Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/08—Peptides having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5421—IL-8
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates generally to the field of cytokine actions and more particularly concerns methods and compositions for inhibiting and modulating the actions of CXC intercrine molecules.
• Disclosed are peptide compositions which inhibit interleukin 8 (IL-8) and, particularly, which preferentially inhibit IL-8- induced release of degradative enzymes by neutrophils. • These compositions may be employed to treat various inflammatory diseases and disorders including the Adult Respiratory Distress Syndrome (ARDS) and cystic fibrosis.
• ARDS Adult Respiratory Distress Syndrome
• IL-8 is a member of the CXC intercrine family of cytokines, so named due to elements of their N-terminal sequences. This family also includes, amongst others, peptide molecules known as growth related oncogene (GRO, or GRO/ GSA) and macrophage inflammatory protein 2/3 (MIP23) .
• GRO growth related oncogene
• MIP23 macrophage inflammatory protein 2/3
• IL-8 is a peptide of approximately 8 kD, and is about 72 amino acids in length, with this length varying according to the post-translational processing in different cell types (Yoshimura et al. , 1989; Hebert et al . , 1990; Strieter et al . , 1989) .
• the IL-8 gene was first identified by analyzing the genes transcribed by human blood mononuclear cells stimulated with Staphylococcal enterotoxin A (Schmid & Weissman, 1987) . IL-8 production is known to be induced by tumor necrosis factor and interleukin 1 (Strieter et al . , 1990) .
• IL-8 interacts with at least two distinct receptors on neutrophils (Holmes et al . , 1991; Murphy & Tiffany, 1991) .
• the receptors are coupled to GTP-binding proteins, allowing transmission of the IL-8 signal into the cell ( u et al . , 1993) .
• GRO and MIP23 While most of the members of the intercrine family, such as GRO and MIP23, bind to one of the receptors, IL-8 binds to both of the IL-8 receptors (LaRosa et al . , 1992; Cerretti et al . , 1993) .
• the three dimensional structure of IL-8 has been elucidated by NMR (Clore et al .
• CXC intercrines have been elucidated by several laboratories (Yoshimura et al . , 1989; Schroder et al . , 1988; Peveri et al . , 1988) .
• the major functions of the IL-8 peptide appear to be related to its ability to stimulate neutrophil chemotaxis and activation (Larsen et al . , 1989; Schroder et al . , 1988; Peveri et al . , 1988; Yoshimura et al . , 1987) and to promote angiogenesis (Koch et al . , 1992) .
• neutrophils are 'primed', e.g., by agents such as surface adherence or E. coli endotoxin (also known as lipopolysaccharide or LPS)
• IL-8 also stimulates the release of neutrophil enzymes such as elastase and mye1operoxidase.
• neutrophil enzymes such as elastase and mye1operoxidase.
• ARDS adult respiratory distress syndrome
• ARDS ARDS attacks between 150,000 and 200,000 Americans per year, with a mortality rate of 50-80% in the best clinical facilities (Balk & Bone, 1983) .
• ARDS is initiated by bacterial infections, sudden severe dropping of the blood pressure (shock) , and many other insults to the body.
• IL-8 is the major neutrophil activator in the lungs of patients with ARDS (Miller et al . , 1992)
• primate models of endotoxin shock also implicate IL-8 as a causative agent (Van Zee et al . , 1991) .
• IL-8 has also been found in . inflammatory exudates in other disorders and pathological conditions in which IL-8 is thought to play an important pathogenic role (Brennan et al . , 1990; Miller &• Idell, 1993; Miller et al. , 1992) .
• IL-8 has also been implicated as a possible mediator of inflammation in rheumatoid arthritis (Brennan et al. , 1990; Seitz et al . , 1991) and pseudogout (Miller & Brelsford, 1993) ; and to have a role in cystic fibrosis (McElvaney et al . , 1992; Nakamura et al . , 1992; Bedard et al . , 1993) . Therefore, modulation of IL-8 function appears to be good strategy to control a variety of pathological conditions.
• IL-8 Some progress has recently been made in identifying compounds capable of reducing IL-8 synthesis. Such compounds include IL-4, oxygen radical scavengers, secretory leukoprotease inhibitor and interferon gamma (Standiford et al . , 1990; DeForge et al. , 1992; McElvaney et al . , 1992; Cassatella et al . , 1993a; 1993b) , however, such studies do not concern IL-8 inhibitors. Other diverse compositions, including protein kinase C inhibitors, IL-4, and anti-IL-8 antibodies, have also been reported to modulate IL-8 actions (Lam et al . , 1990; Standiford et al . , 1992; Mulligan et al . , 1993) . Unfortunately, these compounds are far from ideal as candidates for use as IL-8 inhibitors in a clinical setting.
• the present invention seeks to overcome the drawbacks inherent in the prior art by providing new methods and compositions for modulating and inhibiting the actions of CXC intercrine molecules such as IL-8, GRO (GRO/MGSA) and MIP2/S.
• CXC intercrine molecules such as IL-8, GRO (GRO/MGSA) and MIP2/S.
• the peptides and pharmacological compositions disclosed reduce IL-8, GRO and MIP2/S binding to neutrophils and inhibit IL-8-induced neutrophil activation.
• These peptide formulations are particularly advantageous as they are capable of inhibiting IL-8- induced enzyme release at significantly lower concentrations than is required to inhibit neutrophil chemotaxis .
• Also provided are methods for treating various diseases and disorders, particularly inflammatory diseases, in which the unrestrained actions of CXC intercrines play a role.
• the invention is generally based upon the inventors surprising discovery that relatively small peptides including the amino acid sequence Arg Arg Trp Trp Cys Xaa-, ⁇ (RRWWCX; SEQ ID NO:23) , wherein Xaa 1 is any amino acid residue, are potent inhibitors of CXC intercrine molecules such as IL-8.
• CXC intercrine family molecules and “CXC intercrines” are used collectively to refer to the group of peptide intercrines which include the CXC sequence motif in their N-terminal regions.
• CXC intercrines are known to include IL-8, GRO, MIP2C., MIP2/3 and ENA78, all of which molecules, and any other intercrine polypeptides that include the CXC motif, will be understood to fall within this term as used in the present application.
• inhibitory peptides of the present invention may be termed "antileukinates" .
• Certain hexamer peptides of the sequence RRWWCX (SEQ ID NO:23) have been previously shown to have anti-bacterial activity against Staphylococcal aureus (Houghten et al . , 1991) .
• RRWWCX SEQ ID NO:23
• any such peptides would have the advantageous anti- cytokine/intercrine, anti-neutrophil and anti- inflammatory activities disclosed herein.
• the present invention therefore concerns methods for inhibiting CXC intercrines, such as GRO and MIP2o. or MIP2/3, and most particularly, methods for inhibiting IL-8.
• CXC intercrines such as GRO and MIP2o. or MIP2/3, and most particularly, methods for inhibiting IL-8.
• inhibiting CXC intercrines refers to the processes by which the biological actions of the CXC intercrines are reduced. This may be particularly assessed by inhibiting their binding to one of the IL-8 receptors on their target cells, such as neutrophils, although any mode of determining CXC intercrine inhibition may be employed.
• IL-8 is used to refer to the cytokine compositions previously known as neutrophil-activating factor, monocyte-derived neutrophil-activating peptide, monocyte-derived neutrophil-chemotactic factor and neutrophil-activating peptide-1.
• the term “inhibiting IL-8” generally refers to the processes by which the biological actions of IL-8 are reduced or lessened. This includes the inhibition of any or all of the known actions of IL-8. These actions include modulating sub-cellular effects, such as receptor binding or altering cytosolic calcium levels; modulating cellular effects such as granulocyte recruitment and activation; and also affecting physiological effects, such as inflammation and angiogenesis.
• the inhibition of IL-8 function referred to in this application is the inhibition of IL-8 action on granulocytes such as neutrophils (polymorphonuclear neutrophils, PMN) . This may be determined in many cellular and physiological ways, as disclosed herein.
• IL-8 binding For example, by measuring inhibition of IL-8 binding to purified receptor compositions or neutrophils; by determining the inhibition of IL-8-induced neutrophil chemotaxis or diapedesis; by measuring the inhibition of IL-8- stimulated neutrophil enzyme release (e.g., myeloperoxidase, 3-glucuronidase or elastase release) or superoxide production; or by assaying for anti- inflammatory effects in vivo, e.g., using a rabbit model of dermal inflammation.
• IL-8- stimulated neutrophil enzyme release e.g., myeloperoxidase, 3-glucuronidase or elastase release
• the preferred manner of determining IL-8 inhibition, or indeed GRO or MIP23 inhibition is to assay for a reduction in the intercrine binding to neutrophils, which is the most simple and straightforward method. In addition, binding of the particular intercrine to its receptor(s) must precede any other action that it has on neutrophils or other cell types.
• “Inhibition” of intercrines as exemplified by the inhibition of IL-8, GRO or MIP2Q! or MIP23 binding to neutrophils, refers to the capacity of a given peptide or composition to inhibit intercrine binding to any detectable degree, i.e. to reduce binding below the levels observed in the absence of the peptide or composition.
• the inhibition of CXC intercrine binding to neutrophils may be expressed as a % Binding Inhibition value, with the higher figures representing the more effective inhibitors.
• the preferred peptides will generally have the higher % binding inhibition figures.
• the % binding inhibition calculated will depend upon the precise assay conditions, such as the concentration of CXC in:,arcrine and the concentration of the given peptide or composition. Conditions such as those used to generate the data of Tables 1A, IB, 5A and 5B, may be employed to determine whether a given peptide has any inhibitory activity.
• a peptide or analogue is capable of inhibiting a CXC intercrine, such as IL-8, is a straightforward matter readily achieved using assays such as those disclosed herein.
• the peptide inhibitors of this invention are capable of preferentially inhibiting IL-8-induced neutrophil enzyme release at lower concentrations than IL-8-induced ctnemotaxis.
• the term “inhibiting”, when used in connection with this invention, also means “modulating” in that certain neutrophil functions are more significantly inhibited than others.
• the ability of the peptides to inhibit IL-8-induced neutrophil degradative enzyme release at about a 25 times lower concentration than is required to inhibit IL-8- induced neutrophil chemotaxis is an important discovery that could not have been predicted from prior studies. This means that neutrophils may still be recruited to a site of injury, but that the detrimental effects of the enzymes that they would normally release will be significantly reduced. This property, coupled with their small size, renders these type of peptides ideal for use in various treatment protocols and especially in the treatment of lung injury.
• CXC intercrine inhibition such as IL-8, GRO or MIP2 inhibition
• the "contact" process is the process by which the active peptide or peptides from within the composition contact either the CXC intercrine peptide or one of their receptors present on a target cell, or both, and reduce or inhibit their functional interaction.
• a CXC intercrine or intercrine target cell with a peptide-containing composition one may simply add the peptide or composition to target cells, such as • neutrophils, and intercrines in vi tro .
• target cells such as • neutrophils, and intercrines in vi tro .
• "contact” is achieved simply by administering the composition to the animal .
• Virtually any pharmaceutical peptide formulation may be used, including, but not limited to, formulations for parenteral administration, such as for intravenous, intramuscular and subcutaneous administration; inhalants, aerosols and spray formulations; formulations of peptides for topical use, such as in creams, ointments and gels; and other formulations such as peptides with lipid tails, peptides encapsulated in micelles or liposomes and drug release capsules including the active peptides incorporated within a biocompatible coating designed for slow-release.
• Increased levels of IL-8 are known to be present in lung edema fluids in patients with ARDS (Miller et al .
• the present invention therefore also provides methods for treating a wide variety of diseases and disorders in which CXC intercrines, particularly IL-8, play a role, especially those which have an inflammatory component.
• This includes treating subjects with lung injuries and disorders, including bronchial inflammation, such as chronic bronchitis, cystic fibrosis, pleural effusions, asthma, and ARDS; skin disorders such as psoriasis and dermatitis; diseases of the joints, including rheumatoid arthritis; and generally reducing inflammation in other clinical settings, such as in the treatment of pseudogout, inflammatory bowel disease or reperfusion cardiac damage after myocardial infarction.
• bronchial inflammation such as chronic bronchitis, cystic fibrosis, pleural effusions, asthma, and ARDS
• skin disorders such as psoriasis and dermatitis
• diseases of the joints including rheumatoid arthritis
• generally reducing inflammation in other clinical settings such as in the treatment of pseudo
• any one of the above conditions, or any other disorder influenced by neutrophil activity and characterized by inflammation one would identify a patient having the particular inflammatory or IL-8-linked disease and then administer to the patient, preferably parenterally, a biologically effective amount of a pharmaceutical composition which includes one or more peptides of the family disclosed herein.
• a topical cream or gel formulation would be used, whereas in methods to treat pulmonary disorders, injectable formulations, or even a spray, aerosol or inhalant, may be employed.
• injectable formulations or even a spray, aerosol or inhalant
• methods to reduce inflammation in other areas of the body one may use peptides formulated for parenteral administration or peptides incorporated in a biocompatible coating designed for slow-release. Liposome-encapsulation may be employed, which is known to increase the efficacy and significantly prolong the half-life of administered compounds, particularly those of lower molecular weight such as the peptides disclosed herein.
• IL-8 or CXC intercrine inhibition is achieved by using a biologically effective amount of the inhibitory peptide or peptides.
• a biologically effective amount refers to an amount effective to inhibit the actions of IL-8 or the particular intercrine.
• an appropriate amount would be that effective to reduce neutrophil enzyme release, particularly in comparison to chemotaxis.
• a variety of different peptide concentrations are very effective in vi tro, such as those between about 100 ⁇ M and about 20 ⁇ M. Clinical doses which result in similar a local concentration of peptides are therefore contemplated to be particularly useful .
• the quantity and volume of the peptide composition administered will depend on the host animal and condition to be treated and the route of administration.
• the precise amounts of active peptide required to be administered will depend on the judgment of the practitioner and may be peculiar to each individual.
• the determination of a suitable dosage range for use in humans will be straightforward.
• doses in the order of about 0.83 mg/kg body weight/hour (mg/kg/hr) to about 16.56 mg/kg/hr, preferably about 0.83 mg/kg/hr to about 4.14 mg/kg/hr, and more preferably about 1.66 mg/kg/hr of active ingredient peptide per individual are contemplated.
• compositions for use in inhibiting CXC intercrines, such as IL-8, GRO and MIP2 ⁇ or MIPS, in accordance with the present invention will be compositions that contain a relatively small peptide, generally of from 6 to about 14 residues in length, which includes within its sequence the amino acid sequence RRWWCX (SEQ ID NO:23) .
• a peptide in this sense means at least one peptide, and may refer to one or more such peptides which include a sequence in compliance with the general formula RRWWCX (SEQ ID NO:23) .
• the relatively small peptides encompassed by the present invention may be any length between six residues and about 14 or 15 or so residues in length, with the precise length not being an important feature of the invention.
• There are many advantages to using smaller peptides for example, the cost and relative ease of large scale synthesis, and their improved pharmacological properties, such as the ease with which they can penetrate tissues and their low immunogenicity.
• the peptides may include other short peptidyl sequences of various amino acids.
• the peptides may include a repeat of the
• RRWWCX SEQ ID NO:23
• RRWWCXX SEQ ID NO:57
• additional sequences including, e.g., short targeting sequences, tags, labelled residues, amino acids contemplated to increase the half life or stability of the peptide, or indeed, any additional residue desired for any purpose, so long as they still function to inhibit intercrines such as IL-8 - which can be readily determined by a simple assay such as those described herein.
• any of the so-called rare or modified amino acids may also be incorporated into a peptide of the invention, including the following: 2-Aminoadipic acid, 3-Aminoadipic acid, beta-Alanine (beta-Aminopropionic acid) , 2-Aminobutyric acid, 4-Aminobutyric acid (piperidinic acid) , 6-Aminocaproic acid, 2-Aminoheptanoic acid, 2-Aminoisobutyric acid, 3-Aminoisobutyric acid, 2-Aminopimelic acid, 2, 4-Diaminobutyric acid, Desmosine, 2,2' -Diaminopimelic acid, 2,3-Diaminopropionic acid,
• the inhibitory compositions of the invention may include a peptide modified to render it biologically protected.
• Biologically protected peptides have certain advantages over unprotected peptides when administered to human subjects and, as disclosed in U.S. Patent 5,028,592 (incorporated herein by reference) , protected peptides often exhibit increased pharmacological activity, as was found to be true in the present case.
• the present invention therefore encompasses compositions comprising an acylated peptide or peptides, and preferably, a peptide acylated at the N-terminus.
• an acyl group may be employed in this context, the inventors have found that the addition of an acetyl group to the N-terminus of a given peptide also renders the resultant peptide surprisingly effective at inhibiting intercrines such as IL-8.
• the inhibitory peptide compositions may also include a peptide (s) which is amidated at the C-terminus, i.e., to which an NH 2 group has been added.
• peptides which have both an acylated N- terminal and an amidated C-terminal residue are preferred as they are believed to most closely mimic natural protein and peptide structure.
• compositions for use in the present invention may also comprise peptides which include all L-amino acids, all D-amino acids or a mixture thereof.
• peptides which include all L-amino acids, all D-amino acids or a mixture thereof.
• the finding that peptides composed entirely of D-amino acids have potent inhibitory activity is particularly important as such peptides are known to be resistant to proteases naturally found within the human body and are less immunogenic and can therefore be expected to have longer biological half lives.
• the anti-intercrine and anti-IL-8 compositions of, the present invention will generally comprise one or more peptides which include an amino acid sequence in accordance with those set forth in SEQ ID NO:l or SEQ ID NOS:24 through 42.
• short hexamer peptides may be preferred.
• the inhibitory compositions will generally comprise one or more peptides which have an amino acid sequence in accordance with those set forth in SEQ ID NO:l or SEQ ID NOS:24 through 42, presented below:
• the inhibitory compositions of the invention may include one or more peptides which • include a sequence in accordance with the amino acid sequence Arg Arg Trp Trp Cys Arg Xaa 2 (SEQ ID NO:2) .
• one of the variable positions has been defined as arginine and the remaining Xaa 2 may be any amino acid residue.
• sequences are exemplified by those set forth in SEQ ID NOS:3 through 22.
• the compositions will generally comprise one or more peptides which have an amino acid sequence in accordance with those set forth below:
• the invention also contemplates the use of peptides having the amino acid sequence Gin He Pro Arg Arg Ser
• Trp Cys Arg Phe Leu Phe (SEQ ID NO:52) , either alone, or more preferably, in combination with one or more of the other peptides described above.
• the successful use of this dodecamer illustrates both the fact that longer peptides are successful and that certain. biologically functional equivalent peptides are active. All such active equivalents therefore fall under the scope of the present invention.
• compositions for use in the inhibitory methods described herein may contain only a single active peptidyl species. Alternatively, they may contain more than one peptide, up to and including about 40 or 45 or so distinct peptides. Any and all of the various combinations are contemplated, such as compositions comprising 2, 3, 5, 10, 15, 20, 30 or 45 or so distinct peptides .
• compositions comprising peptides having the amino acid sequence Arg Arg Trp Trp Cys Arg (SEQ ID NO:l) and/or the amino acid sequence Arg Arg Trp Trp Cys Arg Cys (SEQ ID NO:4) are contemplated to be particularly useful, although the invention is not limited to these peptides in any way.
• considerations other than in vi tro activity such as plasma half life and stability, may be considered in ultimately choosing peptides which are preferred for clinical embodiments.
• the effects of different amino acid substitutions on these parameters may be readily determined and the results used to design the optimum peptide or combination of peptides for use in vivo .
• RRWWCX (SEQ ID NO:23) sequence element is an important feature of the peptides of this invention. However, this does not exclude certain biological functional equivalents from falling within the scope of the invention. For example, the inventors have discovered that the first tryptophan in RRWWCX (SEQ ID NO:23) can be exchanged, e.g., by replacing with serine, with only modest loss of activation. Therefore, one example of equivalents encompassed by the invention are peptides of the sequence RRXWCX (SEQ ID NO:58) . . "Equivalent amino acids" may be defined as amino acids whose hydrophilic or hydropathic index are within ⁇ 2, more preferably, within ⁇ 1, and most preferably, within ⁇ 0.5 of each other. Of course, to be a "functional equivalent", a peptide must still retain its intercrine or IL-8 inhibitory activity, as may be easily determined using assays such as those disclosed herein.
• peptidomimetics may be formulated to mimic the key portions of the peptide structure.
• Such compounds may be used in the same manner as the peptides of the invention and hence are also functional equivalents.
• the generation of a structural functional equivalent may be achieved by the techniques of modelling and chemical design known to those of skill in the art. It will be understood that all such sterically similai constructs fall within the scope of the p. ---sent invention.
• the peptides and compositions for use in the invention may be prepared by any one of a variety of different methods .
• One preferred method for preparing peptides in accordance with the present invention is contemplated to be via automated peptide synthesis.
• a synthetic peptide may be straightforwardly prepared using an automated peptide synthesizer, the operation of which will be generally known to those of skill in the art. This method is one of those generally preferred for preparing large quantities of a given peptide, e.g., once a particular peptide has been chosen for therapeutic use.
• Another preferred method for preparing inhibitory peptides, and the biological functional equivalents thereof, is to use a combinatorial peptide library method, as described by Houghten et al . (1991) and disclosed in International Patent Application PCT WO 92/09300, the entire disclosure of which is specifically incorporated herein by reference. These methods are particularly useful for preparing and analyzing a plurality of peptides having a substantially predetermined sequence, such as RRWWC, to which is appended a variety of different amino acids at one or more positions. These methods may be used to synthesize a peptide mixture for direct use in the formulation of a composition in accordance with the invention or to identify a particularly active peptide for subsequent individual synthesis.
• peptides may also by prepared by molecular biological means and the "recombinant" peptide obtained from recombinant host cells which express the peptide.
• a specific oligonucleotide based upon the sequence of the desired peptide, as is known to those of skill in the art, and then insert the oligonucleotide into an expression vector, such as any one of the many expression vectors currently available commercially.
• This methodology is standard practice in the art (see e.g., Sambrook et al . , 1989) .
• FIG. 1 Binding Inhibition by Ac-RRWWCX (SEQ ID NO:23) Series. Twenty peptides which have structure of Ac-RRWWC (SEQ ID NO:56) plus one of the 20 standard protein amino acids in the sixth position were tested. The notation on the x axis indicates the residue at the carboxy-terminal position (SEQ ID N0:1 and 24 through 42) . In this study, neutrophils were incubated with 1 pM 125 I-labeled IL-8 and 20 ⁇ M of each peptides.
• FIG. 1 IL-8 Binding Inhibition by Ac-RRWWCR-NH 2 (SEQ ID NO:l) .
• Figure 3A Saturation Studies in the Presence of Ac- RRWWCR-NH 2 (SEQ ID NO:l), Binding Isotherms with Best Fit Curve Calculated Using Lundon I .
• Binding assays were performed in the absence of the peptide (O) and in the presence of lO ⁇ M (•) , 20 ⁇ M (v) , or 40 ⁇ M ( ⁇ ) peptide with increasing concentrations of 125 I-labeled IL-8. Each data point represent specific binding which was computed by subtracting nonspecific binding in the presence of excess unlabeled IL-8 from total binding.
• FIG. 3B Saturation Studies in the Presence of Ac- RRW CR-NH 2 (SEQ ID N0:1) , Scatchard Plots. Binding assays were performed in the absence of the peptide (O) and in the presence of lO ⁇ M (•) , 20 ⁇ M (v) , or 40 ⁇ M ( ⁇ ) peptide with increasing concentrations of 125j-labeled IL-8.
• FIG. 4 Binding Inhibition Studies in the Presence of Ac-RRWWCR-NH 2 (SEQ ID N0:1) . Neutrophils were incubated with InM of 125 I-labeled IL-8 and increasing concentration of unlabel'ed IL-8 in the presence (•) or the absence (O) of lO ⁇ M Ac-RRWWCR-NH 2 (SEQ ID NO:l) .
• FIG. 5 Effect of Ac-RRWWCR-NH 2 (SEQ ID N0:1) on Binding of IL-8, C5a, and Leukotriene B 4 to Neutrophils.
• the bindings assays were performed with InM 125 I-labeled IL8 (•) , 0.25nM 125 I-labeled C5a (v) , or 0.4nM 3 H-labeled leukotriene B 4 ( ⁇ ) and increased concentration of Ac- RRWWCR-NH 2 (SEQ ID N0:1) . Analysis of variance was used for multiple comparison. When there was significant difference, the differences between binding without the peptide and those with peptide were tested using Sheffes test; ***, p ⁇ 0.001.
• FIG. 6 Cytotoxicity Test. Chromium-labeled neutrophils were incubated with increasing concentrations of Ac-RRWWCR-NH 2 (SEQ ID NO:l) in PBS containing 0.1% BSA for 90 min at 4°C (•) or in RPMI-1640 media containing 1% BSA for 30 min at 37°C (O) . Analysis of variance was used for multiple comparison. When there was significant difference, the differences between % lysis without the peptide and those with peptide were tested using Sheffes test; ***, p ⁇ 0.001.
• FIG. 8 The Effect of Ac-RRWWCR-NH 2 (SEQ ID NO:l) on jS-glucuronidase Release.
• Neutrophils pretreated with cytochalasin B were incubated with lOOnM IL-8 (•) , lOOnM fMLP ( ⁇ ) , lOOnM C5a (v) or lOOnM leukotriene B 4 ( ⁇ ) or without any stimulant (O) in the presence of increasing concentration of Ac-RRWWCR-NH 2 (SEQ ID NO:l) for 30 min at 37°C.
• 3-glucuronidase activity of supernatants were measured using phenolphthalein-glucuronic acid as substrate. Analysis of variance was used for multiple comparison. When there was significant difference, the differences between distance migrated without the peptide and those with peptide were tested using Sheffes test; *, p ⁇ 0.05, **, p ⁇ 0.01, ***, p ⁇ 0.001.
• FIG. 9 Binding Inhibition by All D-amino Acid Ac- rrwwcrx-NH 2 (SEQ ID NO:2) Series.
• Ac-RRWWCR (SEQ ID NO:l) was synthesized using D-amino .acids and added each of the 20 standard protein D-amino acids were added at the seventh position (SEQ ID NO:3 through 22) . The notation on the x axis indicates the residue at the carboxyterminal position.
• the binding study was performed using 10 ⁇ M of each peptide.
• Ac-RRWWCR-NH 2 (SEQ ID NO:l) made with L-amino acids was used as a control .
• B is bound radioactivity in the presence of the peptide
• T is bound radioactivity in the absence of the peptide
• NSP is bound radioactivity in the presence of excess nonlabelled ligand
• IL-8 has been identified as a neutrophil activating molecule (Schroder et al . , 1988; Peveri et al . , 1988; Yoshimura et al . , 1987) . It is produced mainly by monocyte-macrophage and endothelial cell by the stimuli such as bacterial lipopolysaccharide (LPS) , tumor necrosis factor or interleukin 1, and shares common neutrophil activating properties with chemotactic agonists, such as fMLP, C5a or leukotriene B 4 (Baggiolini et al . , 1992) . IL-8 can stimulate chemotaxis of neutrophils as well as enzyme release and respiratory burst.
• LPS bacterial lipopolysaccharide
• fMLP fMLP
• C5a or leukotriene B 4 Baggiolini et al . , 1992
• IL-8 is one member of the family of peptide molecules termed CXC intercrines, which all have the CXC sequence motif in their N-terminal region.
• the CXC intercrines also include GRO, MIP2 ⁇ or MIP23 and, more recently, ENA78.
• IL-8 The functions of IL-8 are mediated by IL-8 receptors on the neutrophil surface membrane. Recent studies showed that IL-8 binds to at least two distinct receptors, whereas most of the other members of the intercrine family, e.g., GRO and MIP23, bind to one of the receptors with high affinity (Holmes et al . , 1991;
• IL-8 has been found in high concentrations in joint fluids from patients with several kinds of joint disease (Brennan et al . , 1990) , in pleural spaces of some patients with pleural effusions (Miller & Idell, 1993) , and lung edema fluids from patients with the adult respiratory distress syndrome (ARDS) (Miller et al . , 1992) . Increased IL-8 levels have also been clearly documented in various recent studies of patients with cystic fibrosis (Richman-Eisenstat et al. , 1993; McElvaney et al . , 1992; Nakamura et al. , 1992; Bedard et al . , 1993) .
• IL-8 activates neutrophils and, although they are powerful antimicrobial cells, neutrophils can also cause considerable tissue damage through the release of certain enzymes. IL-8 is therefore believed to be important in pathogenesis of these and other inflammatory disorders. The inventors therefore hypothesized that modulation of IL-8 function would be a good strategy to control various diseases and pathological conditions, particularly ARDS and cystic fibrosis.
• IL-8 production has been shown to be suppressed in LPS- stimulated whole human blood by oxygen radical scavengers (DeForge et al . , 1992) .
• two cytokines, interferon gamma (Cassatella et al . , 1993a; 1993b) and interleukin 4 (Standiford et al . , 1990) inhibited the synthesis of IL-8.
• interferon gamma Cassatella et al . , 1993a; 1993b
• interleukin 4 Standiford et al . , 1990
• the present inventors assayed various other peptide compositions for IL-8 inhibitory activity by screening a library of 400 groups of hexapeptides. In these screening assays, 125 I-labeled interleukin-8 (10 M) was mixed with 100 ⁇ M peptide, then added to neutrophils and incubated at 4°C for 90 min. The bound radioactivity was separated from unbound by centrifugation through a dense cushion of a mixture of paraffin and silicon oils
• RRWWCX-type peptides had previously been found to exhibit anti-Staphylococcal properties (Houghten et al . , 1991) , no other functional properties have been reported which would suggest these peptides to have either anti-cytokine or anti-inflammatory activities.
• the present inventors also showed that RRWWCR effectively inhibits other CXC intercrines, such as GRO and MIP2, as evidenced by reducing GRO and MIP2/3 binding to human neutrophils .
• binding isotherms in the presence of the peptide fit two-site model best.
• the analysis of binding isotherms in the presence of Ac-RRWWCR-NH 2 showed that this peptide suppressed the binding of IL-8 to two classes of receptors differently.
• the estimated values of binding parameters showed that affinity of one class of receptors was suppressed by lO ⁇ M peptide, which suggested competitive inhibition. Higher concentration of peptide is needed to inhibit the other class of receptor nori-competitively.
• the activity of the present inhibitory peptides is specific for IL-8.
• Ac-RRWWCR-NH 2 does not inhibit the • binding of C5a or leukotriene B 4 to neutrophils, chemotaxis induced by formyl-L-Met-L-Leu-L-Phe (fMLP) , or S-glucuronidase release induced by fMLP, C5a or leukotriene B 4 . It also has no intrinsic ability to cause neutrophil chemotaxis or enzyme release. These observations suggest that peptides such as Ac-RRWWCR-NH 2 can strongly inhibit human neutrophil activations induced by IL-8 as a result of modulation of its receptor binding.
• hexamer and heptamer peptides based upon the Ac-RRWWCR-NH 2 structure are contemplated to preferentially inhibit enzyme release over chemotaxis.
• Ac-RRWWCR-NH 2 is herein shown to significantly suppress neutrophil chemotaxis induced by lOnM IL-8 at a concentration of 50 ⁇ M and -glucuronidase release at
• the inventors next examined the inhibitory activity of a second set of peptides which contained Ac-rrwwcrx- NH 2 (SEQ ID NO:2) , with all D-amino acids. Again, all RRWWCRX (SEQ ID NO:2) series peptides were found to have anti-IL-8 activity (Tables 5A and 5B) . However, the peptide Ac-rrwwcrc-NH 2 (SEQ ID NO:4) was found to be the best inhibitor, being almost four times as potent an inhibitor as Ac-rrwwcr-NH 2 (SEQ ID NO:l) .
• D-amino acid peptide inhibitors are expected to have a longer half life in vivo .
• the inventors therefore examined the IL-8 homologues Ac-KELRCQ-NH 2 (SEQ ID NO:54) and ELRCQCIKTY (SEQ ID NO:49, including the C-X-C motif characteristic of intercrine peptides) , along with its two non-Cys- containing analogues, ELRSQSKTY (SEQ ID NO:50) and ELRMQMKTY (SEQ ID NO:51) . None of these peptides had the ability to inhibit IL-8 binding to neutrophils. The inventors next searched the protein databases to determine if RRWWCR (SEQ ID NO:l) might occur in other peptides which might have relevant physiologic functions in relation to IL-8.
• RRWWCR SEQ ID NO:l
• RRWWCR RRWWCR
• GWRRWWCDAVLY SEQ ID NO:53
• OIPRRSWCRFLF SEQ ID NO:52
• the former peptide is contained in "cell surface glycoprotein CDllc precursor - human leukocyte adhesion receptor pl50,95 alpha chain" (Corbi et al . , 1990; Accession number, A36534 ⁇ A35543 ⁇ S00864) and the latter is 3',5' -cyclic GMP phosphodiesterase beta chain - bovine (Ovchinnikov et al . , 1987; Accession Number, S00251) .
• RRWWCX SEQ ID NO:23
• RRWWCRX SEQ ID NO:2
• inhibitors have the distinct advantage that they will likely permit neutrophils to enter the lungs, via chemotaxis which is not readily inhibited, but that they will then preferentially prevent the detrimental enzyme release (McGuire et al . , 1982) .
• the peptides and compositions of the present invention have utility in other embodiments. These include, for example, their use in various bioassays, e.g., as positive controls in assays of IL-8 inhibitors or neutrophil inhibitors; uses in further delineating IL-8 receptor interactions and functions; generating antibodies, and the like.
• Bioly functional equivalent protein or peptide is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity and that key active site or structurally vital residues cannot be exchanged.
• Biologically functional equivalent peptides are therefore defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted.
• hexamer or heptamer peptides it is contemplated that only about two, or more preferably, a single amino acid change would be made within a given peptide.
• a plurality of distinct peptides with different substitutions may easily be made and used in accordance with the invention.
• Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
• An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that phenylalanine, tryptophan and tyrosine all have a generally similar shape.
• arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined herein as biologically functional equivalents.
• hydropathic index of amino acids may be considered.
• Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5) ; valine (+4.2) ; leucine (+3.8) ; phenylalanine (+2.8) ; cysteine/cystine (+2.5) ; methionine (+1.9) ; alanine (+1.8) ; glycine (-0.4) ; threonine (-0.7) ; serine (-0.8) ; tryptophan (-0.9) ; tyrosine (-1.3) ; proline (-1.6) ; histidine (-3.2) ; glutamate (glutamic acid) (-3.5) ; glutamine (-3.5) ; aspartate (aspartic acid) (-3.5) ; asparagine (-3.5) ; lysine (-3.9) ; and arginine (-4.5) .
• hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference) . It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those which are within +1 are particularly preferred, and those within +0.5 are even more particularly preferred.
• an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically functional equivalent protein or peptide.
• substitution of amino acids whose hydrophilicity values are within +2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
• the peptides and compositions of the invention may be used for treating a variety of diseases and disorders in which CXC intercrines, such as IL-8, or neutrophils are involved or in which there is an inappropriate or increased inflammatory response.
• the invention is particularly suitable for the treatment of lung inflammation such as that connected with asthma, bronchitis, cystic fibrosis and ARDS.
• parenteral administration such as intravenous, intramuscular or subcutaneous injection is contemplated to be the most preferred route, although one may also use aerosols or inhalants. Sprays, aerosols and inhalants, are only effective if the droplets are sufficiently fine and uniform in size so that the mist reaches the bronchioles.
• Particle size is of major importance in the administration of therapeutic agents via aerosols or inhalants.
• the optimum particle size for penetration into the pulmonary cavity is of the order of 0.5 to 7 ⁇ m.
• Fine mists are produced by pressurized aerosols, their use is considered advantageous.
• Such treatment strategies and therapeutic formulations are described in detail hereinbelow in Example VIII.
• compositions of the present invention will generally comprise an effective amount of a relatively small intercrine- or IL-8-inhibiting peptide or peptides, dissolved or dispersed in a pharmaceutically acceptable medium.
• pharmaceutically acceptable refers to molecular entities and compositions that do not produce an allergic, toxic, or otherwise adverse reaction when administered to a human.
• Pharmaceutically acceptable media or carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
• the intercrine-, IL-8- and neutrophil-inhibiting peptides may also be combined with other agents such as IL-8-derived N-terminal peptides, IFN- ⁇ , oxygen radical scavengers and the like, to create peptide cocktails for treatment.
• agents such as IL-8-derived N-terminal peptides, IFN- ⁇ , oxygen radical scavengers and the like.
• the preparation of pharmaceutical or pharmacological compositions containing an intercrine-, IL-8- and neutrophil-inhibiting peptide or peptides, including dextrorotatory peptides, as an active ingredients will be known to those of skill in the art in light of the present disclosure.
• compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection; as tablets or other solids for oral administration; as time release capsules; or in any other form currently used, including cremes, lotions and mouthwashes, and the inhalents and aerosols of Example VIII.
• Solutions of the active peptides and compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, . and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms .
• Sterile solutions suitable for injection are contemplated to be useful in treating various diseases and may be administered into the blood stream or into the precise site of the inflammation.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists . It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• a peptide or peptides can be formulated into a composition in a neutral or salt form.
• Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like.
• inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
• Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, tri
• the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like) , suitable mixtures thereof, and vegetable oils.
• the proper fluidity can be maintained, for example, by the use of a coating, such as le'cithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• DMSO dimethyl methacrylate
• compositions may include vegetable oils, animal fats, and more preferably, semisolid hydrocarbons obtained from petroleum.
• Particular components used may include white ointment, yellow ointment, cetyl esters wax, oleic acid, olive oil, paraffin, petrolatum, white petrolatum, spermaceti, starch glycerite, white wax, yellow wax, lanolin, anhydrous lanolin and glyceryl monostearate.
• Various water-soluble ointment bases may also be used, including glycol ethers and derivatives, polyethylene glycols, polyoxyl 40 stearate and polysorbates. Even delivery through the skin may be employed if desired, e.g., by using transdermal patches, iontophoresis or electrotransport . Buffered ophthalmic solutions also fall within the scope of the invention. These may be used in connection with patients suffering from disorders connected with increased retinal angiogenesis. The buffering is necessary due to pH changes the peptide may cause.
• Ophthalmic preparations may be created in accordance with conventional pharmaceutical practice, see for example "Remington's Pharmaceutical Sciences” 15th Edition, pages 1488 to 1501 (Mack Publishing Co., Easton, PA) .
• Suitable ophthalmic preparations will generally contain a novel dipeptide, peptide or agent as disclosed herein in a concentration from about 0.01 to about 1% by weight, and preferably from about 0.05 to about 0.5%, in a pharmaceutically acceptable solution, suspension or ointment .
• the ophthalmic preparation will preferably be in the form of a sterile buffered solution containing, if desired, additional ingredients, for example preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonanoic wetting or clarifying agents, viscosity-increasing agents and the like.
• Suitable preservatives for use in such a solution include benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal and the like.
• Suitable buffers include boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, sodium biphosphate and the like, in amounts sufficient to maintain the pH at between about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5.
• Suitable tonicity agents are dextran 40, '• xtran 70, dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride, and the like, such that the sodium chloride equivalent of the ophthalmic solution is in the range 0.9 plus or minus 0.2%.
• Suitable antioxidants and stabilizers include sodium bisulfite, sodium metabisulfite, sodium thiosulfate, thiourea and the like.
• Suitable wetting and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol .
• Suitable viscosity-increasing agents include dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose and the like.
• therapeutics Upon formulation, therapeutics will be administered in a manner compatible with the dosage formulation, and in such amount as is pharmacologically effective.
• the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
• a minimal volume of a composition required to disperse the peptide is typically utilized. Suitable regimes for administration are also variable, but would be typified by initially administering the compound and monitoring the results and then giving further controlled doses at further intervals.
• a suitably buffered, and if necessary, isotonic aqueous solution would be prepared and used for intravenous, intramuscular, subcutaneous or even intraperitoneal administration.
• One dosage could be dissolved in 1 mL of isotonic NaCl solution and either added to lOOOmL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example,
• active compounds may be administered orally. This is contemplated for agents which are generally resistant, or have been rendered resistant, to proteolysis by digestive enzymes. Such compounds are contemplated to include dextrorotatory peptides; chemically designed or modified agents; and peptide and liposomal formulations in time release capsules to avoid peptidase and lipase degradation.
• Oral formulations may include compounds in combination with an inert diluent or an assimilable edible carrier; those enclosed in hard or soft shell gelatin capsules; those compressed into tablets; or those incorporated directly with the food of the diet.
• the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• Such compositions and preparations should generally contain at least 0.1% of active compound.
• the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit.
• the amount of active compounds in such therapeutical!y useful compositions is such that a suitable dosage will be obtained.
• Tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
• a binder as gum tragacanth, acacia, cornstarch, or gelatin
• excipients such as dicalcium phosphate
• a disintegrating agent such as corn starch, potato starch, alginic acid and the like
• a lubricant such as magnesium stearate
• a sweetening agent such as sucrose, lactose or saccharin may be added or a flavor
• tablets, pills, or capsules may be coated' with shellac, sugar or both.
• a syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
• any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
• the active compounds may be incorporated into sustained- release preparation and formulations.
• neutrophils were separated by dextran sedimentation and erythrocyte lysis by the method of
• Recombinant human IL-8 (72 amino acids; Pepro Tech Inc., Rocky Hill, NJ) was radioactively labeled with ⁇ - ⁇ - 5 I by the chloramine T method of Hunter and Greenwood (Hunter & Greenwood, 1962) . Binding studies were performed according to Besemer et al .
• the RRWWCR-type peptides were synthesized by Houghten Pharmaceutical Company in San Diego using tBOC for protection of the a-amino group (Stewart & Young, 1969) . All synthetic peptides were purified on high performance liquid chromatography (HPLC) using a preparative C18 reverse phase column (Waters Co., New Bedford, MA) . Peptides were eluted using a gradient from 0.1% trifluoroacetic acid (TFA) to 80% acetonitrile in 0.1% TFA. The composition of the peptides was confirmed by amino acid analysis and sequencing.
• HPLC high performance liquid chromatography
• TFA trifluoroacetic acid
• the data are expressed as the ⁇ mean and the variation as the standard deviation (S.D.) . Significance was determined by the Sheffes test when the variances were equal and the populations were normally distributed and only 2 groups were compared. Multiple comparisons were made using the analysis of variance and Sheffe's test.
• Example 2 An average EC 5Q was determined from multiple replicates of the binding inhibition curve to be 13.7 + 0.6 ⁇ M (a representative curve from these assays is shown in Figure 2) . If the mechanism of inhibition were purely competitive with a single site model, the estimated K j was approximately 10 ⁇ M. However, the mechanism was more complicated. A steeper binding inhibition curve than usual single site model as well as high Hill coefficient value (1.5 - 1.7) suggested the presence of positive cooperativity. The curve did not fit either one-, two- or three-site model well when it was analyzed using the Lundon II computer program, although IL-8 is known to react with at least two distinct receptors on neutrophils.
• C5a human fifth component of complement
• Enzymobead Bio Rad, Richmond, CA
• Tritiated leukotriene B ⁇ was purchased from Du Pont Co., Wilmington, DE. Binding assays for C5a and leukotriene B 4 were performed as described for IL-8 binding in Example I, except that the buffers used were
• Chemotaxis was performed using the leading front method as described by Zigmond and Hirsh (Zigmond &
• IL-8 or controls were placed in the lower well of a Boyden chamber.
• a five micron pore size, 100 ⁇ m thick cellulose nitrate filter (Sartorius Filter, Inc., San Francisco, CA) was placed on the surface and the chamber was then assembled.
• a 200 ⁇ l aliquot of the neutrophil preparation (lxlO 6 cells/ml) in RPMI-1640 media containing 1% BSA was added to the top of the filter and incubated at 37°C for 30 min.
• the filter was. then fixed, stained and mounted on a glass microscope slide.
• the leading front was determined by the position of the leading two cells. The distance that the leading two cells had moved through the filter was measured for six fields on each filter. The measurements were made with four filters for each set of conditions.
• Cytochalasin B (Sigma Chemical Co.) was stored in dimethyl sulfoxide at a concentration of 5 mg/ml and was diluted to a concentration of 50 ⁇ g/ml in Hank's Balanced Salt Solution (HBSS) immediately before use. Cytochalasin B, 200 ⁇ l, was added to 1 ml of suspension of neutrophils, 6.25xl0 6 cells/ml in HBSS, to achieve a final cytochalasin B concentration of 10 ⁇ g/ml. The solution was then incubated in 96 well plates at room temperature for 10 min.
• HBSS Hank's Balanced Salt Solution
• the stimulant 100 ⁇ l was added, and this cell suspension was incubated for 30 min at 37°C. The plates were centrifuged and 100 ⁇ l of supernatant was removed. Aliquots of 40 ⁇ l of supernatant were mixed with lO ⁇ l of 0.01M phenolphthalein-glucuronic acid (Sigma Chemical Co.) and 40 ⁇ l of 0.1M sodium phosphate pH 4.6 in 96-well plates for / ⁇ -glucuronidase measurement. After 16 hours incubation at 37°C, 200 ⁇ l of 0.2M Glycine in 0.2M NaCl, pH10.4 was added to each well and 0D 54Q was measured as the enzyme activity.
• Figures without parentheses represent mean ⁇ SD of the experimental observations in 20 different fields on 4 filters.
• Figures inside parentheses represent a calculation of migration expected if the cells were responding to the absolute concentration and not to the gradient.
• the inventors also tested several additional peptides which were either related to the amino terminal portion of IL-8 or were found in other proteins and had five of the six residues in RRWWCR (SEQ ID NO:l) .
• the peptides ELRCQCIKTY, ELRSQSIKTY, ELRMQMIKTY, QIPRRSWCRFLF, and GWRRWWCDAVLY (SEQ ID NOS:49 through 53, respectively) were synthesized at The University of Texas Health Center at Tyler utilizing an 431 Peptide Synthesizer (Applied Biosystems, Foster City, CA) , using the 9-fluorenylmethoxycarbonyl (fMOC) group to protect the ⁇ -amino group as described by Meienhofer and coworkers (Meienhofer et al .
• Ac-RRWWCX-NH 2 (SEQ ID NO:23) was also examined for the ability to inhibit other CXC intercrines.
• the present example demonstrates that, in addition to IL-8 inhibition, Ac-RRWWCX-NH 2 (SEQ ID NO:23) effectively inhibits GRO and MIP2/S binding to human neutrophils.
• MIP2jS and GRO/MGSA were radioiodinated using Bolten Hunter reagent. The radioiodinated components were mixed with various concentration of the Ac-RRWWCX-NH 2 (SEQ ID NO:23) peptide and incubated at room temperature for 15 minutes. Neutrophil suspension (1 X 10 cells in
• B is bound radioactivity in the presence of the peptide
• T is bound radioactivity in the absence of the peptide
• NSP is bound radioactivity in the presence of excess nonlabelled ligand
• This example is directed to the techniques contemplated by the inventors for use in further characterizing the in vivo actions of the IL-8 inhibitors and their use in animal or human treatment protocols.
• vi tro stability examinations may be performed on the peptides including, for example, pre-incubation in human serum and plasma; treatment with various proteases; and also temperature- and pH-stability analyses. It is already known that D-amino acid peptides are active and that these would likely have enhanced stability in vivo .
• the inventors propose to examine the in vivo properties and effects of the IL-8 peptide inhibitors in animal models prior to moving onto clinical trials .
• the most suitable form, dose and any possible toxicity of the peptides will be determined in animal studies, as is routinely employed in the art.
• the bio- availability and half lives of the peptides administered in various ways may be determined using radioactively labeled peptides and examining their longevity and tissue distribution. If further stability enhancement was desired, the peptides could also be administered in the form of lipid-tailed peptides, surfactant-like micelles, peptide multimers or in semi-permeable drug release capsules .
• the biological effects of the peptides may be determined in various models of human disease.
• IL-8 has been shown to cause the accumulation of neutrophils and edema in rabbit skin (Rampart et al . , 1989) . Therefore, a rabbit dermal inflammation model will be employed to determine what dose of the peptides can effectively inhibit the neutrophil accumulation and edema. This model is useful because of the ease of assessment of inflammation. The most suitable route of peptide administration may be easily determined by comparative in vivo tests.
• New Zealand albino rabbits may be injected with I-labeled human serum albumin through the lateral ear vein. Certain sites may then be injected intradermally with the test compounds, i.e., an agonist to attract neutrophils and an IL-8 inhibitor peptide; the agonist and a control peptide; and the agonist alone. About two hours later, full thickness skin samples 1 cm in diameter may be punched out, fixed and stained with Wright-Giemsa or for myeloperoxidase and the histology examined for neutrophil accumulation and edema or tissue damage. Other skin biopsies may be counted in a gamma counter to assess the amount of albumin flux into the injected skin. Skin inflammation after administration of the inhibitor can then be compared to the time-matched controls, ideally be performed in the same animal . At least 4 replicates for each experimental arm are recommended.
• ARDS Adult Respiratory Distress Syndrome
• the minipigs will be treated with IL-8 through the most appropriate route to cause neutrophil influx and enzyme release into the lungs as assessed above.
• the peptides of interest will be administered to determine appropriate doses for use in impeding these neutrophil functions, especially the dose with which the peptide suppresses enzyme release, but not the neutrophil influx.
• the acute lung damage model of minipigs caused by gram negative bacteria in the circulation will be employed.
• the peptides of interest will be administered to the animals and the effect of the peptides on the prevention of lung damage will be assessed.
• the number of neutrophils in bronchoalveolar fluids, the amount of enzyme released into lung parenchyma, and the degree of protein leakage from circulation to lung will be used as indicators in this study.
• IL-8 causes neutrophil influx, enzyme release into the lungs, and/or ARDS- ' like tissue damage to the lungs, as expected, the peptides of interest will be administered to determine the appropriate doses for use in impeding these neutrophil functions.
• various intravenous doses of radioactively labelled peptides will be administered initially. Plasma concentrations and forms of the radioactivity will then be determined. From these data, plasma clearance, half life and steady state volume of distribution will be measured and used to determine the most effective dose ranges.
• IL-8 peptide inhibitors and compositions of the present invention have not yet been tested in a clinical setting in human subjects.
• compositions which include peptide inhibitors of IL-8 will prove to be useful in the treatment of various conditions, including pulmonary disorders such as bronchial inflammation, cystic fibrosis, pleural effusions, asthma, bronchitis and ARDS; skin disorders such as psoriasis and dermatitis; diseases of the joints, including rheumatoid arthritis; and in the treatment of pseudogout, inflammatory bowel disease, reperfusion cardiac damage or even in the treatment of cancer and other diseases and disorders associated with increased cellular proliferation.
• pulmonary disorders such as bronchial inflammation, cystic fibrosis, pleural effusions, asthma, bronchitis and ARDS
• skin disorders such as psoriasis and dermatitis
• diseases of the joints including rheumatoid arthritis
• pseudogout inflammatory bowel disease, reperfusion cardiac damage or even in the treatment of cancer and other diseases and disorders associated with increased cellular proliferation.
• peptides are thought to be particularly suitable for the inhibition of pulmonary inflammation, such as occurs in ARDS, chronic bronchitis and cystic fibrosis, suitable treatment methods for these disorders will be described.
• parenteral administration such as by using intravenous, intramuscular or subcutaneous injections.
• aerosols or inhalants are also used.
• MOLECULE TYPE peptide
• SEQUENCE DESCRIPTION SEQ ID NO:1 :
• MOLECULE TYPE peptide
• SEQUENCE DESCRIPTION SEQ ID NO:3 :
• MOLECULE TYPE peptide
• SEQUENCE DESCRIPTION SEQ ID NO:5 :
• MOLECULE TYPE peptide
• SEQUENCE DESCRIPTION SEQ ID NO:7 :
• MOLECULE TYPE peptide
• SEQUENCE DESCRIPTION SEQ ID NO: 9 :
• MOLECULE TYPE peptide
• SEQUENCE DESCRIPTION SEQ ID NO:35:
• MOLECULE TYPE peptide
• SEQUENCE DESCRIPTION SEQ ID NO:45:

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Citations (3)

• 1993
  • 1993-12-15 WO PCT/US1993/012245 patent/WO1995016702A1/en not_active Application Discontinuation
  • 1993-12-15 JP JP7516716A patent/JPH09510688A/ja active Pending
  • 1993-12-15 KR KR1019960703142A patent/KR100290224B1/ko not_active IP Right Cessation
  • 1993-12-15 EP EP94906442A patent/EP0734394A1/en not_active Withdrawn
  • 1993-12-15 AU AU60147/94A patent/AU685079B2/en not_active Ceased
• 1996
  • 1996-06-11 FI FI962413A patent/FI962413A/fi not_active IP Right Cessation
  • 1996-06-14 NO NO962549A patent/NO962549L/no not_active Application Discontinuation

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