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Definitions

• H3 relaxin human 3 relaxin
• the invention relates to H3 relaxin, pro- and prepro- H3 relaxin, the individual peptide chains which comprise these sequences, analogues of H3 relaxin, compositions including pharmaceutical compositions, as well as therapeutic uses and methods of treatment.
• the invention relates to nucleic acids encoding H3 relaxin, H3 pro- and prepro- relaxin, H3 relaxin analogues, and individual peptide chains which comprise these sequences.
• Relaxin genes and the encoded relaxin polypeptides have been identified in many species including man, pig, rat, sheep and shark, hi all these species only one relaxin gene has been characterised in mammals, with the exception of the human and higher primates where two separate genes have been described.
• the separate human genes were identified by the present applicant and designated HI (Hudson et al (1983) Nature 301, 628-631) and H2 (Hudson et al (1984) Embo. J. 3, 2333-2339).
• the peptide encoded by the H2 gene is the major stored and circulating form in the human
• HI relaxin expression is restricted to the decidua, placenta and prostate (Hansell et al (1991) J Clin. Endocrinol. Metab. 72, 899-904), however, the HI peptide has similar biological activity to that of H2 relaxin in a rat atrial bioassay (Tan et al (1998) Br. J. Pharmacol. 123, 762-770).
• relaxin include an ability to inhibit myometrial contractions, to stimulate remodelling of connective tissue and to induce softening of the tissues of the birth canal. Additionally, relaxin increases growth and differentiation of the mammary gland and nipple and induces the breakdown of collagen, one of the main components of connective tissue. Relaxin decreases collagen synthesis and increases the release of collagenases (Unemori et al (1990) J. Biol. Chem. 265, 10682-10685). These findings were recently confirmed by the establishment of the relaxin gene-knockout mouse (Zhao et al (1999) Endocrinology 140, 445-453), which exhibited a number of phenotypic properties associated with pregnancy.
• mice lacking a functionally active relaxin gene failed to relax and elongate the interpubic ligament of the pubic symphysis and could not suckle their pups, which in turn, died within 24 hours unless cross-fostered to relaxin wildtype or relaxin heterozygous foster mothers.
• relaxin causes a widening of blood vessels (vasodilatation) in the kidney, mesocaecum, lung and peripheral vasculature, which leads to increased blood flow or perfusion rates in these tissues (Bani et al (1997) Gen. Pharmacol. 28, 13-22). It also stimulates an increase in heart rate and coronary blood flow, and increases both glomerular filtration rate and renal plasma flow (Bani et al (1997) Gen. Pharmacol. 28, 13-22). The brain is another target tissue for relaxin where the peptide has been shown to bind to receptors (Osheroff et al (1991) Proc. Nal.
• H3 relaxin gene H3
• the production of H3 relaxin and analogues thereof has been made possible, as have uses and therapeutic treatment methods.
• the invention relates to the peptides human H3 relaxin, H3 prorelaxin and H3 preprorelaxin, to the individual peptide chains which comprise these sequences and to analogues thereof, particularly truncated and/or amino acid substituted modifications.
• the peptides are provided as pharmaceutically acceptable compositions for human or animal administration, by various therapeutic routes.
• Peptides are preferably isolated in purified or homogenous form free of contaminating peptides and proteins, or in a form of about 90-99% purity.
• composition comprising human H3 relaxin or a human H3 relaxin analogue having an A chain and a B chain,
• amino acid sequence truncated by up to 9 amino acids from the amino-terminus and/or up to about 5 amino acids from the carboxyl-terminus
• the A and B chains being linked by interchain disulphide bonds at Al 1-B10, and A24-B22, and wherein the human H3 relaxin or analogue thereof has relaxin bioactivity.
• composition comprising a human H3 relaxin analogue having a modified A chain and/or a modified B chain,
• carboxyl-terminus is an amide derivative,. and/or Ala at position 2 is replaced with Pro, and/or Arg at position 8 is replaced with Lys,
• composition comprising human H3 preprorelaxin or human H3 prorelaxin, having a signal, A chain, B chain and C chain in respect of human H3 preprorelaxin, and an A chain, B chain and C chain in respect of human H3 prorelaxin, the said amino acid chains having the amino acid sequences:
• the A chain comprising:
• Lys Ser Glu lie Ser Ser Leu Cys 20 (SEQ ID NO: 4)
• the B chain comprising: Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly Arg Glu Phe lie Arg 1 5 10 15
• the signal sequence comprising:
• composition comprising the C chain of human H3 relaxin, the C chain having the amino acid sequence: Arg Arg Ser Asp lie Leu Ala His Glu Ala Met Gly Asp Thr Phe Pro 1 5 10 15
• nucleic acid sequence encoding human prepro-H3 relaxin comprising the nucleic acid sequence:
• nucleic acid sequence encoding human pro-H3 relaxin including an A chain, B chain and C chain sequence
• a chain sequence comprising:
• nucleic acid sequence encoding human H3 relaxin having an A and B chain having an A and B chain
• a chain sequence comprising:
• nucleic acid sequence encoding the A, B or C peptide chains of human H3 relaxin, the said chains comprising the nucleic acid sequences:
• the nucleic acid sequences are isolated and purified nucleic acids, and may be contained within a vector, such as a plasmid, bacteriophage or virus DNA or RNA, and may be in single or double stranded form, and may include promoters or enhancers or other sequences which confer elevated, enhanced or other effects on expression in a host system such as a bacterial cell.
• a vector such as a plasmid, bacteriophage or virus DNA or RNA
• promoters or enhancers or other sequences which confer elevated, enhanced or other effects on expression in a host system such as a bacterial cell.
• the triplet codons of nucleic acids encode specific amino acids. More than one codon may encode the same amino acid, as is well and established in the art. Moreover, methods of modifying or altering the sequence of nucleic acids are well known in the art. Insofar as this invention pertains in its various aspects to nucleic acids encoding human H3 relaxin, pro- H3 relaxin, prepro- H3 relaxin, and constituent peptide chains thereof, the invention includes nucleic acid variants which encode the same protein products, or a protein product having relaxin activity.
• Nucleotide sequence aspects of this invention also include closely related nucleic acid sequences as defined by stringent hybridization, this being annealing of complimentary sequences under conditions of 0.25M H 2 PO 4 , pH 7.2, lmmol EDTA, 20% SDS at 65°C overnight; followed by 3 washes for 5min in 2xSSC, 0.1% SDS at room temperature; and finally a 30 min wash at 65°C in 0.1% SSC; where 6xSSC is 0.9M NaCl, 0.3M Na 3 CO 2 H 2 O at ph 7.0.
• Such sequences will encode H3 relaxin polypeptides having biological or immunological or other activity corresponding to those of H3 relaxin.
• vascular disease including coronary artery disease, peripheral vascular disease, vasospasm including Raynaud's phenomenon, microvascular disease involving the central and peripheral nervous system, kidney, eye and other organs; treatment of arterial hypertension; diseases related to uncontrolled or abnormal collagen or fibronectin formation such as fibrotic disorders (including fibrosis of lung, heart and cardiovascular system, kidney and genitourinary tract, gastrointestinal system, cutaneous, rheumatologic and hepatobiliary systems); kidney disease associated with vascular disease, interstitial fibrosis, glomerulosclerosis, or other kidney disorders; psychiatric disorders including anxiety states including panic attack, agoraphobia, global anxiety, phobic states; depression or depressive disorders including major depression, dysthymia, bipolar and unipolar depression; neurologic or neurodegenerative diseases (including memory loss or other memory disorders, dementias, Alzheimer's disease); disorders of learning, attention and motivation (including
• vascular disease including coronary artery disease, peripheral vascular disease, vasospasm including Raynaud's phenomenon, micro vascular disease involving the central and peripheral nervous system, kidney, eye and other organs
• vascular disease including coronary artery disease, peripheral vascular disease, vasospasm including Raynaud's phenomenon, micro vascular disease involving the central and peripheral nervous system, kidney, eye and other organs
• diseases related to uncontrolled or abnormal collagen or fibronectin formation such as fibrotic disorders (including fibrosis of lung, heart and cardiovascular system, kidney and genitourinary tract, gastrointestinal system, cutaneous, rheumatologic and hepatobiliary systems); kidney disease associated with vascular disease, interstitial fibrosis, glomerulosclerosis, or other kidney disorders
• psychiatric disorders including anxiety states including panic attack, agoraphobia, global anxiety, phobic states
• depression or depressive disorders including major depression, dysthymia, bipolar and unipolar depression
• SEQ ID NO 4 A chain peptide sequence
• SEQ ID NO 7 A chain nucleic acid sequence
• Fig. 1 Assembled DNA sequence of the H3 (A) and M3 (B) genes.
• Start and Stop codons as well as predicted TATA boxes and polyadenylation sequences are underlined.
• the positions of the putative signal peptide, and B-, C- and A- chain peptide sequences are indicated by arrows.
• A- and B-chain sequences are boxed and the residues implicated in relaxin receptor binding are shaded.
• the intron sequence which is at an identical position in the C-chain in both the human (A) and mouse (B) sequences, is indicated by lower case letters and the exact size of the intron is marked.
• Fig. 4 Ability of a well characterized H2 relaxin antibody to recognize H3 relaxin.
• H2 relaxin antibody was immobilized onto ELISA plates and a competition experiment was performed using HI, H2 and H3 relaxin against 125 I-labeled H2 relaxin. Results are mean ⁇ SEM of the specific binding (%) of triplicate determinations from a representative assay.
• This invention in its various aspects provides: the characterisation of nucleotide sequences encoding human H3 relaxin; the isolation of purified nucleic acid material; amplification of nucleotide sequences encoding H3 relaxin (mRNA, cDNA and DNA); nucleic acid cloning of H3 relaxin sequences; nucleic acid sequence identification, and peptide sequences encoding human H3 preprorelaxin, H3 prorelaxin and H3 relaxin.
• the human H3 relaxin polypeptide comprises disulphide bridged A and B chains.
• the amino acid sequence of human H3 relaxin is set out in SEQ ID NO: 4.
• the amino acid sequence of the B chain of human relaxin is set out in SEQ ID NO: 2.
• the A and B chains of human H3 relaxin are linked through cysteine residues, All -B10, A24-B22 disulphide bond formation taking place between these cysteine linkages.
• amino acid sequence of human H3 relaxin A and B chains are as follows:
• Lys Ser Glu lie Ser Ser Leu Cys 20 (SEQ ID NO: 4)
• a and B chains being linked by disulphide bonds between Al 1-BlO, A24-B22.
• Human H3 relaxin possesses classical relaxin bioactivity. Human relaxins, HI and H2 relaxin, bind to cells expressing relaxin receptors, such as THP-1 cells (Parsell et al (1996) J. Biol. Chem. 271, 27936-27941). H2 relaxin produces a dose dependent increase in cAMP production from these cells. Synthetic H3 relaxin produced according to this invention stimulated a dose dependent increase in cAMP in keeping with human H2 relaxin. The specificity of response in target cells bearing the human relaxin receptor as exhibited by H3 relaxin is demonstrated by the inability of bovine insulin (blNSL) or human insulin (hINSL3) to stimulate cAMP responses at doses up to 1 um in THP-1 cells.
• blNSL bovine insulin
• hINSL3 human insulin
• bioassays used for the measurement of active relaxin during pregnancy and non- pregnancy such as the guinea pig interpubic ligament assay may be used (Steinetz et al (1960) Endocrinology 67, 102-115, and Sirosi et al (1983) American Journal of Obstetrics and Gynaecology 145: 402-405) may be used.
• Other bioassays include cAMP production in THP-1 cells (Parsell et al (1996) J. Biol. Chem 271, 27936-27941).
• H3 relaxin analogues may be prepared where up to 9 amino acids are truncated from the N-terminus of the A chain, and up to 9 amino acids are truncated from the N-terminus of the B chain, and up to 5 amino acids are truncated from the C- terminus of the B chain.
• the resulting relaxin analogues comprise a H3 relaxin A and B chain, the A chain having the anino acid sequence
• the A chain of human H3 relaxin contains an intrachain disulphide bond between Cys residues 10 and 15.
• the H3 relaxin analogues referred to above all elicited cyclic AMP production in a manner which was characteristic of full length, non-truncated human H3 relaxin, and indeed human H2 relaxin.
• such truncated H3 relaxin analogues possess relaxin bioactivity.
• compositions comprising a human H3 relaxin analogue having a modified A chain and/or a modified B chain.
• the carboxyl- terminus of the A chain, and/or the B chain may be an amide derivative.
• Lys at position 12 in the A chain may be replaced with Glu, and/or Glu at position 19 may be replaced with Gin.
• the Ala at position 2 may be replaced with Pro, and/or Arg at position 8 may be replaced with Lys.
• the resulting H3 relaxin analogues having modified amino acids comprise an amino acid sequence which may be depicted as follows:
• a human H3 relaxin analogue having a modified A chain and/or a modified B chain
• carboxyl-terminus is an amide derivative and/or Lys at position 12 is replaced with Glu, and/or Glu at position 19 is replaced with Gin,
• the modified B chain having the amino acid sequence: Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly Arg Glu Phe He Arg 1 5 10 15
• carboxyl-terminus is an amide derivative, and/or Ala at position 2 is replaced with Pro, and/or Arg at position 8 is replaced with Lys,
• the A and B chains being linked by disulphide bonds between Al 1-B10 and A24-B22, and wherein the human H3 relaxin analogue has relaxin bioactivity.
• the isolation, purification and characterisation of nucleic acid sequences encoding human H3 relaxin has allowed the characterisation and production of the signal sequence of human H3 relaxin, and the pro-sequence of human H3 relaxin.
• the identification, purification and characterisation of the signal sequence and C chain of human H3 relaxin enables the prepro- and pro-human H3 relaxin to be produced.
• composition comprising human H3 preprorelaxin or human H3 prorelaxin, having a signal, A chain, B chain and C chain in respect of human H3 preprorelaxin, and an A chain, B chain and C chain in respect of human H3 prorelaxin, the said amino acid chains having the amino acid sequences:
• the A chain comprising:
• the signal sequence comprising:
• the C chain of human H3 relaxin said C chain having the amino acid sequence: Arg Arg Ser Asp He Leu Ala His Glu Ala Met Gly Asp Thr Phe Pro 1 5 10 15
• Human H3 prorelaxin possesses characteristic relaxin bioactivity.
• Human H3 relaxin, prorelaxin, preprorelaxin and constitutive peptide chains may be products using teclmiques previously described as useful in the production of relaxin such as US Patent No. 5,991,997, US Patent No. 4,758,516, US Patent No. 4,871,670, US Patent No. 4,835,251, PCT/US90/02085, and PCT/US94/0699.
• Relaxin analogues and derivatives where amino acids are substituted as indicated above may be produced recombinantly using, for example, site directed mutagenesis techniques as set forth, for example, in Tsurushita et al (1988) Gene Tissue: 135-139.
• the disclosed sequence information for human H3 relaxin, analogues thereof wherein one or more amino acids are truncated from the N- and/or C-terminus of the A and/or B chains, or human H3 relaxin analogues having amino acid substitutions as referred to above, may be synthesised according to the methods of B ⁇ llesbach (1991) J. Biol. Chem. 266, 10754- 10761, for synthesising relaxin. Additionally, well known methods of peptide synthesis may be utilised to produce the various H3 relaxin forms referred to herein. Relaxin has been implicated consequently in the treatment and diagnosis of various diseases and disorders.
• Human H3 relaxin, human H3 relaxin truncated analogues, amino acid modified H3 relaxin analogues, and human prorelaxin bind to the relaxin receptor and possess relaxin biological activity. It directly follows that these human H3 relaxin forms possessing relaxin biological activity may be used for the treatment of the above-identified diseases and other diseases.
• vascular disease including coronary artery disease, peripheral vascular disease, vasospasm including Raynaud's phenomenon, microvascular disease involving the central and peripheral nervous system, kidney, eye and other organs; treatment of arterial hypertension; diseases related to uncontrolled or abnormal collagen or fibronectin formation such as fibrotic disorders (including fibrosis of lung, heart and cardiovascular system, kidney and genitourinary tract, gastrointestinal system, cutaneous, rheumatologic and hepatobiliary systems); kidney disease associated with vascular disease, interstitial fibrosis, glomerulosclerosis, or other kidney disorders; psychiatric disorders including anxiety states including panic attack, agoraphobia, global anxiety, phobic states; depression or depressive disorders including major depression, dysthymia, bipolar and unipolar depression; neurologic or neurodegenerative diseases (including memory loss or other memory disorders, dementias, Alzheimer's disease); disorders of learning, attention
• vascular disease including coronary artery disease, peripheral vascular disease, vasospasm including Raynaud's phenomenon, microvascular disease involving the central and peripheral nervous system, kidney, eye and other organs
• vascular disease including coronary artery disease, peripheral vascular disease, vasospasm including Raynaud's phenomenon, microvascular disease involving the central and peripheral nervous system, kidney, eye and other organs
• diseases related to uncontrolled or abnormal collagen or fibronectin formation such as fibrotic disorders (including fibrosis of lung, heart and cardiovascular system, kidney and genitourinary tract, gastrointestinal system, cutaneous, rheumatologic and hepatobiliary systems); kidney disease associated with vascular disease, interstitial fibrosis, glomerulosclerosis, or other kidney disorders
• psychiatric disorders including anxiety states including panic attack, agoraphobia, global anxiety, phobic states
• depression or depressive disorders including major depression, dysthymia, bipolar and unipolar depression
• H3 relaxin may act as a neurotransmitter or neuroregulator in the brain, and other parts of the body including nerves, for example through inducing eAMP production in cells. H3 relaxin may also allow nutrient uptake by cells, or may be involved in autoregulatory presynaptic and/or conventional postsynaptic actions. Applicant further believes that H3 relaxin may also be axonally transported by nerve projections.
• H3 relaxin has surprisingly been found to be expressed in neuroanatomical region of the pars ventromedialis of the dorsal tegmental nucleus (vmDTg), which may otherwise be referred to as the nucleus incertus (Goto et al (2001) Journal of Comparative Neurology 438: 86-122).
• this region has been proposed as part of a brain stem network that may regulate behavioural activation via influences on attention, motivation, locomotion and learning (Goto et al) and may give rise to the therapeutic treatment modalities herein described.
• H3 relaxin may cross the blood brain barrier, or may be treated to facilitate crossing of the blood brain barrier, by methods known in the art incluidng use of one or more sugars or amino acids, or other substances which open the blood brain barrier or make it leaky allowing coadministered/timed administration with H3 relaxin (see for example Naito US Patent 6,294,520), by intranasal administration according to the methods of Frey (US Paetnt 6,313,093), for example using a lipophilic vehicle, and by methods described in PCT/WO89/10134.
• H3 relaxin and anlogues as herein described may be effective in the treatment of a wide range of what may broadly be described as neurologic diseases including psychiatric disorders, disorders of learning, attention and memory, addictive disorders and movement and locomotor disorders.
• H3 relaxin binds to the relaxin receptor as described hereinafter.
• human H3 relaxin analogues of human H3 relaxin where one or more amino acids are truncated from the N- and/or C-terminus of the A and B chains of human H3 relaxin, analogues of human H3 relaxin where one or more amino acids are modified or substituted with another amino acid as described herein, and human H3 preprorelaxin shall collectively be referred to as human H3 relaxin, unless otherwise specifically indicated.
• compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
• the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC.sub.50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
• a therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.
• Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD.sub.50 (the dose lethal to 50% of the population) and the ED. sub.50 (the dose therapeutically effective in 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD.sub.50 and ED. sub.50.
• Compounds which exhibit high therapeutic indices are preferred.
• the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED. sub.50 with little or no toxicity.
• the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
• the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g. Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 pi).
• Dosage amount and interval may be adjusted individually to provide serum levels of the active moiety which are sufficient to maintain the relaxin activity and effects.
• Administration of H3 relaxin can be via any of the accepted modes of administration for agents that serve similar utilities, preferably by systemic administration.
• a daily dose is from about 0.05 to 500.0 .mu.g/kg of body weight per day, preferably about 5.0 to 200.0 .mu.g/kg, and most preferably about 10.0 to 100.0 .mu.g/kg.
• a serum concentration of the H3 relaxin approximating or greater than normal circulating levels of relaxin in pregnancy i.e., 1.0 ng/ml, such as 1.0 to 20 ng/ml, preferably 1.0 to 20 ng/ml.
• the dosage range would be about 7.0 .mu.g to 3.5 mg per day, preferably about 42.0 .mu.g to 2.1 mg per day, and most preferably about 84.0 to 700.0 .mu.g per day.
• the amount of the H3 relaxin administered will, of course, be dependent on the subject and the severity of the affliction, the manner and schedule of administration and the judgment of the prescribing physician and the biological activity of such analog or derivative.
• One treatment regimen can employ a higher initial dosage level (e.g., 100 to 200 .mu.g/kg/day) followed by decreasing dosages to achieve steady H3 relaxin serum concentration of about 1.0 ng/ml.
• Another treatment regimen entails administration of an amount of H3 relaxin sufficient to attain normal pregnancy levels of relaxin (about 1.0 ng/ml) followed by gradual decreasing dosages until H3 relaxin serum levels are no longer detectable (e.g. less than about 20 picograms/ml), optionally discontinuing treatment upon reaching that dosage level.
• H3 relaxin can be administered either alone or in combination with other pharmaceutically acceptable excipients, including solid, semi-solid, liquid or aerosol dosage forms, such as, for example, tablets, capsules, powders, liquids, gels, suspensions, suppositories, aerosols or the like.
• Such proteins can also be administered in sustained or controlled release dosage forms (e.g., employing a slow release bioerodable delivery system), including depot injections, osmotic pumps (such as the Alzet implant made by Alza), pills, transdermal
• compositions will typically include a conventional pharmaceutical carrier or excipient and/or H3 relaxin, H3 prorelaxin, and H3 preprorelaxin or derivatives thereof.
• these compositions may include other active agents, carriers, adjuvants, etc.
• a sustained/controlled release H3 relaxin formulation was a selectively permeable outer barrier with a drug dispensing opening, and an inner H3 relaxin containing portion designed to deliver dosage of the H3 relaxin progressively diminishing at a predetermined rate (e.g. containing about 30 mg of H3 relaxin in a matrix for delivery of initially about 500 .mu.g per day diminishing as a rate of 10 .mu.g per day.
• a sustained/controlled release of H3 relaxin has a selectively permeable outer barrier with a drug dispensing opening, a first inner H3 containing portion designed for steady state release of H3 relaxin at a therapeutically effective daily dosage (e.g. containing about 50 mg of H3 relaxin in a matrix for continuous delivery of about 500 .mu.g per day), and a second inner H3 relaxin a portion designed to deliver a dosage of H3 relaxin progressively diminishing at a predetermined rate (e.g. containing about 3 mg of H3 relaxin in a matrix for delivery of initially about 500 .mu.g per day diminishing at a rate of 50.mu.g per day) commencing upon exhaustion of the H3 relaxin from the first inner portion.
• a therapeutically effective daily dosage e.g. containing about 50 mg of H3 relaxin in a matrix for continuous delivery of about 500 .mu.g per day
• the pharmaceutically acceptable composition will contain about 0.1% to 90%, preferably about 0.5% to 50%, by weight of H3 relaxin, either alone or in combination with H3 relaxin, the remainder being suitable pharmaceutical excipients, carriers, etc.
• H3 relaxin either alone or in combination with H3 relaxin
• the remainder being suitable pharmaceutical excipients, carriers, etc.
• Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975.
• the effective local concentration of the drug may not be related to plasma concentration.
• compositions administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
• Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration.
• Parenteral administration is generally characterized by injection, either subcutaneously, intradermally, intramuscularly or intravenously, preferably subcutaneously.
• Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
• Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like.
• compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, solubility enhancers, and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, cyclodextrins, and the like.
• auxiliary substances such as wetting or emulsifying agents, pH buffering agents, solubility enhancers, and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, cyclodextrins, and the like.
• a more recently devised approach for parenteral administration employs the implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795.
• the liposomes will be targeted to and taken up selectively by the tumor.
• compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
• the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
• the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
• a suitable vehicle e.g., sterile pyrogen-free water
• the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
• the compounds may also be formulated as a depot preparation.
• Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
• the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
• a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
• the cosolvent system may be the NPD co-solvent system.
• VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.
• the VPD co-solvent system (VPD:5W) consists of NPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration.
• co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics.
• identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrohdone; and other sugars or polysaccharides may substitute for dextrose.
• hydrophobic pharmaceutical compounds may be employed.
• Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
• Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
• the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
• sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
• compositions also may comprise suitable solid or gel phase carriers or excipients.
• suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
• Formulations comprising human H3 relaxin may also be administered to the respiratory tract as a nasal or pulmonary inhalation aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose, or with other pharmaceutically acceptable excipients.
• the particles of the formulation may advantageously have diameters of less than 50 microns, preferably less than 10 microns. See, e.g., U.S. Pat. No. 5,364,838, which discloses a method of administration for insulin that can be adapted for the administration of H3 relaxin.
• H3 relaxin for treatment of such disorders may also be administered topically in a formulation adapted for application to the scalp, such as a shampoo (e.g., as disclosed in U.S. Pat. No. 4,938,953, adapted according to methods known by those skilled in the art, as necessary for the inclusion of protein ingredients) or a gel (e.g., as disclosed in allowed U.S. Ser. No. 08/050,745) optionally with increased H3 relaxin concentrations to facilitate absorption.
• a formulation adapted for application to the scalp such as a shampoo (e.g., as disclosed in U.S. Pat. No. 4,938,953, adapted according to methods known by those skilled in the art, as necessary for the inclusion of protein ingredients) or a gel (e.g., as disclosed in allowed U.S. Ser. No. 08/050,745) optionally with increased H3 relaxin concentrations to facilitate absorption.
• the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
• Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
• Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
• Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and or polyvinylpyrrolidone (PNP).
• disintegrating agents may be added, such as the cross-linked polyvinyl pyrrohdone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• suitable coatings may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrohdone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
• the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Narious aspects of the invention will be described with reference to the following non- limiting examples.
• rats are used as an experimental model to map H3 relaxin expression at the mR ⁇ A and protein level in the rat brain, this allowing human brain mapping.
• the rat brain is a standard comparative anatomical model of the human brain (Goto et al (2001) The Journal of Comparative Neurology 438: 86-122).
• RNA/DNA Extraction and R7 PCR-Human genomic DNA was extracted from human CL using standard protocols (Sambrook et al 1989) In Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbour Laboratory Press, NY). Human CL and mouse tissues were finely diced in the presence of liquid nitrogen and immediately homogenized with RNAWiz reagent (Ambion Inc., Austin, TX), and the RNA extracted according to the manufacturer's instructions. Total RNA (5 ⁇ g) from each sample was used for the reverse transcription (RT) reaction, which was performed using the Superscript II RT-PCR kit (Gibco-BRL, Rockville, MD) in a 20 ⁇ l volume according to the manufacturer's instructions.
• RT reverse transcription
• a 50 ⁇ l reaction containing 100 ng of primers and 150 ng of the cDNA template was used for all PCR reactions.
• Mouse tissues were screened for M3 relaxin expression using specific forward [5' TGCGGAGGCTCACGATGGCGC 3'] and reverse [5' GACAGCAGCTTGCAGGCACGG 3'] primers, which generated a 319-bp product.
• Mouse relaxin (Ml) expression was determined using a specific forward [5' GTGAATATGCCCGTGAATTGATC 3'] and reverse [5' AGCGTCGTATCGAAAGGCTCT 3'] primer based on the published sequence (Evans et al (1993) J. Mol. Endocrinol. 10, 15-23), generating a 150-bp product.
• Human CL cDNA was used in RT-PCR reactions with specific primers for H3 relaxin, forward 1 [5' ACGTTCAAAGCGTCTCCGTCC 3'], forward 2 [5' CGGTGGAGACGATCAGACATC 3'] and reverse [5'
• the mouse PCR reactions were completed in a Perkin Elmer Gene Amplifier using the following (touch-down) annealing temperatures: 64°C (2 cycles), 63°C (2 cycles), 62°C (2 cycles), 61°C (2 cycles), 60°C (32 cycles).
• H3 relaxin expression in human CL cDNA was performed by RT-PCR at the following annealing temperatures: 60°C (2 cycles), 59°C (2 cycles), 58°C (2 cycles), 57°C (2 cycles), 56°C (32 cycles).
• Aliquots of the PCR products were electrophoresed on 2% (w/v) agarose gels stained with ethidium bromide and photographed. Mouse tissue samples were transfered to Hybond NX membranes (Amersham International, Aylesbury, UK) for Southern blot analysis.
• PCR was performed using mouse brain and ovarian cDNA using the reverse M3 primer (above) and a forward primer from in front of the ATG start codon (5' GGG TCGCAGGCATCTCAACTG 3').
• the resulting product contained the full H3 relaxin coding sequence.
• PCR was performed as above, but with the following annealing temperatures: 60°C (2 cycles), 59°C (2 cycles), 58°C (2 cycles), 57°C (2 cycles), 56°C (32 cycles).
• RT-PCR was performed on human genomic DNA (50 ng).
• the bands were subsequently subcloned into the pGEM-T vector (Promega, Madison, WI) and multiple subclones were then sequenced on both strands using the ABI PRISM 377 automatic DNA sequencer, according to the manufacturers instructions (Applied Biosystems, Melbourne, Australia).
• AATTTGGCTCTTGCTACAGCCCCACTCG CAGCAACTGCT 3' cDNA sequences, which had been labeled using T4 polynucleotide kmase and [ ⁇ - P] ATP.
• Hybridization was performed at 55°C overnight in 5 x SSC (1 x SSC; 0.15 M NaCl, 15 mM sodium citrate, pH 7), 5 x Denhardts, 1% SDS and 100 ⁇ g/ml sonicated herring sperm.
• Membranes were washed three times for 5 min in 2 x SSC, 0.1% SDS at room temperature followed by a 30 min wash at 55°C in 0.1 x SSC, 0.1% SDS, before being exposed to BioMAX MR film (Eastman Kodak Co., Rochester, NY) for 24 h at room temperature.
• P-labeled probe that corresponded to the 319-bp PCR product, generated by specific primers to M3 relaxin (see above).
• This product was labeled with [ ⁇ - 32 P]dCTP using the specific reverse primer (above) and T7 polymerase as previously described (31).
• the membrane was hybridized at 65°C overnight in 0.25M NaH 2 PO , pH 7.2, 1 mM EDTA, 20% SDS, followed by three washes for 5 min in 2 x SSC, 0.1% SDS at room temperature, and finally a 30 min wash at 65°C in 0.1 x SSC, 0.1% SDS.
• the 374-bp fragment of the H3 relaxin sequence isolated from genomic DNA was labeled with [ ⁇ - 32 P]dCTP using the H3 relaxin specific reverse primer (described above), and T7 polymerase (Bathgate et al (1999) Biol. Reprod. 61, 1090-1098).
• the membrane was exposed to a phosphoimager plate and BioMAX film as described above.
• Probes were labeled with [ ⁇ - 35 S]dATP (1200 Ci/mmol; NEN, AMRAD-Biotech, Melbourne, Australia) to a specific activity of 1 x 10 9 d.p.m./ ⁇ g using terminal deoxynucleotidyl transferase (Roche Diagnostics; Wisden et al (1994) In In Situ Hybridization Protocols for the Brain (Wisden, W. and Morris, B.J. eds), pp 9-34, Academic Press, London). Screening of the sequences used against gene sequence databases (Celera, EMBL and Genbank; NCBI/NIH Blast Service) revealed homology only with the appropriate Ml and M3 relaxin mRNAs.
• Sections were incubated overnight at 42°C with multiple 35 S-labeled probes (30 frnol each probe/slide) in hybridization buffer containing 50% formamide, 4 x SSC, 10% dextran sulphate and 0.2 M dithiothreitol. Slides were washed in 1 x SSC at 55°C for 1 h, rinsed in 0.1 x SSC, then dehydrated before being apposed to Kodak BioMAX MR for 10 d.
• Solid Phase Synthesis A putative peptide sequence encoded by the H3 gene was assembled by solid phase synthesis procedures based on the predicted signal peptide and proteolytic enzyme cleavage sites between the signal peptide and the B-chain, and the B/C and C/A chain junctions of the H3 relaxin prohormone (see Results for details).
• A- and B-peptides were synthesized on a 0.1 mmol scale by the continuous flow Fmoc solid-phase method as previously described Dawson et al (1999) J. Pept. Res. 53, 542-547.
• cAMP levels were measured in the lysates using the cAMP Biotrak EIA system (Amersham international, Aylesbury,
• THP-1 Cell Binding Assay- THP-1 cells were spun down and resuspended in binding buffer (20 mM HEPES, 50 mM NaCl, 1.5 mM CaCl 2 , 1% BSA, 0.1 mg/ml lysine, 0.01% NaN 4 , pH 7.5) (Parsell et al (1996) J Biol. Chem. Ill, 27936-27941) to give 2 x 10 6 cells/well in a 96-well plate. The cells were incubated in binding buffer with 3 P-labeled H2 (B33) relaxin (100 pM: labeled as previously described (Tan et al (1999) Br. J. Pharmacol.
• Antibody Crossreactivity The ability of well characterized human relaxin antibodies to recognize H3 relaxin was tested in comparison to HI and H2 relaxins by radioimmunoassay. Briefly, goat anti-H2 relaxin (Lucas et al (1989) J. Endocrinol. 120, 449-57) was coated onto 96 well ELISA plates (Disposable Products, Sydney, Australia) at a dilution of 1:1000 with 0.05M sodium carbonate buffer at 4°C overnight.
• the antibody-bound- 125 I-labeled H2 relaxin was collected by the addition of 1M NaOH and decanted into tubes for counting on a Packard 5010 gamma counter (Canberra Packard). Experiments were perfonned at least twice and similar results obtained. Data was plotted as the mean ⁇ SEM from one representative experiment performed in triplicate and plotted using PRISM.
• mice All male and female mice used in these studies were age-matched, and had the same background (C57BLK6J). Animals were housed in a controlled environment and maintained on a 14 h light, 10 h dark schedule with access to rodent lab chow (Barastock Stockfeeds, Melbourne, Australia) and water. Female mice (3.5 months old) were mated and pregnancy timed from the identification of the vaginal plug. At day 7.5, 10.5 and 18.5 of pregnancy, mice were sacrificed for tissue collection. Tissues were also collected from non-pregnant female and male mice (4 months old). These experiments were approved by the Howard Florey Institute's Animal Experimental Ethics Committee, which adheres to the Australian Code of Practice for the care and use of laboratory animals for scientific purposes.
• Tissue Collection-A ms ⁇ s were killed with an overdose of Isofluorane (Abbott Australasia Pty Ltd, Sydney, Australia).
• Human CL from women in early pregnancy undergoing surgery for ectopic pregnancies were utilized with the approval of the Howard Florey Institute Human Ethics Committee and the written consent of the patients.
• Both H3 relaxin sequences in the human and mouse contain features representative of functional genes (Fig. ⁇ A human; ⁇ B mouse). Each contain a putative TATA box for initiation of transcription 65, and 59 bp, upstream of putative ATG start codons for human and mouse, respectively. A polyadenylation signal is present in the 3' untranslated region of both genes, in a position 582 and 448 bp downstream from an inframe TAG stop codon for the human, and mouse genes respectively.
• a single intron interrupts the coding region in an identical position in the sequence of both genes, corresponding to a similar position to that of other relaxin and insulin family members (Hudson et al (1983) Nature 301, 628- 631; Evans et al (1993) J. Mol. Endocrinol. 10, 15-23; Ivell, R (1997) Rev. Reprod. 2, 133- 138).
• the H3 relaxin gene is localized on chromosome 19 at 19pl3.3, whereas the mouse gene is located on chromosome 8 at 8C2.
• the derived coding regions of the H3 and M3 relaxin genes were 142, and 141, amino acids, respectively.
• the human sequence most closely resembles the hTNSL5 peptide sequence on direct amino acid homology, the presence of this binding motif indicates that the peptide is more like a relaxin peptide.
• the M3 relaxin A-chain conforms to the cysteine pattern of family members, whereas the previously characterized Ml relaxin sequence contains an extra tyrosine residue before the final cysteine residue (Fig. 2A).
• H3 and M3 (mouse "3" relaxin) sequences share greater than 70% homology in the coding region at the nucleotide level. However, the homology is most striking in the derived amino acid sequence. Both derived pro-hormone sequences contain a typical signal sequence after the ATG start codon which is likely to be cleaved at an identical position between alanine and arginine in both the human and mouse peptides (Nielsen et al (1997) Prot. Engineer. 10, 1-6). The arginine-arginine pair of basic amino acids at the B/C junction found with other members of the relaxin family strongly suggests cleavage between tryptophan and arginine.
• cleavage at the C/A junction is most likely to occur between the arginine and aspartic acid as indicated in Figs. ⁇ A and IB, as this corresponds to a weak furin (proprotein convertase) cleavage site (Nakayama, K. (1997) Biochem. J. 327, 625-635. Therefore, it is believed that both H3 and M3 relaxins comprise a B-chain of 27 amino acids, a C-peptide of 66 amino acids and an A-chain of 24 amino acids.
• FIG. 2A A comparison of the A- and B-chain sequences of H3 and M3 relaxin with HI, H2 and Ml relaxin is outlined in Fig. 2A.
• the homology between Ml and H2 relaxin is only 42% and 45% in the A-, and B-, chains respectively.
• H2 and H3 relaxin there is very little homology between H2 and H3 relaxin, and between Ml and M3 relaxin.
• H3 and M3 relaxin show high homology of the C-peptide domain (73%), compared with less than 20% homology in this region of other insulin/relaxin family members.
• the C-peptide lengths of H3 and M3 relaxin are 65, and 66 amino acids, respectively, and are much shorter than that of other relaxins (102 amino acids for HI and H2 and 99 amino acids for Ml relaxin).
• the C-peptide chain length and sequence homology is most similar to INSL5 (24%).
• H3 relaxin was prepared by solid phase synthesis in low overall yield (0.7%).
• MALDITOF MS showed a single product with an MH + of 5,494.7 (theoretical value: 5,497.5). Amino acid analysis also confirmed its correct composition.
• Both peptides were prepared on a 0.1 mmol scale as peptide-carboxyl terminal amides using Fmoc peptide amide linker polyethylene glycol polystyrene (Fmoc-PAL-PEG-PS) supports (Applied Biosystems).
• Couplings were generally of 30 minutes duration, with the exception of double couplings and extended times for A-chain residues Ser 7 ' 8,21 and all cysteines, and double couplings of B-chain residues Arg 1 ' 12 ' 16 , Ala 2 ' 3 ' 17 and Cys 10 .
• Purified A-chain peptide 3 (1.0 mg, 0.38 ⁇ mol) and purified B-chain peptide 2 (1.2 mg, 0.38 ⁇ mol) were dissolved separately in 1.0 ml and 0.5 ml 0.1M NH 4 HCO 3 respectively.
• the B-chain solution was then slowly added to A-chain and the reaction mixture was stirred vigorously at room temperature for 30 min.
• the solution was acidified with 0.5 ml glacial acetic acid and then subjected to RP-HPLC, as detailed below, to isolate the bis- disulfide bonded chain combined product. (Alternative to RP-HPLC purification, the resulting A/B product, peptide 4, was desalted on a Sephadex G-25 gel filtration column in 20% aq acetic acid).
• the peptide was dissolved in a solution of 80mM HC1 and acetic acid. 20mM iodine in 95% aqueous acetic acid was then added dropwise (25 equivs of iodine per Acm group). The reaction was performed for 1 hour in the dark at room temperature after which excess oxidant was quenched with 20mM aqueous ascorbic acid. Purification of the relaxin was by RP-HPLC, with a final yield, relative to peptide 3 starting material, of 0.74%.
• the separate crude chains and intermediate peptides were purified by RP-HPLC, using a Waters 600 multisolvent delivery system connected to a model 996 photodiode array detector.
• a 10x250mm Nydac 218 TP column packed with C 4 silica gel (330A pore size, lO ⁇ m particle size) was used.
• the peptides were eluted with a solvent system of (A) 0.1% aq. TFA (v/v) and (B) 0.1% TFA in acetonitrile (v/v) in a linear gradient mode (25-50% B over 30 minutes).
• the target fractions were collected and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) and lyophihzed.
• MALDI-TOF MS matrix-assisted laser desorption ionization mass spectrometry
• Peptide characterisation Peptide quantitation was by duplicate amino acid analysis of 24 hr acid hydrolyzates on a GBC automatic analyser (Melbourne, Aust).
• MALDITOF MS was performed in the linear mode at 19.5kv on a Bruker Biflex instrument (Bremen, Germany) equipped with delayed ion extraction.
• the specificity of this response was demonstrated by the inability of bovine insulin (blNSL), or human insulin 3 (hTNSL3), to stimulate cAMP responses at doses up to 1 ⁇ M.
• H2 relaxin was able to displace I-labeled H2 relaxin binding to the anti-H2 relaxin antibody with high specificity.
• HI and H3 relaxin showed poor cross reactivity with the antisera as determined by their poor ability to displace 125 I-labeled H2 relaxin binding.
• the non-parallellism of the displacement curves indicates that not all the antibody epitopes are recognized by the two peptides.
• M3 relaxin mRNA was expressed in a number of tissues in C57BLK6J mice where Ml relaxin was found, but the pattern of expression, between the two mouse relaxins was different.
• M3 relaxin expression was detected at highest levels in brain, however, it was expressed at moderate levels in the thymus, lung and spleen, only at very low levels in the heart and liver, and not at all in the kidney, skin and gut.
• Female mice showed an almost identical pattern of expression for both genes in these tissues, h male reproductive tissues M3 relaxin mRNA was significantly expressed only in the testis whereas, Ml relaxin mRNA was detected in the testis, epididymis and prostate.
• Both relaxins were also detected in female reproductive organs in the mammary gland, ovaries of non-pregnant, pregnant and lactating mice, and the endometrium and myometrium of pregnant mice. Significant expression of M3 relaxin mRNA was observed in all ovarian stages, while Ml relaxin expression was higher in ovaries of late gestation compared to ovaries from non-pregnant and lactating mice. High levels of M3 relaxin mRNA were detected in the brain and further analysis of this tissue revealed that both relaxins were expressed in several distinct regions.
• M3 relaxin mRNA was found to be highly expressed in the thalamus and pons/medulla, thus suggesting, that the two relaxins may play distinct roles in the mouse.
• RNA 5-25 ⁇ g
• RNA from the heart, brain, lung, thymus, spleen, ovary, endometrium, myometrium, cervix and vagina were initially probed with a 32 P -labeled M3 relaxin specific probe, but no specific hybridizing bands were found in any tissue.
• RNA from the brain 15 ⁇ g
• spleen 5 ⁇ g
• liver 5 ⁇ g
• testis 25 ⁇ g
• the obtained transcript size was consistent with the predicted size based on the M3 relaxin transcript sequence ( ⁇ lkb) plus apoly-A tail ( ⁇ 200-bp).
• H3 Relaxin in Human Tissues-A Clonetech Multi Tissue Expression Array was used to examine sites of expression of H3 relaxin in human tissues.
• the array contained normalized poly-A RNA (50-750 ng) from 76 different human tissues including 8 different control RNAs and DNAs, spotted onto a nylon membrane.
• the array was probed with a 32 P-labeled 374-bp H3 relaxin specific gene fragment from the 3' end of the H3 relaxin transcript, generated from genomic DNA. This DNA fragment was sequenced on both strands. Very weak hybridizing signals were observed in spleen, thymus, peripheral blood leukocytes, lymph node and testis however, these signals were barely discernable above background and hence, the data is not shown.
• RT-PCR was also performed on human CL from early pregnancy using two different primer combinations based on the H3 relaxin sequence. No specific bands were observed in any PCR reaction even after changing the PCR conditions, whereas transcripts for H2 relaxin and GAPDH were easily amplified (data not shown), confirming the integrity of the cDNA.
• M3 relaxin mRNA The strongest level of M3 relaxin mRNA was present in the pars ventromediahs of the dorsal tegmental nucleus, h addition, M3 relaxin mRNA was also detected, albeit at far lower levels, in the hippocampus and olfactory regions. Brain regions containing low levels of mRNA encoding M3 relaxin may not have been detected in the current study due to sensitivity limitations associated with in situ hybridization histochemistry. The distribution of M3 relaxin mRNA in the brain differs from that of Ml relaxin mRNA, as no Ml relaxin mRNA was detected in the pars ventromediahs of the dorsal tegmental nucleus (data not shown).
• Prorelaxin H3 cDNA sequences from human, mouse and rat are expressed in both prokaryotic and eukaryotic cell systems using appropriate expression transfer vectors. These systems include appropriate mammalian host cells, other higher eukaryotic cells including insect cells, plant cells and avian cells as well as bacterial and yeast expression systems. Additionally, fusion protein products of these three sequences are produced by linking a portion of a prokaryotic or eukaryotic protein characteristic of the host cell. The fusion products facilitate the purification of the protein product such that the fusion product may be subsequently removed. All transfer vectors may also be modified by codon substitutions/deletions/additions with the modifications giving rise to shortened C peptide prorelaxins with B/C and C/A junction modifications to facilitate the removal of the modified C peptide sequence.

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