Patent Abstract:
new polypeptide agonists of at2r are disclosed , as well as pharmaceutical compositions comprising the agonists , methods of their use in the treatment of diseases , conditions or disorders characterized by insufficient at2r activity or excessive at1r activity , and methods of their use as laboratory reagents for research purposes .

Detailed Description:
at2r activation is suppressed in a variety of disease states including hypertension , diabetes , cancers and various neurodegenerative diseases . suppression of at2r leads to increased activity of the at1r which is a major contributor to metabolic diseases ( e . g ., cardiovascular and renal diseases , type 2 diabetes ) and cancers . therefore , the present invention provides a new class of laboratory reagents and therapeutic polypeptides which can be used to characterize and treat such disorders . the patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art . the issued u . s . patents , pending u . s . applications , published foreign patents and applications , and references , including protein and nucleotide database sequences , that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference . as used herein , the terms “ about ” or “ approximately ” mean within twenty percent ( 20 %) of the numerical amount cited . as used herein , the terms “ increased ” or “ decreased ” mean at least 10 % more or less , respectively , relative to pre - treatment with an agonist of the invention . as used herein , a “ pharmaceutical composition ” includes an active agent and a pharmaceutically acceptable carrier . as used herein , the phrase “ pharmaceutically acceptable ” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce a severe allergic , pyrogenic or similarly undesired reaction when administered to a mammal . as used herein , the term “ carrier ” refers to a diluent , adjuvant , excipient , or vehicle with which a compound is administered . such pharmaceutical carriers can be sterile liquids , such as water and oils , including those of petroleum , animal , vegetable or synthetic origin , such as peanut oil , soybean oil , mineral oil , sesame oil and the like . water or other aqueous solutions , saline solutions , aqueous dextrose and glycerol solutions may be employed as carriers , particularly for injectable solutions . suitable pharmaceutical carriers are described in “ remington &# 39 ; s pharmaceutical sciences ” by e . w . martin ( 61 ). the invention provides a new class of agonists which are polypeptides ( or polypeptide derivatives or analogues ) comprising 6 amino acid residues with the generic sequence a1 - a2 - a3 - a4 - a5 - a6 , where a1 is lys ; a2 is pro , 3hyp or 4hyp ; a3 is leu or ile ; a4 is lys , a5 is pro , 3hyp or 4hyp , and a6 is trp . this new class is referred to as np - 6ak agonists . sequences with hyp in at least one position may be preferred due to increased stability . other derivatives or analogues of these agonists may include chemical modifications that increase stability in the bloodstream for use as a pharmaceutical reagent . one possible modification is formation of non - natural peptide bonds for additional stability or the attachment of the side chain atoms to a different atom of the residue , an example of such chemistry is cited in hook et al . ( 60 ). the authors describe beta amino acids , wherein the side chains are attached to the beta carbon , whereas natural amino acid side chains are attached to the alpha carbon . various studies have shown that these “ beta peptides ” are less likely to be degraded by non - specific peptidases compared to natural peptides . any such chemistry that modifies the natural peptide for additional stability could be used , including peptoids in which the side chain is attached to the nitrogen . another method of stabilizing the polypeptides of the invention is covalent or non - covalent association with an inert water - soluble polymer . when administered systemically , therapeutic compositions are often cleared rapidly from the circulation and may therefore elicit relatively short - lived pharmacological activity . consequently , frequent injections of relatively large doses of bioactive compounds may be required to sustain therapeutic efficacy . any water - soluble ( e . g ., at least about 0 . 01 mg / ml ) inert polymer which provides the conjugate with the desired increase in stability or half - life is suitable for use in the invention . non - proteinaceous polymers are particularly preferred . the polymer is preferably a hydrophilic synthetic polymer , such as a polyvinyl polymer ( e . g ., polyvinylalcohol and polyvinylpyrrolidone ), polyalkylene ether ( e . g ., polyethylene glycol ( peg )); polyoxyalkylene ( e . g ., polyoxyethylene , polyoxypropylene , and block copolymers of polyoxyethylene and polyoxypropylene ( pluronics )); polymethacrylate ; or carbomer . however , natural polymers are also useful , such as branched or unbranched polysaccharides which comprise the saccharide monomers d - mannose , d - and l - galactose , fucose , fructose , d - xylose , l - arabinose , d - glucuronic acid , sialic acid , d - galacturonic acid , d - mannuronic acid ( e . g ., polymannuronic acid , or alginic acid ), d - glucosamine , d - galactosamine , d - glucose and neuraminic acid including , for example , lactose , amylopectin , starch , hydroxyethyl starch , amylose , dextran sulfate , dextran , dextrins , glycogen , or polymers of sugar alcohols such as polysorbitol and polymannitol , heparin or heparan . the molecular weight of the polymer can range from about 10 , 000 to 500 , 000 daltons ( d ), and may typically be about 20 , 000 d , about 30 , 000 d , about 40 , 000 d , or about 50 , 000 d . compounds modified by the covalent attachment of water - soluble polymers such as polyethylene glycol ( peg ), copolymers of polyethylene glycol and polypropylene glycol , or monomethoxypolyethylene glycol ( mpeg ), carboxymethyl cellulose , dextran , polyvinyl alcohol , polyvinylpyrrolidone or polyproline are known to exhibit substantially longer half - lives in blood following intravenous injection than do the corresponding unmodified compounds . such modifications may also increase the composition &# 39 ; s solubility in aqueous solution , reduce aggregation , increase the physical or chemical stability of the compound , and reduce the immunogenicity and reactivity of the composition . attachment of polyethylene glycol ( peg ) to agonist compositions of the invention is particularly useful because peg has very low toxicity in mammals and may reduce the immunogenicity or antigenicity of the agonist compositions . numerous activated forms of peg suitable for direct reaction with proteins have been described . useful peg reagents for reaction with protein amino groups include active esters of carboxylic acid or carbonate derivatives , particularly those in which the leaving groups are n - hydroxysuccinimide , p - nitrophenol , imidazole or 1 - hydroxy - 2 - nitrobenzene - 4 - sulfonate . peg derivatives containing maleimido or haloacetyl groups are useful reagents for the modification of protein free sulfhydryl groups . likewise , peg reagents containing amino hydrazine or hydrazide groups are useful for reaction with aldehydes generated by periodate oxidation of carbohydrate groups in proteins . the agonist compositions of the present invention may be delivered in a microencapsulation device so as to reduce or prevent a host immune response against the polypeptide or against cells which may produce the polypeptide . the polypeptide or compositions of the present invention may also be delivered microencapsulated in a membrane , such as a liposome . as an example , polymers such as peg may be conveniently attached to one or more reactive amino acid residues in a polypeptide of the agonist compositions , such as the alpha - amino group of the amino terminal amino acid , the epsilon amino groups of lysine side chains , the sulfhydryl groups of cysteine side chains , the carboxyl groups of aspartyl and glutamyl side chains , the alpha - carboxyl group of the carboxy - terminal amino acid , tyrosine side chains , or to activated derivatives of glycosyl chains attached to certain asparagine , serine or threonine residues . another method of modifying the polypeptide agonists of the invention is to add a signal sequence to the n - or c - terminus . the term “ signal sequence ,” as used herein , refers to any short peptide that directs the trafficking of a protein in the cell . signal sequences may , for example , direct secretion of a polypeptide , or localization within an intracellular compartment . signal sequences also frequently determine the orientation of a peptide across a cell membrane . one example is an n - terminal sequence of about 20 amino acids that directs secretory and transmembrane proteins to the endoplasmic reticulum ( er ) ( see , e . g ., von heijne ( 1985 ), j . mol . biol . 184 : 99 - 105 ). signal sequences may also be engineered to include one or more specific protease recognition sites , such that the signal sequences will be removed by endogenous proteases after trafficking . activation of at2r in laboratory testing is of high interest due to the many effects of at2r described above . therefore , in one aspect , the invention provides at2r agonists as in vitro or in vivo reagents for laboratory research . thus an np - 6ak agonist to at2r has utility as a control to characterize and quantify downstream effects of the receptor activation , such as its effects on the mammalian target of rapamycin ( mtor ), nhe6 , erbb3 and nitric oxide synthase . for example , cho cells expressing at2r and mtor are treated with an np - 6ak agonist and insulin to activate both mtor and at2r . at2r suppresses mtor - mediated phosphorylation of ribosomal protein s6 ( rps6 ). western blotting can be used to determine rps6 phosphorylation state which is decreased by at least 10 % in response to at2r activation by the agonist np - 6ak . the same cell line can then be treated with a different at2r agonist candidate to assess the efficacy of the agonist candidate , or the same cell line can be treated with both an at2r agonist of the invention and an at2r antagonist candidate ( e . g ., ema300 , smith et al . ( 2013 ), pain medicine 14 ( 10 ): 1557 - 68 ; pd123319 , chakrabarty et al . ( 2008 ), endocrinology 149 ( 7 ): 3452 ) to assess the efficacy and mode of action ( i . e ., competitive , non - competitive ) of the antagonist candidate . an np - 6ak agonist increases expression levels of at least one , and in some embodiments all three , mcl - 1 isoforms . in order to treat an at1r - mediated inflammatory response or an inflammatory response arising from under - activation of the at2r , and / or the symptoms arising therefrom , an np - 6ak agonist is administered by any route that will permit delivery of the active agent to the affected cells . in some embodiments , administration is subcutaneous , intramuscular or intraperitoneal , but may also be by inhalation , intra - arterial , intravenous , intradermal , topical , oral , parenteral , intraventricular , or intracranial administration . alternatively , the active agent may be delivered locally to the system or the affected cells by any suitable means . in therapeutic treatments of the invention , a therapeutically effective amount of the pharmaceutical composition is administered to a mammalian patient . as used herein , the term “ therapeutically effective amount ” means an amount sufficient to reduce by at least 15 percent , preferably by at least 50 percent , more preferably by at least 90 percent , and most preferably substantially eliminate or prevent , a clinically significant metric or deficit in the activity , function and response of the patient . specifically , a therapeutically effective amount will cause one or more of the following : decreased at1r activation , increased at2r activation ; decreased cortisol levels ; stabilized insulin levels ; decreased pro - inflammatory cytokines , decreased pro - inflammatory interleukins , increased function of dopaminergic neurons , decreased reactive oxygen species , decreased mucous production , or a decrease or increase in any other relevant markers as discussed herein or that would be known to one of ordinary skill in the art as it relates to cystic fibrosis ( cf ), diabetes , metabolic x syndrome ; hyperglycemia , autism , alzheimer &# 39 ; s disease , inflammation or cancer . the frequency and dosage of the therapy can be titrated by the ordinary physician or veterinarian using standard dose - to - response techniques that are well known in the art . liquid forms , such as lotions suitable for topical administration or for cosmetic application , may include a suitable aqueous or non - aqueous vehicle with buffers , suspending and dispensing agents , thickeners , penetration enhancers , and the like . solid forms such as creams or pastes or the like may include , for example , any of the following ingredients , water , oil , alcohol or grease as a substrate with surfactant , polymers such as polyethylene glycol , thickeners , solids and the like . liquid or solid formulations may include enhanced delivery technologies such as liposomes , microsomes , microsponges and the like . the above - described components for liquid , semisolid and solid topical compositions are merely representative . other materials as well as processing techniques and the like are set forth in part 8 of remington &# 39 ; s pharmaceutical sciences , 17th edition , 1985 , mack publishing company , easton , pa ., which is incorporated herein by reference . when pharmaceutical compositions are to be administered transdermally they typically are employed as liquid solutions or as gels . in these settings the concentration of agonists of the present invention range from about 0 . 1 % to about 20 %, and preferably from about 0 . 1 % to about 10 %, of the composition with the remainder being aqueous mixed or non - aqueous vehicle , such as alcohols and the like , suspending agents , gelling agents , surfactant , and the like . examples of suitable such materials are described below . the agonist - containing compositions of this invention can also be administered in sustained release transdermal forms or from transdermal sustained release drug delivery systems . a description of representative sustained release materials can be found in the incorporated materials in remington &# 39 ; s pharmaceutical sciences , supra . the agonist compositions for systemic administration include compositions for oral administration , that is liquids and solids , and compositions for injection . compositions for oral administration can take the form of bulk liquid solutions or suspensions , or bulk powders . more commonly , however , the compositions are presented in unit dosage forms to facilitate accurate dosing . the term “ unit dosage forms ” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals , each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect , in association with a suitable pharmaceutical solvent . typical unit dosage forms include profiled , premeasured ampules or syringes of the liquid compositions or pills , tablets , capsules or the like in the case of solid compositions . according to one embodiment , an agonist composition of the present invention is usually a minor component ( from about 0 . 01 to about 20 % by weight or preferably from about 0 . 1 to about 15 % by weight ) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form . liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers , suspending and dispensing agents , colorants , flavors and the like . solid forms may include , for example , any of the following ingredients , or compounds of a similar nature including a binder such as microcrystalline cellulose , gum tragacanth or gelatin ; an solvent such as starch or lactose , a disintegrating agent such as alginic acid , primogel , or corn starch ; a lubricant such as magnesium stearate ; a glidant such as colloidal silicon dioxide ; a sweetening agent such as sucrose or saccharin ; or a flavoring agent such as peppermint , methyl salicylate , or orange flavoring . according to another embodiment , injectable compositions are typically based upon injectable sterile saline or phosphate - buffered saline or other injectable carriers known in the art . a compound of the present invention in such compositions is typically a minor component , about 0 . 1 - 30 % by weight , with the remainder being the injectable carrier and the like . the above - described components for orally administrable or injectable compositions are merely representative . other materials as well as processing techniques and the like are set forth in the part of remington &# 39 ; s pharmaceutical sciences noted above . the np - 6ak agonists if the invention , including polypeptides comprising , consisting of , or consisting essentially of the sequences lys - pro - leu - lys - pro - trp ; lys - 3hyp - leu - lys - pro - trp ; lys - 4hyp - leu - lys - pro - trp ; lys - pro - ile - lys - pro - trp ; lys - 3hyp - ile - lys - pro - trp ; lys - 4hyp - ile - lys - pro - trp ; lys - pro - leu - lys - 3hyp - trp ; lys - 3hyp - leu - lys - 3hyp - trp ; lys - 4hyp - leu - lys - 3hyp - trp ; lys - pro - ile - lys - 3hyp - trp ; lys - 3hyp - ile - lys - 3hyp - trp ; lys - 4hyp - ile - lys - 3hyp - trp ; lys - pro - leu - lys - 4hyp - trp ; lys - 3hyp - leu - lys - 4hyp - trp ; lys - 4hyp - leu - lys - 4hyp - trp ; lys - pro - ile - lys - 4hyp - trp ; lys - 3hyp - ile - lys - 4hyp - trp ; and lys - 4hyp - ile - lys - 4hyp - trp , can be tested in various cell - based systems to demonstrate at2r agonist activity , and utility in the methods described herein . thus , an np - 6ak agonist comprising the sequence lys - pro - leu - lys - pro - trp promoted cell survival across mouse hl - 1 cardiomyocytes and human smooth vascular muscle cells by acting through at2r activation . cell survival was assessed by measurement of cell index ( ci ) using a xcelligence ™ real - time cell analyzer ( westburg b v , leusden , n l ) to quantitatively determine the effect of different treatments on cell survival , adhesion , and proliferation . cell lines treated with the np - 6ak agonist ( 300 nm ) demonstrated significantly better survival and more proliferation than those treated with cgp42112a ( 300 nm ) ( sigma - aldrich , co . llc , st . louis , mo . ), a partial agonist of at2r . cgp42112a and the np - 6ak agonist ( 300 nm ) increased ci of serum starved hl - 1 cells in the order cgp42112a (≧ 14 %), np - 6ak (≧ 25 %). increase in ci is a direct effect of at2r activation . this was verified by addition of an at2r antagonist , pd123319 ( sigma - aldrich , co . llc , st . louis , mo .). in the presence of this antagonist , the cell survival otherwise observed using the np - 6ak agonist was not present . an np - 6ak agonist upregulated mcl - 1 by selectively activating at2r across several cell lines . this effect was observed in cardiomyocytes , human vascular smooth muscle cells , sh - sy5y cells ( atcc , manassas , va . ), and pc - 12 neuronal cells ( atcc , manassas , va .) in conditions of serum starvation and / or toxicity . when incubated with an np - 6ak agonist at a concentration of 300 nm , cardiomyocytes displayed 45 % higher mcl - 1 expression and human smooth vascular muscle cells displayed 22 % higher mcl - 1 expression ( fig1 ). when any of these cell lines was pre - treated with an at2r antagonist such as pd123319 , the mcl - 1 upregulation was not observed , implying that np - 6ak activation of at2r is necessary for mcl - 1 upregulation . cells treated with the np - 6ak agonist generated higher mcl - 1 upregulation relative to those treated with a partial agonist such as cgp42112a . the sh - sy5y cell line is a neuronal cell line with both dopaminergic and adrenergic receptors . when this cell line was treated with rapamycin , mcl - 1 expression was reduced . addition of an np - 6ak agonist to this cell line after treatment with rapamycin was able to recover mcl - 1 expression . when just the np - 6ak agonist was added at a concentration of 300 nm , these cells displayed 2 - fold increase in mcl - 1 expression . see fig2 . serum - starvation reduced survival of sh - sy5y cells and pc - 12 cells . when these cell lines were treated with an np - 6ak agonist , they had a higher survival rate and demonstrated increased mcl - 1 expression and neurite elongation . see fig3 . when pc - 12 cells were treated with at2r antagonist pd123319 , the protective effects of the np - 6ak agonist were lost , indicating that the np - 6ak agonist &# 39 ; s direct action is through at2r activation . treatment of this cell line with 300 nm of np - 6ak agonist led to increased cell viability by over 30 % relative to treatment with native ligand of at2r ang ii ( 300 nm ). relative to treatment with 300 nm cgp42112a , cells treated with an np - 6ak agonist displayed over 70 % increased survival . treatment with angii or cgp42112a was more detrimental relative to results obtained when cells are treated with at2r antagonist pd123319 . at2r activation by np - 6ak agonist promotes cell survival whereas at2r activation by other ligands are harmful to it . these results were assessed using an mts cell proliferation assay ( biovision inc ., milpitas , calif .). ex vivo neurons were obtained and treated with an np - 6ak agonist in different conditions to assess neuroprotective properties . normal primary murine embryonic cortical neurons in culture ( 14 days in vitro ) were challenged with nutrient deprivation by incubation in glucose - free locke &# 39 ; s medium ( schnapf et al . ( 1990 )). these cultures were then supplemented with 300 nm np - 6ak agonist or no np - 6ak agonist . cultures that were supplemented 300 nm np - 6ak agonist demonstrated 60 % increased activity ( p & lt ; 0 . 05 ) compared with negative controls . see fig4 . zucker obese ( zo ) rats ( charles river laboratories , inc ., wilmington , mass . ), a diabetic animal model displaying signs of cardiovascular disease were treated for 2 weeks ( dose of 0 . 9 mg / kg / day ) via subcutaneous injection with an np - 6ak agonist and assessed for several markers of cardiovascular health including blood markers and structural parameters of the heart . controls were zo rats that received saline . fasting plasma and urine profiles demonstrated that animals receiving the np - 6ak agonist had reduced triglycerides (˜ 50 %), urine protein (˜ 68 %), reduced urine n - acetyl - beta - a - glucosaminidase (˜ 60 %), and increased hdl by 12 % on average . echocardiography performed using the vevo ® 2100 platform ( visualsonics , toronto , ontario , ca ) indicated that animals treated with the np - 6ak agonist had improved structural cardiac parameters including circumferential strain of endocardium ( short axis view ), and myocardial performance index ( mpi ) ( p ≦ 0 . 005 ), and e / e ′ ratio ( p ≦ 0 . 002 ), a powerful predictor of primary cardiac events . see table 1 . although the invention has been described in detail with particular reference to these preferred embodiments , other embodiments can achieve the same results . variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents . the entire disclosures of all references , applications , patents , and publications cited herein are hereby incorporated by reference . 1 . kalupahana and moustaid - moussa ( 2012 ), “ the renin - angiotensin system : a link between obesity , inflammation and insulin resistance ,” obes . rev . 13 ( 2 ): 136 - 49 . 2 . santos et al . 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