/or one or more growth factors are provided in lyophilized form, they can conveniently reconstituted prior to use, for example, in sterile water or in an aliquot of a base medium (e.g., UW solution), prior to addition to a base medium (e.g., hyaluronic acid, UW solution).
Alternatively, the at least one microbial polypeptide and/or one or more trophic factor can be provided as a separate composition (i.e., a “bullet”) that is added to a base medium. In preferred embodiments, the bullet contains an antimicrobial peptide and/or a substance having an antimicrobial peptide effect and/or one or more trophic factor as described above. In some embodiments, the bullet contains an antimicrobial peptide and/or a substance having an antimicrobial peptide effect and/or one or more of the trophic factor as described above in concentrations that provide the appropriate concentration when added to a specific volume of the base medium, where used.
It is contemplated that the trophic factor combination can be provided in a pre-formulated form, such as in a kit format. The kit can include (1) at least one of an antimicrobial peptide and a substance having an antimicrobial peptide effect and (2) a neurotrophin. The kit can also include a viscous substance. At least one of a growth factor and a neuropeptide can also be included.
II. Uses of Trophic Factor Combinations and Their Individual Components
It is contemplated that the trophic factor combinations and their individual components described above may be utilized in a variety of procedures related to injury to the nervous system and other medical procedures. It is contemplated that the trophic factor combinations and their individual components can be used for the treatment of any part of the nervous system, including the central nervous system and the peripheral nervous system.
In one embodiment, the trophic factor combinations or one or more of their individual components are used during surgery of the disc and/or other portions of the nervous system. In an embodiment, a trophic factor combination or one or more of their individual components applied to surgical hardware and/or other implants, such as surgical screws, plates, pins, clamps, wires, pins, rods, nails, probes, spinal fixation devices, and the like. In another embodiment, a trophic factor combination or one or more of their individual components is applied directly during surgery, such as to a surgical opening, for example, an incision, a section, or any other opening. In one embodiment, a trophic factor combination or one or more of their individual components is applied to one or more tissue, nerve, organ, or cavity. A trophic factor combination or one or more of their individual components can also be applied to a surgical instrument such that when the instrument is used, the trophic factor combination or one or more of their individual components is delivered to injury and/or surrounding tissue, fluid, organ, and the like.
In use, an injury to the nervous system is identified. At least one component of the trophic factor combination is applied to the injury to the nervous system.
In some embodiments, the trophic factor combinations can be utilized to reduce body weight loss post injury in injured animals treated with the combination when compared to injured animals not treated with the trophic factor combination. Preferably, the decrease in loss of body weight is improved by at least 25% and more preferably by at least 50% as compared to animals not receiving the trophic factor combination. In some embodiments, the trophic factor combinations are used to strengthen motor recovery in injured animals treated with the trophic factor combination when compared to injured animals not treated with the trophic factor combination. In some embodiments, the trophic factor combinations are used to increase evoked potential amplitudes in injured animals treated with the trophic factor combination when compared to injured animals not treated with the trophic factor combination. In some embodiments, the trophic factor combinations are used to lower the current required to evoke a response (threshold current) in injured animals treated with the trophic factor combination when compared to injured animals not treated with the trophic factor combination. Application of the trophic factor combination according to the invention can also have at least one of the following additional effects: reduced pain in the animal, a neuroprotective effect, triggered neuronal plasticity, reduced inflammation, and growth of new cells.
The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
Experiments were performed on 3-5 month old male Sprague-Dawley (SD) and Lewis rats that were housed individually with free access to food and water. Rats were placed into four groups: 1) spinally injured SD rats without a trophic factor combination administered (n=8), 2) spinally injured SD rats with a trophic factor combination (n=2); 3) spinally injured Lewis rats without a trophic factor combination administered (n=5), and 4) spinally injured Lewis rats with a trophic factor combination administered (n=2).
Spinal cord injury. Rats were anesthetized with medetomidine (75 μg/kg i.m.) and isoflurane in oxygen. After oro-tracheal intubation, anesthesia was maintained with isoflurane in oxygen and rats were mechanically ventilated. A laminectomy was made at the second cervical vertebral level to allow the second cervical spinal segment and the cranial segment of the third cervical spinal segment to be exposed. A 1-mm-long left-sided hemisection was made in the cranial segment of C2 and the section aspirated with a fine tipped glass pipette. The surgical wound was closed using standard techniques. All animals were allowed to recover and received atipamezole (0.1 mg/kg i.v.) to antagonize the anesthetic effects of medetomidine. Buprenorphine (50 μg/kg i.v.) and carprofen (5 mg/kg i.v.) were administered for postsurgical pain control. Analgesics were repeated as required over the next 2 days.
Trophic Factor Combination. The trophic factor combination (also referred to as the trophic factor combination) was made by adding 10 ug of BNP-1 (bactenecin), 100 ng of insulin-like growth factor (IGF-1), and 25 mg of Substance P to 200 ul of distilled water.
Trophic factor combination administration. Prior to closure of the surgical wound, hyaluronic acid (Hylartin V, sodium hyalurate) (10%) was added to the neurotrophin mixture to thicken the solution and improve retention at the site of spinal injury. Two ug of BDNF was added to 0.4-0.45 ml of the mixture. The mixture (0.4-0.45 ml) was then administered using a syringe and 22-gauge needle into the hemisection cavity. The wound was closed immediately after injection.
Experimental preparation. Two weeks after surgical spinal injury, respiratory motor output was measured from both phrenic nerves using two distinct experimental techniques. First, spontaneous (brain-stem driven) phrenic motor activity was measured in anesthetized rats during standardized conditions. Second, spontaneous activity was removed by hyperventilating the rats and evoke potentials were elicited by spinal stimulation to evaluate the strength of the spinal pathways contributing to motor recovery.
Isoflurane anesthesia was induced in a closed chamber and maintained (2.5-3.5%) via nose cone while rats were tracheotomized. Rats were mechanically ventilated following tracheal cannulation. Following femoral venous catheterization rats were converted to urethane anesthesia (1.6 g/kg) then bilaterally vagotomized and paralyzed with pancuronium bromide (2.5 mg/kg, i.v.). Blood pressure was monitored via a femoral arterial catheter and pressure transducer (Gould P23ID, Valley View, Ohio). End-tidal CO2 was monitored with a rapidly responding analyzer (Novametrix, Wallingford, Conn.). Arterial partial pressures of O2 (PaO2) and CO2 (PaCO2) as well as pH were determined from 0.2 ml blood samples (ABL-500, Radiometer, Copenhagen, Denmark); unused blood was returned to the animal. Rectal temperature was maintained (37-39° C.) with a heated table. Phrenic nerves were isolated with a dorsal approach, cut distally, desheathed, bathed in mineral oil and placed on bipolar silver electrodes. Nerve activity was amplified (1000-10,000×) and filtered (100-10,000 Hz bandpass; model 1800, A-M Systems, Carlsborg, Wash.).
Spontaneous phrenic motor output. In all rats, the CO2 apneic threshold for inspiratory activity in the phrenic nerve contralateral to hemisection was determined after waiting a minimum of one hour following conversion to urethane anesthesia. This delay allowed blood pressure and respiratory motor output to stabilize. The procedure to establish the apneic threshold began by increasing the ventilator frequency until inspiratory activity ceased. Ventilator rate was then decreased slowly until inspiratory activity re-appeared. The end-tidal CO2 partial pressure (PETCO2) corresponding to the onset of inspiratory bursting was defined as the CO2 apneic threshold. PETCO2 was maintained 3 mmHg above the apneic threshold by adjusting the ventilator pump rate and inspired CO2 content. After the CO2 apneic threshold and baseline PaCO2 levels were established, 30-45 minutes were allowed to attain stable baseline conditions.
Evoked phrenic potentials. Rats were hyperventilated (PaCO2<30 mmhg) to prevent spontaneous inspiratory efforts. A monopolar tungsten electrode (5 MΩ, A-M Systems) was inserted contralateral to the spinal hemisection and adjacent to the C2 dorsal roots. The electrode tip was placed in or in close proximity to the ventrolateral finiculus (1.8-2.3 mm below the dorsal root entry zone). Electrode position was selected by maximizing the amplitude of a short latency (<1.0 ms) evoked potential in the phrenic nerve contralateral to SCI. Stimulus-response relationships were obtained by applying current pulses (20-1000 μA, 0.2 ms duration) with a stimulator (model S88, Grass Instruments, Quincy, Mass.) and stimulus isolation unit (model PSIU6E, Grass Instruments). Phrenic potentials were digitized and analyzed with P-CLAMP software (Axon Instruments, Foster City, Calif.).
Results:
Body Weight. Body weight decreased by 2 weeks post-injury in rats that had received a spinal hemisection (
Spontaneous Phrenic Nerve Activity. Spontaneous recovery of phrenic motor function on the injured side was evident as inspiratory bursts that were in synchrony with phrenic motor activity on the uninjured side. Phrenic motor recovery was present in all spinally injured rats regardless of treatment. However, the magnitude of this recovery differed between groups (
Evoked Phrenic Nerve Potentials. Evoked potentials were recorded from the phrenic nerve on the side of injury (
In addition, a strong trend existed for the current required to evoke a response (threshold current) to be lower after trophic factor combination administration compared to the control group (
This study was performed to determine whether application of a trophic factor combination can improve motor function after spinal cord injury (SCI). In this study, the trophic factor combination of Example 1 was applied and included insulin-like growth factor (IGF-1), brain-derived neurotrophic factor (BDNF), bactenesin (BNP-1), and substance P. The trophic factor combination was applied to test whether this combination would augment spontaneous respiratory motor recovery in a well-defined model of high cervical incomplete spinal cord injury (C2 hemisection). The trophic factor combination was applied to the injured spinal cord at the time of surgical injury. At 2 weeks post-injury, respiratory motor output was recorded bilaterally from phrenic nerves in urethane anesthetized, vagotomized, and mechanically ventilated spinally injured Lewis male rats (SCI-only: n=6; SCI+trophic factor combination; n=6, with some of these rats being the same as the rats in Example 1). Body weight decreased in all rats after injury. However, the change in body weight was significantly less after trophic factor combination treatment (see
Subtractive studies. Experiments can be performed on rats in accordance with the methods described in Example 2 except that fewer than all four components, i.e., insulin-like growth factor (IGF-1), brain-derived neurotrophic factor (BDNF), bactenesin (BNP-1), and substance P, of the trophic factor combination can be used (except for one or more controls using all four components). Different components can also be used. For example, a different growth factor (and/or neurotrophin and/or neuropeptide and/or antimicrobial peptide) can be used than the one listed above. Studies can also be run using only one component, i.e., either IGF-1, BDNF, BNP-1, or substance P or any other trophic factor to determine the effects of the individual components. Studies can also be performed using combinations of two of the components and using combinations of three of the components to determine whether all four components are needed to achieve the desired results.
Experiments can be performed on dogs having herniated discs. Traditionally, many dogs undergo surgical treatment of disc herniation, but no trophic factor combination has been administered during such surgery. Four naïve dogs can be first treated to test for unanticipated common severe negative effects of the trophic factor combination. Once this is done, 50 dogs presenting to the Veterinary Medical Teaching Hospital (VTMH) at the University of Wisconsin with severe spinal cord dysfunction can be tested.
Trophic factor combination. The trophic factor combination can be formulated of insulin like growth factor-1 (IGF-1) (10 ng/ml), substance P (2.5 μg/ml), bactenecin (1 μg/ml) and brain derived neurotrophic factor (BDNF) (2 μg/ml). The factors can be dissolved in a 1% hyaluronic acid solution. The hyaluronic acid is used in order to increase the contact time of the factors with the tissues.
Dogs that are clinical patients. Surgery can be performed under general anesthesia. A hemilaminectomy can be done at the site of the disc herniation. A 22-gauge catheter can be placed through the dura mater and arachnoid membrane and inserted in the subarachnoid space just caudal to the disc herniation. One ml of the trophic factor combination can be injected in the subarachnoid space. The surgery site can be closed routinely.
After recovery from anesthesia, intravenous lactated Ringer's solution and analgesics can be continued until the dog is able to drink on its own and does not appear painful. Neurologic examinations can be done twice a day. The dogs can be discharged to the owner when they are considered not to need pain medication, can urinate on their own, and are eating and drinking. Follow up examinations can be scheduled as appropriate clinically.
Pain or discomfort during surgery can be alleviated by maintenance of a surgical plane of anesthesia and constant rate infusion (CRI) of fentanyl 10 μg/kg/hr. The fentanyl CRI can be continued up to 12 hours post operatively at a dose of 2-5 μg/kg/hr. A Fentanyl Patch (50 mcg/hr, 5 mcg/kg/hr for total of 72 hours) can be administered as a routine postoperative treatment. Butorphanol can be further administered if the dogs demonstrate discomfort and can be given as long as clinical signs of pain, as indicated by abnormal posturing, vocalization, or discomfort upon palpation of the surgical wound site are present.
Immediately after surgery, the dogs can be monitored continuously until the animals are able to drink water on their own sufficient to maintain their hydration. After this recovery period, the animals can be checked a minimum of 3 times daily to determine if they are experiencing pain or discomfort. The dogs can be evaluated by physical exam, neurological exam, and direct palpation of the surgical wound. The dogs can receive routine recumbent care.
Dogs can be monitored post-surgically for cardiovascular stability by physical exam, pulse character, capillary refill time, heart rate, respiratory rate, and packed cell volume, if needed. Fluids can be administered if needed to maintain hydration. Postoperative discomfort can be alleviated by administration of fentanyl CRI (10 μg/kg/hr) during surgery and fentanyl CRI (2-5 μg/kg/hr) after surgery or butorphanol (0.2-0.4 mg/kg/IV or SQ) every 4-6 hours thereafter and a Fentanyl Patch (50 mcg/hr, 5 mcg/kg/hr for total of 72 hours).
Safety trial of trophic factor combination on dogs. The toxicity of the trophic factor combination described in Example 4 was tested on dogs. Four beagle dogs were studied over a three-day period. While the dogs were anesthetized, the trophic factor combination described in Example 4 in hyaluronic acid was injected into 1) the lumbar cerebrospinal fluid (2 dogs) and 2) the cistema magna cerebrospinal fluid (2 dogs). In all four cases, the dogs recovered easily and showed no signs of toxic reactions. There was no evidence for chronic pain on neurological exam. All dogs were euthanized on the third day of the study. In summary, no adverse reactions were observed in any animal.
It is understood that the various preferred embodiments are shown and described above to illustrate different possible features of the invention and the varying ways in which these features may be combined. Apart from combining the different features of the above embodiments in varying ways, other modifications are also considered to be within the scope of the invention.
The invention is not intended to be limited to the preferred embodiments described above, but rather is intended to be limited only by the claims set out below. Thus, the invention encompasses all alternate embodiments that fall literally or equivalently within the scope of these claims.
This application is a divisional application of patent application Ser. No. 11/214,372, filed Aug. 29, 2005 now abandoned, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 60/604,912, filed Aug. 27, 2004, the entirety of which is incorporated by reference herein.
This invention was made with United States government support awarded by the National Institutes of Health, Grant # HL069064. The United States has certain rights in this invention.
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| Number | Date | Country |
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| 0209738 | Feb 2002 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20090298741 A1 | Dec 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60604912 | Aug 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11214372 | Aug 2005 | US |
| Child | 12123366 | US |
