NO319848B1 - Synthetic peptide analogues for PTH and PTHrp, methods for solid phase synthesis thereof, pharmaceutical compositions comprising them, and use thereof for the manufacture of medicaments for the treatment of bone mass reductions. - Google Patents

Description

The background of the invention

a) The scope of the invention

This invention relates to novel analogs of parathyroid hormone and parathyroid hormone-related peptide, their synthesis by solid-phase and recombinant techniques, and their use in increasing bone mass in mammalian subjects.

b) Description of related art

Osteoporosis is the most common form of metabolic bone disease and can be considered the symptomatic breaking stage of bone loss (osteopenia). Although osteoporosis can occur secondary to a number of underlying diseases, 90% of all cases appear to be idiopathic. Women after menopause are at particular risk of idiopathic osteoporosis (postmenopausal or type I osteoporosis). Another high-risk group for idiopathic osteoporosis is the elderly of both sexes (senile or type II osteoporosis). Osteoporosis has also been related to corticosteroid use, immobilization or prolonged bed rest, alcoholism, diabetes, gonadotoxic chemotherapy, hyperprolactinemia, anorexia nervosa, primary and secondary amenorrhea, and oophorectomy.

In the various forms of osteoporosis, bone fractures often occur as a result of bone loss that has reached the point of mechanical failure. Postmenopausal osteoporosis is characterized by fractures in the wrist and spine, while femoral neck fractures seem to be the dominant feature of senile osteoporosis.

The mechanism by which bone is lost in osteoporotics is thought to involve an imbalance in the process by which the skeleton renews itself. This process has been called bone remodeling. It enters into a series of separate activity pockets.

These pockets arise spontaneously in the bone mass on a specific bone surface as a seat for bone resorption. Osteoclasts (bone-dissolving or resorbing cells) are responsible for the resorption of a portion of bone of generally constant dimension. This resorption process is followed by the appearance of osteoblasts (bone-forming cells) which then refill with new bone the cavity left by the osteoclasts.

In a healthy, adult individual, the rate at which osteoclasts and osteoblasts are formed is such that bone formation and bone resorption are in balance. In osteoporetics, however, an imbalance develops in the bone remodeling process which results in bone being lost at a greater rate than it is being made. Although this imbalance occurs to some extent in most individuals as they age, it is much more severe and occurs at a younger age in postmenopausal osteoporetics or after oophorectomy.

There have been many attempts to treat osteoporosis with the goal of either slowing further bone loss or, more desirably, producing a net gain in bone mass. Certain agents, such as estrogen and the bisphosphonates, appear to reduce further bone loss in osteoporotics. Agents that reduce bone loss due to the different durations of bone resorption and formation may appear to increase bone mass (on the order of 3 to 7%). However, this apparent increase is limited in time, not progressive and is due to a reduction in "remodeling space". Due to the close link between resorption and formation, treatments that make bone resorption more difficult will also ultimately make bone formation more difficult.

It has been suggested that treatment with parathyroid hormone (PTH) would lead to both increased bone turnover and a positive calcium balance. However, human clinical trials have shown that any increase in trabecular bone is offset by a decrease in cortical bone so that there is no net increase in total bone.

Hefti et al. in Clinical Science 62, 389-396 (1982), have reported that daily subcutaneous doses of either bPTH(l-84) or hPTH(l-34) increased the total body calcium content and the ash weight of individual bones in both normal and osteoporotic ethical adult female rats.

Liu et al. in J. Bone Miner. Res. 6:10, 1071-1080

(1991), have noted that ovariectomy of adult female rats induced a 47% loss in the percentage of trabecular bone in the proximal tibial metaphysis, accompanied by a significant increase in the number of osteoblasts and trabecular osteoclasts. Daily subcutaneous injections of hPTH(1-34) completely reversed the loss of trabecular bone and resulted in amounts of trabecular bone exceeding that of sham-operated controls. The number of osteoblasts increased, and the number of osteoclasts decreased.

Hock et al. in J. Bone Min. Res. 7:1, 65-71 (1992), have reported that daily subcutaneous injections of hPTH(1-34) into healthy adult male rats for 12 days increased trabecular and cortical bone calcium and dry weight. Total bone mass, trabecular bone volume, trabecular thickness and number, and osteoblastic surfaces were increased.

The mammalian parathyroid hormones, e.g. human (hPTH), bovine (bPTH) and porcine (pPTH), are single polypeptide chains of 84 amino acid residues with molecular weights of approximately 9500. Biological activity is associated with the N-terminal portion, with residues (1-34) apparently being the minimally required ones.

The N-terminal segment of human PTH differs from the N-terminal segment of the bovine and porcine hormones by only three and two amino acid residues, respectively:

The primary function of PTH is to trigger the adaptive changes that serve to maintain a constant concentration of Ca<2*> in the extracellular fluid. PTH acts on the kidneys so that the tubular reabsorption of Ca<2*> from the urine increases, and that the conversion of calcifediol to calcitriol, which is responsible for the absorption of Ca<2*> from the small intestine, is stimulated. One prominent effect is to increase the mobilization of Ca<2*> from bone. PTH acts on bones so that the resorption rate for Ca<2*> and phosphate is increased. PTH stimulates the rate of bone resorption by osteoclasts, increases the rate of differentiation of mesenchymal cells into osteoclasts, and prolongs the half-life of these latter cells. With prolonged action of PTH, the number of bone-forming osteoblasts also increases; thereby increasing the speed of bony replacement and remodeling. However, individual osteoblasts appear to be less active than normal.

Rosenblatt et al. in US Patent No. 4,423,037,

4,968,669 and 5,001,223 have described PTH antagonists obtained

by means of the deletion of the N-terminal (1-6) amino acids and the selective replacement of Phe<7>, Met<818> and Gly<12>. Tyr<34->NH2ø<k>t demonstrably increased the activity and stability of these compounds.

Parathyroid hormone-related peptide (PTHrp), a 140+ amino acid protein, and fragments thereof, reproduce the major biological actions of PTH. PTHrp is produced by a variety of human and animal tumors and other tissues and may play a role in hypercalcemia in malignant cancer. The sequence of hPTHrp (1-34) is as follows:

The sequence homology between hPTH and hPTHrp is largely limited to the 13 N-terminal residues, of which 8 are identical; only 1 of 10 amino acids in the (25-34) receptor binding region of hPTH is conserved in hPTHrp. Conformational similarity may be the reason for the common activity. Cohen et al. in J. Biol. Chera. 266:3, 1997-2004 (1991), have proposed that much of the sequence of PTH(l-34) and PTHrp(l-34), particularly the regions (5-18) and (21-34), occupy an a- helical configuration, while they note that there is some uncertainty as to whether this configuration overcomes the carboxyl terminus under physiological conditions. Such a secondary structure can be important for lipid interaction, receptor interaction and/or structural stabilization.

We have synthesized analogues of PTH and of PTHrp with

the purpose of developing improved therapeutic agents for the restoration of bone mass in mammalian subjects, including those suffering from osteoporosis.

Summary of the invention

This invention provides synthetic polypeptide analogues of parathyroid hormone (PTHrp), parathyroid hormone-related peptide (PTHrp) or salts thereof, characterized in that the amino acid residues (22-31) of PTH or PTHrp are modified so that they form an amphipathic α-helix, where the sequence of the residues ( 22-31) is selected from:

Xaa<22> and Xaa<25> are independently Glu, Glu(OCH3), His or Phe; Xaa<23> is Leu or Phe; Xaa<26> is Lys or His; Xaa<28> and Xaa<31> are independently Leu or Ile; Xaa<29> is Ala, Arg or Glu; and Xaa<30> is Lys or Glu (SEQ ID NO: 85); wherein in a compound where Xaa<22>is Phe; Xaa<23> is Phe; Xaa<25> is His; Xaa<26> is Lys or His; Xaa28 is Ile; Xaa<29> is Ala; and Xaa<30> is Glu; is Xaa31Leu;

Xaa<22> and Xaa<25> are independently Glu, Glu(OCH3), His or Phe; Xaa<23> is Leu or Phe; Xaa<29> is Glu, Lys or Lys(COCH2PEGX) and PEGX is a poly-(ethylene glycol methyl ether) radical with a molecular weight of 100 to 10,000 (SEQ ID NO: 86);

Pharmaceutical preparations are also provided which are characterized in that they comprise an effective bone mass-increasing amount of a polypeptide analogue of parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrp) or salts thereof, in admixture with a pharmaceutically acceptable carrier.

This invention also includes a method for the solid-phase synthesis of polypeptide analogues of PTH, PTHrp and salts thereof, where the amino acid residues (22-31) are modified so that they form an amphipathic α-helix, the residues (22-31) being selected from

Xaa<22> and Xaa<25> are independently Glu, Glu(OCH3), His or Phe; Xaa<23> is Leu or Phe; Xaa<26> is Lys or His; Xaa<2B>and Xaa<31> are independently Leu or Ile; Xaa<29> is Ala, Arg or Glu; and Xaa<30> is Lys or Glu (SEQ ID NO: 85); wherein in a compound where Xaa22 is Phe; Xaa<23> is Phe; Xaa25 is His; Xaa<26> is Lys or His; Xaa<28> is Ile; Xaa<29> is Ala; and Xaa<30> is Glu; is Xaa<31>Leu;

Xaa<22> and Xaa<25> are independently Glu, Glu(OCH3), His or Phe; Xaa<23> is Leu or Phe; Xaa<29> is Glu, Lys or Lys(COCH2PEGX) and PEGX is a poly-(ethylene glycol methyl ether) radical of molecular weight 100 to 10,000 (SEQ ID NO: 86);

where the method is characterized by sequential linking of protected amino acids on a suitable resin carrier, removal of the side chain and the Na<-> protecting groups, and cleavage of the polypeptide from the resin.

Also included is the use of a compound according to claims 1-9 or a pharmaceutically acceptable salt thereof, for the preparation of a drug for the treatment of mammalian conditions characterized by reductions in bone mass, in particular for the treatment of osteoporosis.

Brief description of the drawings

Figure 1 shows the DNA sequence and enzyme restriction sites of a synthetic gene encoding a PTHrp(1-34) analog according to this invention (hPTH-PCR amplification). Figure 2 outlines the preparation of a plasmid in which a PTHrp(1-34) analog {Trp LE 18 hPTHrp(1-34)1 construct) is incorporated. Figure 3 outlines the preparation of a plasmid where two copies of a PTHrp(1-34)-analogous gene (Trp LE 18 hPTHrp(1-34)2 construct) are incorporated. Figure 4 outlines the preparation of a plasmid in which four copies of a PTHrp (1-34) analogue (Trp LE 18 hPTHrpd-34)4 construct) are incorporated.

Detailed description of the invention

Abbreviations and definitions

The one- and three-letter abbreviations for the various common nucleotide bases and amino acids are as recommended in Pure Appl. Chem. 31, 639-645 (1972) and 40, 277-290 (1974), and Complies with 37 CFR §1.822 (55 FR 18245, May 1, 1990), and the PCT Rules (WIPO Standard ST.23: Recommendation for the Presentation of Nucleotide and Amino Acid Sequences in Patent Applications and in Published Patent Documents). The one- and three-letter abbreviations are as follows:

Aminos<y>reror Abbreviations

The abbreviations represent L-amino acids unless designated differently as D- or D,L-. Certain amino acids, both natural and non-natural, are achiral, e.g. glycine. All peptide sequences are shown with the N-terminal amino acid on the left and the C-terminal amino acid on the right.

Additional abbreviations for other amino acids and compounds used herein are:

"Hydrophilic amino acid (Haa)" refers to an amino acid with at least one hydrophilic functional group in addition to those required for peptide bond formation, such as arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, serine, threonine and homologues hence.

"Lipophilic amino acid (Laa)" refers to an uncharged, aliphatic or aromatic amino acid, such as isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, valine, and homologues thereof.

For the purposes of this invention, alanine is classified as "amphiphilic", ie capable of acting as either hydrophilic or lipophilic.

"Physiologically active, truncated homologue or analog of PTH or PTHrp" refers to a polypeptide having a sequence that comprises less than the full complement of amino acids found in PTH or PTHrp, but which elicits a similar physiological response. The truncated PTH or PTHrp need not be completely homologous to PTH or PTHrp to elicit a similar physiological response. PTH(1-34) and PTHrpfl-34) are preferred, but not the only representatives of this group.

"Amphipathic α-helix" refers to the secondary structure exhibited by certain polypeptides where the amino acids adopt an α-helical configuration with opposing polar and non-polar surfaces oriented along the long axis of the helix. The possibility of α-helical structure in the polypeptide in question can to a certain extent be investigated by means of the construction of a "Schiffer-Edmundson turn" (M. Schiffer and A. B. Edmundson, Biophys. J. 7, 121 (1967)), with the appropriate tilt angle and by noting the deposition of the hydrophilic and lipophilic residues on opposing surfaces of the cylinder surrounding the helix. Alternatively, empirical evidence, such as circular dichroism or X-ray diffraction data, may be available and indicate the presence of an α-helical region in a particular polypeptide. An ideal α-helix has 3.6 amino acid residues per turn with adjacent side chains separated by an arc angle of 100°. Eisenberg et al. in Nature 299:371-374 (1982) and Proe. Nat. Acad. Pollock. USA 81:140-144

(1984) have combined a hydrophobicity scale with the helical twist to quantify the concept of amphipathic helices. The average hydrophobic moment is defined as the vector sum of the hydrophobicities of the component amino acids that make up the helix. The following hydrophobicities for the amino acids are those reported by Eisenberg (1984) as the "consensus scale: Ile 0.73; Phe 0.61; Val 0.54; Leu 0.53; Trp 0.37;

Met 0.26; Ala 0.25; Gly 0.16; Cys 0.04; Taurus 0.02;

Pro -0.07; Thr -0.18; Ser -0.26; His -0.40;

Glu -0.62; Asn -0.64; Gin -0.69; Asp -0.72;

Light -1.10; Arg -1.76.

The hydrophobic moment, u„, for an ideal α-helix with 3.6 residues per revolution (or a 100° arc angle ( = 360°/3.6) between the side chains), can be calculated from:

where H„ is the hydrophobicity value of the N-th amino acid and the sums are taken over the N-amino acids in the sequence with the periodicity 6=100°. The hydrophobic moment can be expressed as the average hydrophobic moment per remainder by dividing u„ by N, which gives <Ph>. A value for <uH> at 100° ± 20° of approx. 0.20 or more indicates amphipathic helix formation. The <uH> values at 100° for hPTHrp (22-31) and hPTH (22-31) are 0.19 and 0.37, respectively.

Cornett et al., in J. Mol. Biol., 195:659-685 (1987), has further expanded the investigation of amphipathic α-helices by introducing the "amphipatic index" as a predictor of amphipathicity. They concluded that approximately half of all known α-helices are amphipathic, and that the dominant frequency is 97.5" and not 100°, the number of residues per turn being closer to 3.7 than 3.6. Although such subtleties are scientifically interesting, the basic approach of Eisenberg et al is sufficient to classify a particular sequence as amphipathic, particularly when designing a sequence ab initio to form an amphipathic α-helix.

An amphipathic α-helical substitute amino acid sequence may lack homology to the sequence of a particular segment in a naturally occurring polypeptide, but elicits a similar secondary structure, i.e. an α-helix with opposing polar and non-polar faces, in the physiological environment. Replacement of the naturally occurring amino acid sequence with an alternative sequence can favorably affect the physiological activity, stability or other properties of the altered parent polypeptide. Guidance regarding the design and selection of such sequences is provided in, among others, J. L. Krstenansky et al., FEBS Letters 242:2, 409-413 (1989), and J.

P. Segrest et al., Proteins: Structure, Function, and Genetics 8:103-117 (1990).

The amphipathic thi-amino acid α-helix according to this invention has the formula:

wherein the Haa's are selected from the group of hydrophilic amino acids and

The Laa's are selected from the group of polyunsaturated amino acids, as defined above. Assuming an idealized α-helix, the residues 1, 4, 5, 8 and 9 are distributed along one side (A) of the helix within an arc angle of approx. 140" apart, while residues 2, 3, 6, 7, and 10 occupy an opposite arc angle of 140° on the other side (B) of the helix. All residues on one side preferably have the same polarity, while all those on the other side, has the opposite polarity, i.e. if side A is totally hydrophilic, side B is totally lipophilic and vice versa.Those skilled in the art will appreciate that although the helices of this invention are described by means of will the opposite sequence.

also fulfill the residue distribution criteria and is an equivalent descriptor for the helices according to this invention.

Either hydrophilic or lipophilic amino acids can be replaced by alanine as Ala can easily be on both sides of an amphipathic α-helix even though Ala10 does not form an amphipathic α-helix. In general, proline, cysteine and tyrosine are not used; however, their presence and other arbitrary errors in the sequence can be tolerated, e.g. a hydrophilic residue on the lipophilic side as long as the remaining amino acids in the segment essentially join after the division between hydrophilic side and lipophilic side. A suitable method for determining whether a sequence is sufficiently amphipathic to be a sequence of this invention is to calculate the average hydrophobic moment, as defined above. If the average moment for the peak per rest at 100" ± 20" exceeds approx. 0.20, then the sequence will form an amphipathic helix and is a sequence according to this invention.

The average hydrophobic moment per residue at 100° for (sequence identity no.: 26), Xaa = Glu, is calculated e.g. in the following way:

For this sequence, the average hydrophobic peak moment occurs at 92° and has a value of 0.48.

When this concept is applied to parathyroid hormone and parathyroid hormone-related peptide, the hypothesis is made that one or both of the regions (7-16) and (22-31) may exhibit α-helical secondary structure and will be able to be replaced with a non -homologous sequence with similar structural propensities without loss of biological activity or induction of immunological reaction.

Preferred embodiments

In one aspect, this invention provides the polypeptides of the formula:

In another aspect, the polypeptides of the formula are provided:

Xaa is Glu, Lys or Lys (COCH2PEGX) and PEGX is a poly-(ethylene glycol methyl ether) radical of molecular weight 100-10,000 (SEQ ID NO: 27).

In another aspect, this invention provides polypeptides of the formula:

Xaa<1> is absent or is Ala;

Xaa2 is absent or is Val;

Xaa3 is absent or is Ser;

Xaa4 is absent or is Glu or Glu(0CH3);

Xaa<5> is absent or is His or Ala;

Xaa<6> is absent or is Gin;

Xaa' is absent or is Leu;

Xaa<10>and Xaa<17> are independently Asp or Asp(OCH3);

Xaa<11> is Lys, Arg or Leu;

Xaa<13> is Lys, Arg, Tyr, Cys, Leu, Cys (CH2CONH (CH2) 2NH(biotinyl)), Lys(7-dimethylamino-2-oxo-2H-1-benzopyran-4-acetyl) or Lys( dihydrocinnamoyl);

Xaa<20> is Arg or Leu;

Xaa<19>and Xaa<21> are independently Lys, Ala or Arg; Xaa<22*31> is selected from (SEQ ID NOs: 26, 27, 28, 29 or 30);

Xaa<32> is His, Pro or Lys;

Xaa<33> is absent or is Pro, Thr, Glu or Ala;

Xaa<34> is absent or is Pro, Arg, Met, Ala, hSer, hSerlactone, Tyr, Leu or 1,4-diaminobutyryl lactam;

Xaa<35> is absent or is Pro, Glu, Ser, Ala or Gly;

Xaa<3fi> is absent or is Ala, Arg or Ile;

Xaa<37> is absent or is Arg, Trp or 3-(2-naphthyl)-L-alanine;

Xaa<38>is absent or is Ala or hSer, or Xaa<38>"<42>is Thr Arg Ser Ala Trp; and Term is OR or NR2wherein each R is independently H, (C^-C^alkyl or phenyl [C 1 -C 1 ) alkyl; and the pharmaceutically acceptable salts thereof.

In yet another aspect, this invention encompasses polypeptide analogs of the physiologically active truncated homolog hPTHrp(1-34) as shown in formula (I): Ala Val Ser Glu Xaa5 Gin Leu Leu His Asp Xaa<11>Gly Xaa<13>Ser Ile Gin Asp Leu Xaa<19>Arg Xaa<21>Xaa<22>"<31>Xaa<32>Xaa<33>Xaa34 Term, where: Xaa<5> is His or Ala;

Xaa<11> and Xaa<13> are independently Lys, Arg or Leu; Xaa<19> and Xaa<21> are independently Ala or Arg;

Xaa<22>"<31> is selected from:

Xaa is Glu, Lys or Lys (COCH2PEGX) and PEGX is a poly(ethylene glycol methyl ether) radical of molecular weight 100-10,000 (SEQ ID NO: 27);

Xaa<32> is His or Lys;

Xaa<33> is Thr, Glu or Ala;

Xaa<34> is Ala, hSer, Tyr or Leu;

and Term is Gly Arg Arg, lactone, OH or NR 2 , where each R is H or (C 1 -C 4 )alkyl; and their pharmaceutically acceptable salts.

(Formula I).

A more specific aspect of the invention comprises those polypeptides of formula (I) where Xaa<22>"<31>is (SEQ ID NO: 26) for which </iH> at 100° exceeds 0.45. An even more specific aspect of the invention comprises those polypeptides of formula (I) where Xaa<22>"<31> is (sequence identity no: 26); Xaa11 and Xaa<13> are both Light; and Xaa<19> and Xaa<21> are both Arg.

Representative polypeptides include, but are not limited to:

Another aspect of this invention comprises those polypeptides of formula (I) wherein Xaa<22>"<31>is (SEQ ID NO: 26); Xaa<11>and Xaa<13> are both Lys; and one of Xaa< 19> and Xaa<21> is Arg and the other is Ala. Representative polypeptides of this subfamily include, but are not limited to:

In another aspect, this invention encompasses the polypeptides of formula (I) wherein Xaa<22>-<31> is (SEQ ID NO: 26); one of Xaa<11> and Xaa<13> is Leu and the other is Lys; and Xaa<19> and Xaa<21> are both Arg. Representative polypeptides of this subfamily include, but are not limited to:

In another aspect, this invention encompasses those polypeptides of formula (I) where Xaa<22>-<31> are (SEQ ID NO: 27), for which <uH> at 100° exceeds 0.50. A further aspect of this invention comprises those polypeptides of formula (I) wherein Xaa<22>"<31>is (SEQ ID NO: 27); Xaa<11>and Xaa<13> are both Lys or both are Arg; and Xaa<19>and Xaa<21>are both Arg. Representative polypeptides of this subfamily include, but are not limited to:

In another aspect, this invention encompasses polypeptides of formula (I) wherein Xaa<22>"<31>is {SEQ ID NO: 28), for which <uH> at 100° is about 0.25. Representative polypeptides of this the subgenus includes, but is not limited to":

In another aspect, this invention comprises polypeptides of formula (I) where Xaa<22>'<31> is (SEQ ID NO: 29), for which <u„> at 100° is approx. 0.28. Representative polypeptides of this subfamily include, but are not limited to:

In another aspect, this invention comprises polypeptides of formula (I) where Xaa<22>'<31> is (SEQ ID NO: 30), for which <uH> at 100° is approx. 0.29. Representative polypeptides of this subfamily include, but are not limited to:

Still another aspect of this invention comprises polypeptide analogs of the physiologically active truncated homolog bPTH{1-34), as shown in formula (II):

Xaa<1> is Ser or Ala;

Xaa<7> is Leu or Phe;

Xaa<8> is Met or Nie;

Xaa<16> is Asn or Ser;

Xaa<18> is Leu, Met or Nie;

Xaa<19> is Glu or Arg;

Xaa<21> is Val or Arg;

Xaa<22>-<31> are selected from (SEQ ID NOs: 26, 27, 28, 29 and 30);

Xaa<3*> is Phe or Tyr;

Term is OH or NR 2 , where each R is H or (C 1 -C 2 )-alkyl; and the pharmaceutically acceptable salts thereof. (Formula II). Representative polypeptides include, but are not limited to:

In yet another aspect of this invention, it has surprisingly been found that homologs and analogs of PTH and PTHrP having less than 34 amino acids are also potent bone remodeling agents. These compounds have the general formula: Ala Val Ser Glu Xaa<5>Gin Leu Leu His Asp Xaa<11>Gly Xaa<13>Ser Ile Gin Asp Leu Xaa<19>Arg Xaa<21>Xaa<22>"<31> Xaa<32>Xaa<33>Xaa34 Term.

Representative polypeptides include, but are not limited to: Compound 41: AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHP-NH2

(SEQ ID NO: 55)

Physical data

Compound 42: AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LP-NH,

(SEQ ID NO: 56)

Physical data

Those skilled in the art will appreciate that a large number of permutations of the polypeptide analogs can be synthesized that will have the desirable advantages of those described herein, provided that an amino acid sequence having an average hydrophobic moment per rest at 100° ±20° which is greater than approx. 0.20, is inserted in the positions (22-31).

Classic synthesis of the polypeptides

The polypeptides of the present invention can be synthesized by methods such as those set forth in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, Illinois

(1984) and J. Meienhofer, Hormonal Proteins and Peptides, vol.

2, Academic Press, New York, (1973) for solid phase synthesis and E. Schroder and K. Lubke, The Peptides, vol. 1, Academic Press, New York, (1965) for solution synthesis.

In general, these methods involve the sequential addition of protected amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain groups are protected. This protected amino acid is then either attached to an inert, solid support or used in solution, and the next amino acid in the sequence, which is also suitably protected, is added under conditions which are favorable for the formation of the amide bond. After all the desired amino acids have been bound in the correct sequence, protective groups and any solid carrier are removed, whereby the crude polypeptide is obtained. The polypeptide is desalted and purified, preferably chromatographically, whereby the final product is obtained.

A preferred method for producing the analogues of the physiologically active, truncated polypeptides, which have fewer than approx. 40 amino acids, includes solid-phase peptide synthesis. In this method, the o-amino functions (N°) and any reactive side chains are protected using acid- or base-sensitive groups. The protecting group should be stable to the conditions of peptide bond formation, while being easily removable without affecting the polypeptide chain that is still intact. Suitable α-amino protecting groups include, but are not limited to, t-butoxycarbonyl (Boe), benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl, biphenylisopropyloxycarbonyl, t-amyloxycarbonyl (Amoc), isobomyoxycarbonyl, α,α-dimethyl-3 ,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsul-phenyl, 2-cyano-t-butoxycarbonyl, 9-fluorenylmethoxycarbonyl (Pmoc) and the like, preferably t-butoxycarbonyl (Boe). Suitable side chain protecting groups include, but are not limited to: acetyl, benzyl (Bzl), benzyloxymethyl (Bom), o-bromo-benzyloxycarbonyl, t-butyl, t-butyldimethylsilyl, 2-chlorobenzyl

(Cl-z), 2,6-dichlorobenzyl, cyclohexyl, cyclopentyl, isopropyl, pivalyl, tetrahydropyran-2-yl, tosyl (Tos), trimethylsilyl and trityl.

In solid-phase synthesis, the C-terrainal amino acid is first attached to a suitable resin carrier. Suitable resin carriers are those materials which are inert to the reagents and reaction conditions for the stepwise condensation and deprotection reactions, and are insoluble in the media used. Examples of commercially available resins include styrene/divinylbenzene resins modified with a reactive group, e.g. chloromethylated copoly(styrene-divinylbenzene), hydroxymethylated copoly(styrene-divinylbenzene) and the like. Benzylated, hydroxymethylated phenylacetamidomethyl resin (PAM) is preferred. When the C-terminus of the compound is an amide, a preferred resin is p-methylbenzhydrylamino-copoly(styrene-divinylbenzene) resin.

Attachment to the PAM resin can be accomplished by reacting the JT-protected amino acid, preferably the Boc amino acid, as its ammonium, cesium, triethylammonium, 1,5-diazabicyclo-[5.4.0]undec-5-ene -, tetramethylammonium or similar salt in ethanol, acetonitrile, N,N-dimethylformamide (DMF) and the like, preferably the cesium salt in DMF, with the resin at an elevated temperature, e.g. between approx. 40 and 60°C, preferably about 50°C, for from about 12 to 72 hours, preferably about 48 hours.

The N°-Boc amino acid can be attached to the benzhydrylamine resin by means of e.g. a coupling mediated by N,N'-di-isopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) in from ca. 2 to approx. 24 hours, preferably approx. 2 hours at a temperature between approx. 10 and 50°C, preferably 25°C, in such a solvent as dichloromethane or dimethylformamide, preferably dichloromethane.

The successive coupling of protected amino acids can be carried out using methods well known in the art, typically in an automated peptide synthesizer. After neutralization with triethylamine or a similar base, each protected amino acid is preferably introduced in an approximately 1.5 to 2.5 times molar excess, and the coupling is carried out in such an inert, non-aqueous, polar solvent as dichloromethane, DMF or mixtures thereof, preferably in dichloromethane, at ambient temperature. Representative coupling agents are N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) or other carbodiimide, either alone or in the presence of 1-hydroxybenzotriazole (HOBt), O-acyl-urea compounds, benzotriazole-l -yl-oxytris(pyrrolidino)phosphonium-hexafluoro-phosphate (PyBop), N-hydroxysuccinimide, other N-hydroxyimides or oximes. Alternatively, protected amino-acid-active esters (eg p-nitrophenyl, pentafluorophenyl and the like) or symmetrical anhydrides can be used.

At the end of the solid-phase synthesis, the fully protected peptide is removed from the resin. When the bond to the resin support is of the benzyl ester type, cleavage can be carried out by means of aminolysis with an alkylamine or fluoroalkylamine for peptides with an alkylamide C-terminus, or by means of aminolysis with e.g. ammonia/methanol or ammonia/ethanol for peptides with an unsubstituted amide C-terminus, at a temperature between approx. -10 and 50°C, preferably approx. 25°C, for between about 12 and 24 hours, preferably about 18 hours. Peptides with a hydroxy C-terminus can be cleaved by HF or other strong acid deprotection treatment or by saponification. Alternatively, the peptide can be removed from the resin by transesterification, eg with methanol, followed by aminolysis or saponification The protected peptide can be purified by silica gel chromatography.

The side chain protecting groups can be removed from the peptide by treating the aminolysis product with e.g. anhydrous, liquid hydrogen fluoride in the presence of anisole or other carbonium ion remover, treatment with a complex of hydrogen fluoride and pyridine, treatment with tris(trifluoroacetyl)boron and trifluoroacetic acid, by reduction with hydrogen and palladium-on-carbon or polyvinylpyrrolidone, or by reduction with sodium in liquid ammonia, preferably with liquid hydrogen fluoride and anisole at a temperature between approx. -10 and +10"C, preferably at about 0<8>C, for between about 15 minutes and 2 hours, preferably about 1.5 hours.

For peptides on the benzhydrylamine resin, the resin cleavage and deprotection steps can be combined in a single step using liquid hydrogen fluoride and anisole as described above.

The solution can be desalted (e.g. with "BioRad AG-3"

anion exchange resin) and the peptide is purified by a series of chromatographic steps using any of the following types: ion exchange on a weakly basic resin in acetate form; hydrophobic adsorption chromatography on underivatized copoly(styrene-divinylbenzene), e.g. "Amberlite" XAD; silica gel adsorption chromatography; ion exchange chromatography on carboxymethyl cellulose; partition chromatography, e.g. on "Sephadex" G-25; countercurrent distribution; or high performance liquid chromatography (HPLC), especially reverse phase HPLC on octyl or octadecylsilyl silica (ODS) bonded phase column packing.

Another aspect of the present invention thus relates to methods for the production of polypeptides and pharmaceutically acceptable salts thereof, where the methods comprise sequential condensation of protected amino acids on a suitable resin carrier, removal of the protective groups and the resin carrier, and purification of the product, thereby obtaining analogues of the physiologically active, truncated homologs and analogues of PTH and PTHrp, preferably of PTH(1-34) and PTHrp(1-34), where the amino acids in positions (22-31) form an amphipathic α-helical peptide sequence, as defined above.

Recombinant synthesis of the polypeptides

Alternatively, the polypeptides according to this invention can be produced by cloning and expression of a gene that codes for the desired polypeptide. With this method, a plasmid containing the desired DNA sequence is produced and inserted into a suitable host microorganism, usually a bacterium, such as E. coli, or a yeast, such as Saccharomyces cerevisiae, whereby the host microorganism is induced to produce multiple copies of the plasmid, and also of the cDNA which codes for the polypeptide analogues according to this invention.

First, a synthetic gene encoding the selected PTH or PTHrp analog is designed, with appropriate restriction enzyme cleavage sites to facilitate subsequent modifications. Polymerase chain reaction (PCR) as described by Mullis in US Patent Nos. 4,683,195 and 4,683,202 can be used to amplify the sequence.

The amplified, synthetic gene can be isolated and ligated into a suitable plasmid, such as a Trp-LE plasmid, into which four copies of the gene can be inserted sequentially. Production of Trp-LE plasmids is described in US Patent No. 4,738,921 and European Patent Publication No. 0212532. Trp-LE plasmids generally produce 8-10 times more protein than Trp-E plasmids. The multicopy gene can then be expressed in a suitable host, such as E. coli or S. cerevisiae.

The particular expression vector used here was Trp-LE-18-Prot-(Ile<3>, Pro<5>) containing the following elements: a pBR322 fragment (EcoRI-BamHI) containing the ampicillin resistance gene and the plasmid origin of replication; an EcoRI-SacII fragment containing the trp promoter and the trpE gene; an HIV protease (Ile<3>,Pro<5>) gene fragment (Sacll-Hindlll);

a bGRF gene fragment (HindIII-BamHI); and a transcription terminator from the E. coli rpoc gene. The HIV protease and the bGRF gene fragments are not of decisive importance and can, if desired, be replaced by other coding sequences.

The expressed multimer fusion proteins accumulate intracellularly in stable inclusion bodies and can be separated by centrifugation from the rest of the cellular protein. The isolated fusion protein is converted to the monomeric PTH or PTHrp analogue and can be purified using cation exchange and/or reverse phase HPLC.

Alternative methods of cloning, amplification, expression and purification will be apparent to those skilled in the art. Representative methods are described in Maniatis et al., Molecular Cloning, a Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory (1989).

Applicability and administration

The polypeptides according to this invention can be used for the prevention and treatment of many different mammalian conditions manifested by loss of bone mass. The compounds according to this invention are particularly indicated for the prophylaxis and therapeutic treatment of osteoporosis and osteopenia in humans.

In general, the polypeptides of this invention, or salts thereof, are administered in amounts between approx. 0.002 and 1 ug/kg body weight per day, preferably from approx. 0.04 to approx. 0.2 ug/kg body weight per day. For a 50 kg female human individual, the daily dose of active ingredient is from approx. 0.1 to approx. 50 ug, preferably from approx. 2.0 to approx. 10 ug. In other mammals, such as horses, dogs and cattle, higher doses may be required. This dosage can be delivered in a conventional pharmaceutical preparation by means of a single administration, by means of multiple applications or via controlled release, depending on what is needed to achieve the most effective results, preferably once or more times a day by injection.

The selection of the precise dose and composition, and the most appropriate method of delivery will be influenced by, among other things, the pharmacological properties of the selected polypeptide, the type and severity of the condition being treated, and the physical condition and mental clarity of the recipient.

Representative delivery regimens include oral, parenteral (including subcutaneous, intramuscular and intravenous), rectal, buccal (including sublingual), pulmonary, transdermal and intranasal.

Pharmaceutically acceptable salts retain the desired biological activity of the parent polypeptide without toxic side effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids, such as e.g. acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, embonic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenesulfonic acids, polygalacturonic acid and the like; (b) base addition salts formed with polyvalent metal cations, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like; or with an organic cation formed from N,N'-dibenzylethylenediamine or ethylendiamine; or (c) combinations of (a) and (b), e.g. a zinc tannate salt and the like.

A further aspect of the present invention relates to pharmaceutical preparations which comprise as an active ingredient a polypeptide according to the present invention or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable, non-toxic carrier. As mentioned above, such preparations can be prepared for parenteral (subcutaneous, intramuscular or intravenous) administration, preferably in the form of liquid solutions or suspensions; for oral or buccal administration, particularly in the form of tablets or capsules; for pulmonary or intranasal administration, especially in the form of powders, nasal drops or aerosols; and for rectal or transdermal administration.

The preparations may conveniently be administered in single-dose form and may be prepared by any of the methods well known in the pharmaceutical art, e.g. as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA., (1985). Formulations for parenteral administration may contain as excipients sterile water or physiological saline, alkylene glycols, such as propylene glycol, polyalkylene glycols, such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. For oral administration, the formulation can be strengthened by adding bile salts or acylcarnitines. Formulations for nasal administration may be solid and may contain excipients, e.g. lactose or dextran, or can be water or oil solutions for use in the form of nasal drops or metered spray. For buccal administration, typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch and the like.

When formulated for nasal administration, absorption across the nasal mucosa can be enhanced with the help of surface-active acids, such as e.g. glycocholic acid, cholic acid, taurocholic acid, etocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid, cyclodextrins and the like, in an amount in the range between approx. 0.2 and 15% by weight, preferably between approx. 0.5 and 4% by weight, preferably approx. 2% by weight.

Delivery of the compounds according to the present invention to the individual over long periods of time, e.g. for periods of one week to one year, can be carried out by means of a single administration of a controlled release system containing sufficient active ingredient for the desired release period. Various controlled release systems, such as monolith or reservoir type microcapsules, depot implants, osmotic pumps, vesicles, micelles, liposomes, transdermal patches, iontophoretic devices and

alternative, injectable dosage forms can be used for this purpose. Localization at the site where delivery of the active ingredient is desired is a further feature of some controlled release devices which may prove beneficial in the treatment of certain diseases.

One form of controlled release preparation contains the polypeptide or its salt dispersed or encapsulated in a slowly degrading, non-toxic, non-antigenic polymer, such as copoly(lactic/glycolic) acid, as described in the pioneering work of Kent, Lewis, Sanders and Tice, US Patent No. 4,675,189. The compounds or preferably their relatively insoluble salts can also be formulated into cholesterol or other lipid matrix pellets, or silastomer matrix implants. Additional slow-release formulations, depot implant formulations, or injectable formulations

will be obvious to those skilled in the art. See e.g. Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson ed.,

Marcel Dekker, Inc., New York, 1978, and R.W. Baker, Controlled Release of Biologically Active Agents, John Wiley & Sons, New York, 1987.

The following specific examples are intended to illustrate the synthesis and testing of representative compounds of the invention and should not be considered as limiting the scope of the claims. In the examples, "mp." melting point,<n>[a]D<25>" is the optical activity at 25"C and the determined concentration in

the solvent indicated, "FAB" is fast atomic bombardment-Q ment mass spectrometry, and "AAA" is amino acid analysis with the expected values in parentheses after the observed values. The amino acid analyzes were performed on a Hewlett-Packard AminoQuant analyzer following the manufacturer's recommended procedures. Primary amino acids were derivatized with o-phthalaldehyde; 5 secondary amino acids with Fmoc. Fluorescence detection of the derivatized amino acids was used for quantification. The protected amino acids were obtained from Applied Biosystems Inc.

(Foster City, CA).

Example I

Compound 1 (SEQ ID NO: 7) was prepared on a 0.5 mmol scale by the solid phase method on 4-methylbenz-hydrylamine resin using an automated Applied Biosystems model 430A peptide synthesizer. The α-amino groups were protected with t-butoxycarbonyl (Boe). The side chain protecting groups were: benzyl (Bzl) for Asp, Glu and Ser; tosyl (Tos) for Arg; benzyloxymethyl (Bom) for His, and 2-chlorobenzyl (Cl-z) for Lys. The amino acids were linked sequentially using N,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBt) following Stewart and Young (supra). After each amino acid coupling, the peptide was acetylated using a mixture of acetic anhydride and diisopropylethylamine in N-methylpyrrolidone. The full-length peptide was cleaved from the resin with simultaneous deprotection of the side chain protecting groups using anhydrous HF (25 mL) in the presence of anisole (2.5 mL) at -10°C for 30 min, and at 0°C for 60 min. After evaporation of HF under vacuum, the residue was washed with anhydrous ether and the crude peptide was extracted with 10% acetic acid. Lyophilization of the 10% acetic acid extract gave 900 mg of crude product. The peptide was purified by ODS reverse phase column chromatography at medium pressure using a gradient of 22-45% CH 3 CN in 0.1% TFA. The product eluted in three fractions which were concentrated and lyophilized, whereby 130 mg of white solid with >98% purity was obtained.

Connection 1:

AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA- NHT

(sequence identity no. : 7)

Physical data: m.p. 150-159°C [a]D<35>-34.88° (c 0.16, H20) F AB <Cl7SH300NS6051): [M+H]<+>4005.5

AAA: Asp, 1.9(2); Glu, 5.6(6); See, 1.6(2); His, 2.7(3);

Gly, 1.0(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.8(3);

Choice, 1.0(1); Ile, 0.9(1); Leu, 7.3(8); Light, 4.0(4).

Likewise, compounds 2, 5-18, 21-27, 29-36, 38-48, 50-54, 58-64 and 66-70 were prepared and characterized, PAM resin being replaced as needed for the synthesis of hydroxy-terminal polypeptides.

Connection 2:

AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA- OH

( sequence identity no. : 6)

Physical data: mp. 154-170'C [o]D<25>-49.35° (c 0.46, H2O) FAB (<C>175<H>301N57<0>50): [M+H]<*>4005 ,0

AAA: Asp, 2.1(2); Glu, 5.9(6); See, 1.7(2); His, 2.9(3);

Gly, 1.1{1); Thr, 1.0(1); Ala, 1.9(2); Arg, 3.0(3);

Choice, 1,2(1); Ile, 1.0(1); Leu, 7.8(8); Light, 4.2(4).

Compound 5:

AVSEHQLLHDKGKSIQDLRRRELLERLLERLHTA- OH

(sequence identity no.: 15)

Physical data: m.p. 147-165°C [α]0<2S>-49.17° (c 0.66, H2O) FAB (C175H299<N>59<0>52): [M<+>H]<+>4061

AAA: Asp, 2.1(2); Glu, 6.1(6); See, 1.8(2); His, 3.1(3);

Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 5.0(5);

Choice, 1.0(1); Ile, 0.9(1); Leu, 7.7(8); Light, 1.9(2).

Compound 6:

AVSEHQLLHDRGRSIQDLRRRELLERLLERLHTA- OH

sequence identity no.: 16)

Physical data: mp. 150-170'C [a]D<25>-48.65" (c 0.54, H2O) FAB (C175H299N63052 ): [M+H]<+>4118.0

AAA: Asp, 2.1(2); Glu, 6.1(6); See, 1.8(2); His, 3.2(3);

Gly, 1.2(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 6.9(7);

Choice, 1.0(1); Ile, 1.0(1); Leu, 7.8(8).

Compound 7:

AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA- OH

(sequence identity no.: 17)

Physical data: mp. 177-182<C>C [ct]D25 -46.17° (c 0.14, H2O) FAB (C176<H>304N64050): [M+H]<+>4117

AAA: Asp, 2.0(2); Glu, 4.8(5); See, 1.8(2); His, 3.2(3);

Gly, 1.1(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 6.7(7);

Choice, 1.0(1); Ile, 1.0(1); Leu, 7.7(8); Light, 0.9(1).

Compound 8:

AVSEHQLLHDKGKSIQDLRRRELLEKLLRKLHTA- OH

( sequence identity no. : 5)

Physical data: m.p. 147-165"C [α]D<25>-49.17° (c 0.66, H2O) FAB (C176H305N5g049): [M+H]<+>4033.0

AAA: Asp, 2.0(2); Glu, 4.8(5); See, 1.8(2); His, 2.7(3);

Gly, 1.1(1); Thr, 0.9(1); Ala, 2.0(2); Arg, 3.9(4);

Choice, 1.0(1); Ile, 1.0(1); Leu, 7.9(8); Light, 4.0(4).

Compound 9:

AVSEHQLLHDKGKSIQDLRRRELELLEKLKLHTAGRR- OH

(sequence identity no. : 10)

Physical data: m.p. 158-160°C [a]D<25>-44.76" (c 0.1, H2O) FAB (C189H326<N>M<0>5S): [M]<*>4375.0

AAA: Asp, 2.0(2); Glu, 5.9(6); See, 1.7(2); His, 2.9(3);

Gly, 2.3(2); Thr, 1.0(1); Ala, 1.9(2); Arg, 5.0(5);

Choice, 1,2(1); Ile, 1.0(1); Leu, 7.8(8); Light, 4.3(4).

Compound 10:

AVSEAQLLHDLGKSIQDLRRRELLEKLKLHAL- OH

(sequence identity no.: 14)

Physical data: m.p. 170-175"C [α]D<25>-31.59° (c 0.54, H2O) FAB (<C>174<H>300N52<0>51): [M+H]<+>3936 ,0

AAA: Asp, 2.0(2); Glu, 6.0(6); See, 1.8(2); His, 2.0(2);

Gly, 1.2(1); Ala, 3.0(3); Arg, 2.8(3); Choice, 1.1(1);

Ile, 1.0(1); Leu, 9.9(10); Light, 3.0(3).

Compound 11:

AVSEHQLLHDKGKSIQDLRRRELLEKLLELLKEL- NH,

(sequence identity no.: 11)

Physical data: m.p. 172-174°C [a]D<25>-43.29° (c 0.2, H2O) FAB (<C>179<H>311N55<0>52): [M+H]<+>4065 ,8

AAA: Asp, 2.2(2); Glu, 7.7(7); See, 1.7(2); His, 2.0(2);

Gly, 1.0(1); Ala, 1.0(1); Arg, 3.0(3); Choice, 1.1(1);

Ile, 1.0(1); Leu, 9.3(9); Light, 5.1(5).

Compound 12:

AVSEIQFXHNLGKHLSSXERVELELLEKLHNY- NHT

(X=Nle. sequence identity no. : 23)

Physical data: mp. 178°C [o]D<25>-36.88°C (c 0.4, H2O) FAB (C1B2<H>2<g>sNsoO5l): [M+H]<+>4001.6

AAA: Asp, 2.1(2); Glu, 6.5(6); See, 2.7(3); His, 3.1(3);

Gly, 1.1(1); Ala, 1.0(1); Arg, 1.0(1); Taurus, 0.8(1);

Choice, 2.0(2); Phe, 1.0(1); Ile, 0.9(1); Leu+Nle, 8.5(7+2); Light, 3.1(3).

Compound 13:

AVSEIQFXHNLGKHLSSXRRRELELLEKLHNY- NH7

(X=Nle. sequence identity no. : 24)

Physical data: mp. 260°C [a]D<25>-37.02° (c 0.2, H20) FAB (C1M<H>304N56049): [M+H]<*>4084

AAA: Asp, 2.1(2); Glu, 5.5(5); See, 2.6(3); His, 3.1(3);

Ala, 1.0(1); Gly, 1.1(1); Arg, 3.2(3); Taurus, 1.0(1);

Choice, 1.0(1); Phe, 1.0(1), Ile, 1.0(1); Leu, 9.0(9);

Light, 3.0(3).

Compound 14:

AVSEHQLLHDKGKSIQDLRRRALAEALAEALHTA- NH3

(sequence identity no. : 20)

Physical data: m.p. 190-225'C [α]D<25>-56.58° (c 0.36, H2O) FAB (C161H272N540„): [M+H]<+>3747.0

AAA: Asp, 2.1(2); Glu, 4.9(5); See, 1.7(2); His, 2.6(3);

Gly, 1.1(1); Thr, 1.0(1); Ala, 7.6(7); Arg, 2.8(3);

Choice, 1,2(1); Ile, 1.0(1); Leu, 6.6(6); Light, 1.9(2).

Compound 15:

AVSEHOLLHDKGKSIQDLARRELLEKLLEKLHTA- NH7

(sequence identity no.: 12)

Physical data: m.p. 170-180°C [o]D<25>-48.19° (c 0.2, H2O) FAB (<C>172<H>293N53<0>51): [M+H]<+>3919 ,0

AAA: Asp, 2.1(2); Glu, 6.1(6); See, 1.7(2); His, 3.1(3);

Gly, 1.1(1); Thr, 1.0(1); Ala, 3.0(3); Arg, 2.1(2);

Choice, 1.1(1); Huh, 1.0(1); Leu, 8.0(8); Light, 4.4(4).

Compound 16:

AVSEHOLLHDKGKSIQDLRRAELLEKLLEKLHTA- NH,

(sequence identity no.: 13)

Physical data: m.p. 190-195°C [α]D<25>-50.50° (c 0.4, H2O) FAB (C172H293N„051): [M+H]<+>3919.0

AAA: Asp, 2.1(2); Glu, 6.0(6); See, 1.8(2); His, 3.1(3);

Gly, 1.1(1); Thr, 1.0(1); Ala, 3.0(3); Arg, 2.1(2);

Choice, 1.1(1); Ile, 1.0(1), Leu, 7.5(8); Light, 4.2(4).

Compound 17: AVSEHQLLHDKGKSIQDLRRRSLLSSLLSSLHTA- NH- ;

(sequence identity no.: 21)

Physical data: mp. 195-204°C [α]D<25>-67.11" (c 0.3, H2O) FAB (<C>163<H>280<N>54050): [M+H]<+>3796 ,0

AAA: Asp, 2.1(2); Glu, 2.9(3); See, 6.8(7); His, 3.1(3);

Gly, 1.2(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.0(3);

Choice, 1.0(1); Ile, 1.0(1); Leu, 8.2(8); Light, 2.0(2).

Compound 18:

AVSEHQLLHDKGKSIQDLRRRAFYDKVAEKLHTA- NH2

(sequence identity no.: 22)

Physical data: mp. 200-207°C [α]D<25>-60.26° (c 0.6, H2O) FAB (<C>174H284NS6050): [M+H]<+>3960.0

AAA: Asp, 2.9(3); Glu, 3.5(4); See, 1.4(2); His, 2.6(3);

Gly, 0.9(1); Thr, 1.0(1); Ala, 4.0(4); Arg, 3.0(3);

Taurus, 0.9(1); Choice, 1.9(2); Phe, 1.1(1); Ile, 0.9(1);

Leu, 3.6(4); Light, 4.1(4).

Compound 21:

AVSEIQFLHN LGKHLSSLRR RELLEKLLEK LHNY-NH,

(sequence identity no: 351

Physical data: m.p. 148-155'C [α]D<25>-45.97 (c 0.26, H2O) FAB (C184H304N56049): [M+H]<+>4084

AAA: Asx, 2.1(2); Glx, 5.0(5); See, 2.7(3); His, 3.0(3);

Gly, 1.0(1); Ala, 0.9(1); Arg, 3.1(3); Taurus, 0.9(1);

Choice, 1.0(1); Phe, 0.9(1); Ile, 0.9(1); Leu, 9.3(9);

Light, 3.2(3).

Compound 24:

AVSEHQLLHD KGKSIQDLKL RELLEKLLEK LHTA- NH,

(sequence identity no.: 38)

Physical data: m.p. 175-182°C [cc]D25 -49.99 (c 0.47, H20) FAB (C175H299N49<0>51)<:>[M+H]* 3906.5

AAA: Asx, 2.1(2); Glx, 6.5(6); See, 1.8(2); His, 3.1(3);

Gly, 1.1(1); Thr, 1.0(1); Ala, 2.1(2); Choice, 1.1(1);

Ile, 1.0(1); Leu, 9.1(9); Light, 6.5(6).

Compound 25:

AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTA- NH,

(sequence identity no: 39)

Physical data: mp. 136.5-153.5°C [a]D<25>-32.57 (c 0.13, H20) FAB (C175H300N60<0>51): [M+H]<+>4060.8

AAA: Asx, 2.2(2); Glx, 6.2(6); See, 1.8(2); His, 3.2(3);

Gly, 1.1(1); Thr, 1.0(1); Ala, 2.1(2); Arg, 5.2(5);

Choice, 1.1(1); Ile, 1.1(1); Leu, 8.4(8); Light, 2,2(2).

Compound 26:

AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAP- OH

f sequence identity no.: 40)

Physical data: m.p. 125.8-127.2°C [a]D<25>-54.62 (c 0.23, H2O) FAB (C180H306<N>60053): [M+H]<+>4158.0

AAA: Asx, 2.1(2); Glu, 6.2(6); See, 1.8(2); His, 2.9(3);

Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 5.1(5);

Choice, 1.0(1); Ile, 1.0(1); Leu, 8.0(8); Light, 2.1(2);

Pro, 1.1(1).

Compound 27:

AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAGRR- OH

(sequence identity no.: 41)

Physical data: m.p. 106-137.3<8>C [a]D<25>-39.55 (c 0.67, H2O) FAB (<C>189H326<N>68055): [M+H]* 4430.5

AAA: Asx, 2.1(2); Glx, 5.9(6); See, 1.6(2); His, 2.7(3);

Gly, 2.2(2); Thr, 1.0(1); Ala, 1.8(2); Arg, 7.3(7);

Choice, 0.8(1); Ile, 1.0(1); Leu, 8.1(8); Light, 2.1(2).

Compound 29:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTY- NH,

(sequence identity no.: 43)

Physical data: mp. 160-172"C [a]B<2S>-49.85 (c 0.34, H 2 O) FAB (<C>181<H>3M<N>56052): [M+H] + 4096.9

AAA: Asx, 2.0(2); Glx, 5.6(6); See, 1.7(2); His, 3.1(3);

Gly, 1.1(1); Thr, 0.9(1); Ala, 0.9(1); Arg, 3.0(3);

Taurus, 0.9(1); Choice, 1.0(1); Ile, 1.0(1); Leu, 7.7(8);

Light, 4.4(4).

Compound 30:

AVSEHQLLHD KGYSIQDLRR RELLEKLLEK LHTA- NH^

(sequence identity no.: 44)

Physical data: mp. 130-171°C [ct]D25 -40.65 (c 0.34, H20) FAB (C178H297N„0„): [M+H] + 4039.4

AAA: Asx, 2.0(2); Glx, 5.5(6); See, 1.8(2); His, 3.4(3);

Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 2.9(3);

Taurus, 0.8(1); Choice, 1.0(1); Ile; 0.9(1), Leu, 7.9(8);

Light, 3,4(3).

Compound 31:

AVSEHQLLHD KGCSIQDLRR RELLEKLLEK LHTA- NH,

(sequence identity no.: 45)

Leu His Thr Ala nh, C (SEQ ID NO: 45)

Physical data: mp. 140-160'C [a]D<25>-44.48 (c 0.25, H 2 O) FAB (C^jH^<N>ssOsiSi): [M+H]* 3979

AAA: Asx+Cys, 3.0(2+1); Glx, 5.6(6); See, 1.7(2); Hiss,

3.0(3); Gly, 1.0(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.5(3); Choice, 1.0(1); Ile, 0.9(1); Leu, 7.5(8); Light, 3.3(3).

Compound 32:

AVSEHQLLHD KGXSIODLRR RELLEKLLEK LHTA- NH7

(sequence identity no.: 46)

f X - Cvsf CH?CONH( CH?) 7NH( biotinvl)))

Physical data: mp. (not determined) [<x]D<zs>(not determined) FAB (<C>186<H>3l6N„0MSz): [M+H]<*>4306.6

AAA: Asx, 2.2(2); Glx, 6.1(6); See, 1.8(2); His, 3.8(3);

Gly, 1.0(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3);

Choice, 1.1(1); Ile, 0.9(1); Leu, 8.3(3); Light, 3.3(3).

Compound 33:

AVSEHQLLHD KGXSIQDLRR RELLEKLLEK LHTA- NH7

(sequence identity no: 47)

( X = Lys( 7- dimethylamino- 2- oxo- 2H- 1- benzopyran- 4- acetyl))

Physical data: mp. 135-205°C [a]D<25>-26.92 (c 0.104, 50%

aqueous HOAc)

FAB (C188<H>311N57054): [M+H]<+>4233

AAA: Asx, 1.9(2); Glx, 6.3(6); See, 1.7(2); His, 3.2(3);

Gly, 1.0(1); Thr, 1.1(1); Ala, 2.0(2); Arg, 3.2(3);

Choice, 1.1(1); Ile, 0.9(1); Leu, 8.2(8); Light, 4.5(4).

Compound 34:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAG- OH

(sequence identity no.: 48)

Physical data: m.p. 92.1-146.6°C [α]D<25>-40.76 (c 0.34, H2O) FAB (<C>177<H>302N56O53): [M+H]<+>4062, 0

AAA: Asx, 2.0(2); Glx, 5.7(6); See, 1.8(2); His, 3.0(3);

Gly, 2.2(2); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.8(3);

Choice, 1,2(1); Ile, 0.9(1); Leu, 7.5(8); Light, 4.2(4).

Compound 35:

AVSXjHQLLHX-, KGKSI0X7LRR RX1LLX, KLLX1 K LHA- OH

(sequence identity no: 49)

(X1 o GlufOCH^ : X7= Asp( 0CH,))

Physical data: m.p. (not determined) [a]D<25>-21.96 (c 0.132,

H20)

FAB (<C>1B1<H>311<N>55052): [M+H]<+>4089.0

AAA: Asx, 2.1(2); Glx, 6.3(6); See, 1.8(2); His, 3.3(3);

Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3);

Choice, 1.1(1); Ile, 0.9(1); Leu, 8.0(8); Light, 4.2(4).

Compound 36:

AVSX^ OLLHXt KGKSIQX, LRR RX, LLX1 KLLX1K LHA- OCH,

(sequence identity no. : 50)

(Xt = GlufOCH,) ; X7= Asp( 0CH,n

Physical data: m.p. (not determined) [a]D<25>-46.80 (c 0.07,

H20)

FAB (C182H313N55<0>52): [M+H]<+>4103

AAA: Asx, 2.1(2); Glx, 6.2(6); See, 1.4(2); His, 3.0(3);

Gly, 1.1(1); Thr, 1.1(1); Ala, 1.7(2); Arg, 3.2(3);

Choice, 0.6(1); Ile, 0.9(1); Leu, 8.0(8); Light, 4.1(4).

Compound 38:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAP- OH

(sequence identity no.: 52)

Physical data: mp. 152.1-186.5°C [a]D<25>-55.91 (c 0.33, H2O) FAB (<C>180<H>306N56OS3): [M+H]<+>4102, 6

AAA: Asx, 2.0(2); Glx, 5.6(6); See, 1.7(2); His, 2.9(3);

Gly, 1.1(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.9(3);

Choice, 1,2(1); Ile, 1.0(1); Leu, 7.7(8); Light, 4.3(4).

Compound 39:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTP- OH

(sequence identity no.: 53)

Physical data: m.p. 120-148.2°C [a]D<25>-52.78 (c 0.14, H2O) FAB (C177H301<N>550S2): [M+H]<+>4031.0

AAA: Asx, 2.0(2); Glx, 5.5(6); See, 1.8(2); His, 2.9(3);

Gly, 1.0(1); Thr, 1.0(1); Ala, 0.9(1); Arg, 2.9(3);

Choice, 1,2(1); Ile, 0.9(1); Leu, 7.5(8); Light, 3.6(3);

Pro, 0.9(1).

Compound 40:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTP- NH,

(sequence identity no.: 54)

Physical data: m.p. 133.9-155.1°C [α]D<25>-54.22 (c 0.37, H2O) FAB (<C>177<H>302N56051): [M+H]<+>4030, 7

AAA: Asx, 2.0(2); Glx, 5.6(6); See, 1.9(2); His, 2.9(3);

Gly, 1.1(1); Thr, 0.9(1); Ala, 0.9(1); Arg, 2.8(3);

Choice, 1,2(1); Ile, 1.1(1); Leu, 7.8(8); Light, 4.2(4);

Pro, 0.9(1).

Compound 41:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHP- NH-,

(sequence identity no.: 55)

Physical data: m.p. 142.8-166.1°C [a]D<25>-53.80 (c 0.38, H2O) FAB (<C>173<H>295N55049): [M+H]<*>3929

AAA: Asx, 2.0(2); Glx, 5.7(6); See, 1.8(2); His, 3.0(3);

Gly, 1.1(1); Ala, 0.9(1); Arg, 2.8(3); Choice, 1,2(1);

Ile, 0.9(1); Leu, 7.4(8); Light, 4.4(4); Pro, 0.9(1).

Compound 42:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LP- NH?

(sequence identity no.: 56)

Physical data: m.p. 161.0-177.0°C [a]D<25>-61.97 (c 0.19, H2O) FAB (<C>167H288N52048): [M+H]<+>3792.0

AAA: Asx, 2.2(2); Glx, 5.9(6); See, 1.9(2); His, 2.1(2);

Gly, 1.1(1); Ala, 1.0(1); Arg, 3.0(3); Choice, 1.1(1);

Ile, 1.0(1); Leu, 7.9(8); Light, 4.3(4); Pro, 0.9(1).

Compound 43:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAW- OH

(sequence identity no.: 57)

Physical data: m.p. 181-202'C [a]D<2S>-45.14 (c 0.19, H2O) FAB (C195H326N62056): [M+H]* 4435.2

AAA: Asx, 2.0(2); Glx, 5.8(6); See, 2.8(3); His, 2.8(3);

Gly, 1.1(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 3.7(4);

Ile, 0.9(1); Leu, 7.5(8); Light, 4.3(4); Trp, 0.9(1).

Compound 44:

AVSEHQLLHD RGRSIQDLRR RELLERLLER LHTAGRRTRSAW- OH

(sequence identity no: 58)

Physical data: m.p. 130-132.2°C [a]D<a5>-46.66 (c 0.195, H2O) FAB (<C>216<H>365N81062): [M+H]<+>5088.8

AAA: Asx, 2.2(2); Glx, 6.0(6); See, 2.7(3); His, 3.0(3);

Gly, 2.2(2); Thr, 2.1(2); Ala, 3.0(3); Arg, 10.5(10);

Choice, 0.9(1).; Ile, 1.0(1); Leu, 8.2(8); Trp 1.0(1).

Compound 45:

AVSEHQLLHD RGRSIQDLRR RELLERLLER LHTÅGRRTRSAW- NH7

(sequence identity no.: 59)

Physical data: m.p. 158-174°C [a]D<zs>-43.57 (c 0.53, H2O) FAB (C216<H>366<N>8206l): [M+H]<+>5087.4

AAA: Asx, 1.9(2); Glx, 5.6(6); See, 2.6(3); His, 3.3(3);

Gly, 2.1(2); Thr, 2.0(2); Ala, 2.9(3); Arg, 10.1(10);

Choice, 0.9(1); Ile, 1.0(1); Leu, 8.3(8); Trp, 1.1(1).

Compound 46:

AVSEHQLLHD RGXSIQDLRR RELLERLLER LHTAGRRTRSAW- OH

(sequence identity no.: 60)

Physical data: mp. 165.4-175.2'C [α]D<25>-40.43 (c 0.20, H20) FAB (C225<H>374<N>80062): [M+H]<*>5191

AAA: Asx, 2.1(2); Glx, 6.3(6); See, 2.8(3); His, 3.2(3);

Gly, 2.1(2); Thr, 2.0(2); Ala, 3.2(3); Arg, 9.9(9);

Choice, 1.0(1); Ile, 0.9(1); Leu, 8.6(8); Light, 1.1(1);

Trp, 1.1(1).

Compound 47:

AVSEIQFXHN LGKHLSSXTR SAWLRKKLQD VHNY- NH;

(sequence identity no.: 61)

(X » norleucine)

Physical data: mp. 140-160°C [o]D<25>-56.88 (c 0.16, H2O) FAB (C180H287NS5058): [M+H]<+>3989.8

AAA: Asx, 3.0(3); Glx, 2.9(3); See, 3.7(4); His, 2.8(3);

Gly, 1.1(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.0(2);

Taurus, 1.0(1); Choice, 1.7(2); Phe, 0.9(1); Ile; 0.9(1), Leu+Nle, 5.8(2+4); Light, 3.4(3); Trp, 1.1(1).

Compound 48:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTMA- NH-,

(sequence identity no.: 62)

Physical data: mp. 140-210°C [α]D<25>-47.75 (c 0.178, HaO) FAB (C^^O^): [M+Hr 4135.0

AAA: Asx, 2,3{2); Glx, 6.6(6); See, 1.4(2); His, 3.2(3);

Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3);

Choice, 0.9(1); Met, 1.1(1), Ile, 1.0(1); Leu, 8.8(8);

Light, 4.4(4).

Compound 50:

AVSEHQLLHD KGKSIQDLRR RFFLEKLLEK LHTA- NH-,

(sequence identity no.: 64)

Physical data: mp. 136.5-156.8°C [a]D<25>-49.89 (c 0.24, H2O) FAB (C182H300N56<0>49): [M+H]<+>4056.0

AAA: Asx, 2.2(2); Glx, 5.0(5); See, 1.9(2); His, 3.3(3);

Gly, 1.0(1); Thr, 1.0(1); Ala, 2.1(2); Arg, 3.1(3);

Choice, 1.0(1); Phe, 2.0(2); Ile, 0.9(1); Leu, 7.2(7);

Light, 3.5(4).

Compound 51:

AVSEHQLLHD KGKSIQDLRR RELLHKLLEK LHTA- NH-,

(sequence identity no.: 65)

Physical data: m.p. 80.7-141.0'C [a]0<Z5>-55.38 (c 0.23, H2O) FAB (C176H300N58O„): [M+H]<+>4012.8

AAA: Asx, 2.2(2); Glx, 4.9(5); See, 1.8(2); His, 4.3(4);

Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3);

Choice, 1.1(1); Ile, 1.0(1); Leu, 8.1(8); Light, 3.9(4).

Compound 52:

AVSEHQLLHD KGKSIQDLRR RELLEHLLEK LHTA- NH,

(sequence identity no.: 66)

Physical data: m.p. 134.3-157.9°C [a]D<25>-50.72 (c 0.45, H2O) FAB (C175H295<N>57051): [M+H]<+>4012.8

AAA: Asx 2.1(2); Glx, 5.9(6); See, 1.8(2); His, 4.2(4);

Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.0(3);

Choice, 1.1(1); Ile, 0.9(1); Leu, 8.1(8); Light, 3.1(3).

Compound 53:

AVSEHQLLHD KGKSIQDLRR RELLEKLIAK LHTA- NH,

(sequence identity no.: 67)

Physical data: m.p. 142.7-159.8<e>C [<x]D25 -54.01 (c 0.21, H20) FAB (C173H29BN560„): [M+H]<+>3946.0

AAA: Asx, 2.2(2); Glx, 4.9(5); See, 1.8(2); His, 3.1(3);

Gly, 1.1(1); Thr, 1.0(1); Ala, 3.1(3); Arg, 3.1(3);

Choice, 1.0(1); Ile, 1.9(2); Leu, 7.0(7); Light, 4.3(4).

Compound 54:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEE IHTA- NH,

(sequence identity no.: 66)

Physical data: mp. 138-185°C [a]D<25>-50.17 (c 0.14, H2O) FAB (C174H295N550„): [M+H]<+>4005

AAA: Asx, 2.2(2); Glx, 7.1(7); See, 1.7(2); His, 2.8(3);

Gly, 1.0(1); Thr, 1.0(1); Ala, 2.1(2); Arg, 3.1(3);

Choice, 1.1(1); Ile, 1.7(2); Leu, 7.1(7); Light, 2.7(3).

Compound 58:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAW-NH,

(sequence identity no.: 72)

Physical data: mp. 158-163°C [a]D<Z5>-46.06 (c 0.17, H2O) FAB (C195H327N63<0>55): [M+H]<+>4434.8

AAA: Asx, 2.0(2); Glx, 5.5(6); See, 2.7(3); His, 3.1(3);

Gly, 1.0(1); Ala, 1.8(2); Arg, 4.0(4); Thr, 0.9(1);

Choice, 0.9(1); Ile, 0.9(1); Leu, 7.5(8); Light, 3.9(4);

Trp, 1.0(1).

Compound 59:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAX- OH

(sequence identity no.: 73)

( X = Nal( 2) = 3-( 2- naphthyl)- L- alanln)

Physical data: m.p. 156-162'C [a]D<25>-44.44 (c 0.189, Hz0) FAB (C197H328<N>62<0>5S): [M+H]<+>4445.6

AAA: Asx, 2.1(2); Glx, 5.5(6); See, 2.8(3); His, 2.9(3);

Gly, 1.0(1); Ala, 2.0(2); Arg, 4.0(4); Thr, 0.9(1);

Choice, 1.0(1); Ile, 0.9(1); Leu, 7.5(8); Light, 4.2(4);

Nail, 1.1(1).

Compound 60:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTASAW- OH

(sequence identity no.: 74)

Physical data: mp. 159-164'C [cc]D25 -50.94.(c 0.29, H20) FAB (C192H320N60<O>55): [M+H]<+>4349.0

AAA: Asx, 2.0(2); Glx, 5.6(6); See, 2.7(3); His, 3.2(3);

Gly, 1.0(1); Ala, 3.1(3); Arg, 2.8(3); Thr, 1.0(1);

Choice, 1.1(1); Ile, 0.9(1); Leu, 7.6(8); Light, 4.0(4);

Trp, 1.0(1).

Compound 61:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEIRA- OH

(sequence identity no.: 75)

Physical data: m.p. 155-210°C [a]D<25>-46.15 (c 0.12, H2O) FAB (C195H334<N>620sa): [M+H]<+>4475.8

AAA: Asx, 2.2(2); Glx, 6.9(7); See, 1.7(2); His, 3.2(3);

Gly, 1.1(1); Ala, 3.1(3); Arg, 4.0(4); Thr, 0.9(1);

Choice, 1.1(1); Ile, 1.9(2); Leu, 8.1(8); Light, 4.1(4).

Connection 62

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEIR- OH

(sequence identity no.: 76)

Physical data: mp. 186-218'C [α]D<25>-52.73 (c 0.265, H20) FAB (C192H329N61057): [M+H]<+>4404.4

AAA: Asx, 2.0(2); Glx, 6.6(7); See, 1.9(2); His, 3.4(3);

Gly, 1.1(1); Ala, 2.0(2); Arg, 3.8(4); Thr, 1.0(1);

Choice, 1.1(1); Ile, 1.7(2); Leu, 7.9(8); Light, 4.0(4).

Compound 63:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEI- OH

(sequence identity no.: 77)

Physical data: m.p. 169-205<e>C [a]„" -50.78 (c 0.51, H2O) FAB (C186<H>317<N>57056): [M+H]<+>4248.0

AAA: Asx, 2.2(2); Glx, 6.8(7); See, 1.8(2); His, 3.3(3);

Gly, 1.0(1); Ala, 2.0(2); Arg, 3.0(3); Thr, 1.0(1);

Choice, 1.0(1); Ile, 1.8(2); Leu, 7.8(8); Light, 3.6(4).

Compound 64:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAE- OH

(sequence identity no.: 78)

Physical data: m.p. 199-205'C [α]D<25>-52.47 (c 0.41, H2O) FAB (<C>180<H>306<N>56<0>55): [M+H]< +>4135.0

AAA: Asx, 2.0(2); Glx, 6.6(7); See, 1.9(2); His, 3.3(3);

Gly, 1.1(1); Ala, 2.0(2); Arg, 2.9(3); Thr, 1.0(1);

Choice, 1.1(1); Ile, 1.0(1); Leu, 8.2(8); Light, 3.8(4).

Compound 66:

SEHQLLHD KGKSIQDLRR RELLEKLLEK LHTA- NH,

(sequence identity no.: 80)

Physical data: mp. 134.2°C [a]„<25>-48.12 (c 0.36, H2O) FAB (C167H286N5t0„): [M+H]<+>3834.4

AAA: Asx, 2.0(2); Glx, 5.7(6); See, 1.7(2); His, 2.9(3);

Gly, 1.0(1); Thr, 0.9(1); Ala, 1.0(1); Arg, 2.8(3);

Ile, 0.9(1); Leu, 7.4(8); Light, 4.3(4).

Compound 67:

LLHD KGKSIQDLRR RELLEKLLEK LHTA- NH?

(sequence identity no.: 81)

Physical data: m.p. 128.5-184.5°C [o]D<25>-6.53 (c 0.69,

MeOH)

FAB (C148<H>259N47<Q>41): [M+H]<+>3353

AAA: Asx, 2.0(2); Glx, 4.1(4); Ser, 0.9(1); His, 2.1(2);

Gly, 1.0(1); Thr, 0.9(1); Ala, 1.0(1); Arg, 3.0(3);

Ile, 1.0(1); Leu, 8.1(8); Light, 4.2(4).

Compound 68:

LHD KGKSIQDLRR RELLEKLLEK LHTA- NH,

(sequence identity no.: 82)

Physical data: mp. 165-210°C [a]D<25>-36.05 (c 0.12, Ha0) FAB (<C>142<H>248N46<0>40): [M+H]<+>3239, 0

AAA: Asx, 2.0(2); Glx, 3.9(4); Ser, 0.9(1); His, 1.9(2);

Gly, 1.0(1); Thr, 1.0(1); Ala, 1.0(1); Arg, 2.9(3);

Ile, 0.9(1); Leu, 6.8(7); Light, 4.2(4).

Compound 69:

SEHQLLHD RGRSIQDLRR RELLERLLER LHAGRRTRSAW- OH

(sequence identity no.: 83)

Physical data: m.p. 150-210<8>C [a]D<25>-18.0 (c 0.64, H2O) FAB (C206H351N79<0>60): [M+H]* 4918.6

AAA: Asx, 2.2(2); Glx, 6.2(6); See, 2.8(3); His, 3.1(3);

Gly, 2.2(2); Thr, 2.2(2); Ala, 2.2(2); Arg, 10.4(10);

Ile, 1.0(1); Leu, 8.0(8); Trp, 1.1(1).

Compound 70:

LLHD RGRSIQDLRR RELLERLLER LHAGRRTRSAW- OH

sequence identity no: 84)

Physical data: mp. 150-210°C [a]D<25>-41.70 (c 0.36, H2O) FAB (<C>189<H>324N72<0>52): [M+H]<+>4437, 14

AAA: Asx, 2.1(2); Glx, 4.1(4); See, 1.9(2); His, 2.0(2);

Gly, 2.1(2); Thr, 2.0(2); Ala, 2.1(2); Arg, 9.7(10);

Ile, 0.9(1); Leu, 7.4(8).

The side-chain cyclized analog (compound 57) was synthesized as above, except that N°-Boc-NE<->Fmoc-Lys and tf-Boc-N^-Fmoc-Asp were placed at positions 13 and 17, respectively. After completion of For the Boc amino acid synthesis, the resin was treated with 20% piperidine in DMF at room temperature for 30 minutes. The resin was filtered and washed with DMF, MeOH and CH 2 Cl 2 . The resin (1.1 g) was slurried in 10 mL DMF containing 250 mg PyBOP. The pH was adjusted to 8-9 with DIEA and the resin stirred for 1 hour. The resin was filtered, washed with DMF and CH 2 Cl 2 , and then reslurried in DMF. The coupling was repeated using 125 mg of PyBOP. The resin was filtered, washed with DMF, MeOH and CH 2 Cl 2 and dried. The resin was then treated with HF and the peptide purified as mentioned above.

Compound 57:

IN 1

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTA- NH,

(sequence identity no.: 71)

Physical data: m.p. 142.5-163.5°C [a]D<25>-34.31 (c 0.17, H2O) FAB (<C>17s<H>2g8<N>56050): [M+H]< +>3986.4

AAA: Asx, 1.9(2); Glx, 5.9(6); See, 1.8(2); His, 3.2(3);

Gly, 1.1(1); Ala, 2.0(2); Arg, 3.0(3); Thr, 1.0(1);

Choice, 1.1(1); Ile, 0.9(1); Leu, 8.0(8); Light, 4.0(4).

Example II

[Met<3*>, Ala35] compound 1, AVSEHQLLHDKGKSIQDLRRREL-LEK-LLEKLHTMA-NH2, was prepared and purified following the procedures of Example I. This polypeptide was converted

to the homoserine lactone in the following manner. The purified peptide (160 mg) was dissolved in 44% formic acid (4 ml). This solution was combined with a premixed solution of cyanobromide (700 mg) and phenol (1.6 mg) in 44% formic acid (4 ml) at 0<8>C. The solution was stirred at 0°C for 2 hours and at room temperature for 2 hours. The formation of the product was monitored by HPLC ("Vydac" C-18, 300 A, 4.6 x 250 mm), flow at

1.2 ml/min, gradient 25-45% acetonitrile in 0.1% TFA over 10 minutes. The turnover was complete within 4 hours. Half of the sample was concentrated and purified by preparative RP-HPLC ("Vydac" C-18, gradient 25-45% acetonitrile in 0.1% TFA). The homoserine lactone peptide fractions were combined and lyophilized, yielding 28 mg of white solid with >95% purity, compound 4.

Connection 4

AVSEHQLLHDKGKSIQDLRRRELELLEKLHTX

( X = hSerlac. sequence identity no. : 9)

Physical data: m.p. 138-142'C [α]D<25>-50.66° (c 0.1, H2O) FAB (C176H299N55052): [M+H]<*>4017.61

AAA: Asp, 2.1(2); Glu, 6.1(6); See, 1.8(2); His, 3.0(3);

Thr, 1.1(1); Ala, 1.1(1); Arg, 2.7(3); Choice, 1.0(1);

Ile, 1.0(1); Leu, 8.2(8); Light, 3.8(4); Gly, 1.09(1);

hSer, 1.09(1).

Likewise, compound 65 was prepared in accordance with this procedure.

Compound 65:

AVSEIQFX^ N KGKHLSSXjER VEWLRKKLQD VHNX,

sequence identity no: 79)

fX t = L-norleucine: X2■ = homoserine lactone)

Physical data: m.p. 166-176"C [a]D<25>-52.22 (c 0.25, H2O) FAB (C180HZB8NS4<0>50): [M+H]<+>4008.6

AAA: Asx, 3.1(3); Glx, 4.8(5); See, 2.9(3); His, 2.9(3);

Gly, 1.1(1); Ala, 1.1(1); Arg, 2.0(2); Choice, 2.7(3);

Phe, 1.0(1); Ile, 1.0(1); Leu+Nle 5.9(4+2); Light, 2.8(3).

Example III

To prepare the homoserinamide, the crude hSerlactone analog, compound 4, was concentrated and treated with 25 mL of saturated NH 3 in methanol. The solution was stirred at 0°C for 2 hours and at room temperature for 16 hours. The reaction was monitored by HPLC ("Vydac" C-18, 300 A, 4.6 x 250 mm, flow rate of 1.2 ml/min, gradient 20-45% acetonitrile in 0.1% TFA) and was complete within 18 hours. The solution was concentrated and purified by preparative RP-HPLC ("Vydac" C-18, gradient of 25-45% acetonitrile in 0.1% TFA). The homoserinamide peptide fractions were pooled and lyophilized to give 30 mg of white solid with >98% purity, compound 3.

Connection 3

AVSEHQLLHDKGKSIQDLRRRELELLEKLHTX- NH-,

( X = hSer. Sequence Identity No. : 8)

Physical data: m.p. 138-142'C [a]D<25>-45.97<*>(c 0.25, H2O) FAB <C176H30JNS6052): [M+H]<+>4033.9

AAA: Asp, 2.1(2); Glu, 6.1(6); See, 1.6(2); His, 2.8(3);

Gly, 0.97(1); hSer, 0.97(1); Thr, 1.0(1); Ala, 1.0(1); Arg, 2.9(3); Choice, 1.0(1); Ile, 1.0(1); Leu, 7.6(8); Light, 3.9(4).

Likewise, compounds 22, 23 and 28 were prepared by following this procedure.

Compound 22:

AVSEIQFLHN LGKHLSSLRR RELLEKLLEK LHNX-NH,

( sequence identity no. : 36) ( X°homoserine)

Physical data: mp. 69.4-128"C [a]D<2S>-43.93. (c 0.15, H2O) FAB (C179H302N560„): [M+H]<*>4022.9

AAA: Asx, 2.0(2); Glx, 4.9(5); See, 2.6(3); His, 2.8(3);

Gly, 1.0(1); Ala, 1.0(1); Arg, 3.0(3); Choice, 1.0(1);

Phe, 1.0(1); Ile, 0.9(1); Leu, 8.8(9); Light, 3,4(3).

Compound 23:

AVSEIQFLHN KGKHLSSLRR RELLEKLLEK LHNX- NH,

( sequence identity no. : 37) ( X => homoserine)

Physical data: mp. 87.1-142.1'C [a]D<25>-52.14 (c 0.41, H2O) FAB (Cl79H303<N>57049): [M+H]<*>4038

AAA: Asx, 2.1(2); Glx, 4.9(5); See, 2.7(3); His, 2.8(3);

Gly, 1.0(1); hSer, 1.0(1); Ala, 1.0(1); Arg, 3.0(3);

Choice, 1.1(1); Phe, 0.9(1); Ile, 0.9(1); Leu, 7.9(8);

Light, 3.7(4).

Compound 28:

AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAGRRX-NH,

f sequence identity no: 42) (X = homoserine)

Physical data: m.p. 80°C [a]D<25>-48.64 (c 0.09, H2O)

FAB (C193H334<N>70056): [M+H]<*>4530.0

AAA: Asx, 2.2(2); Glx, 6.1(6); See, 1.7(2); His, 3.0(3);

Gly, 1.9(2); hSer, 1.0(1); Thr, 1.0(1); Ala, 2.1(2);

Arg, 7.2(7); Choice, 0.8(1); Ile, 1.0(1); Leu, 8.4(8);

Light, 2.1(2).

The homoserine alkylamides were likewise prepared from the homoserine lactone by dissolving it in DMF containing an excess of the corresponding alkylamine. After stirring at room temperature for several days (the reaction was monitored by analytical HPLC), the mixture was evaporated to dryness and the peptide purified by preparative HPLC. Representative homoserine alkyl amides are compounds 55 and 56.

Compound 55:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX- NHCHXH,

fsequence identity no: 69) (X = homoserine)

Physical data: mp. (not determined) [a]„<25>(not determined) FAB (C1V8H306<N>56<O>52): [M<+>H]<+>4063.0

AAA: Asx, 2.1(2); Glx, 5.8(6); See, 1.7(2); His, 3.1(3);

Gly, 0.9(1); Thr, 1.0(1); Ala, 0.9(1); Arg, 3.0(3);

Choice, 1.1(1); Ile, 1.0(1); Leu, 8.4(8); Light, 3.7(4);

hSer, 0.9(1).

Compound 56:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX- NHCH7CHTCffH«

( sequence identity no : 70) ( X = homoserine)

Physical data: m.p. (not determined) [a]D<25>(not determined) FAB (C1M<H>310N56052): [M+H]<+>4138.8

AAA: Asx, 2.0(2); Glx, 5.9(6); See, 1.7(2); His, 2.9(3);

Gly, 0.9(1); Thr, 1.0(1); Ala, 0.9(1); Arg, 3.0(3);

Choice, 1.0(1); Ile, 0.9(1); Leu, 8.0(8); Light, 4.1(4);

hSer, 0.9(1).

Example IV

The cesium salt of Na-Boc-NT-Fmoc-L-2,4-dlaminobutyric acid was attached to Merrifield resin (DMF, 50°C, 48 h) and used in a solid phase synthesis in place of the Boc-Ala resin as in Example I After completion of the synthesis, the peptide was treated with 20% piperidine in DMF at room temperature for 30 minutes to remove the FMOC side chain protecting group. The protected peptide spontaneously cyclized to the lactam, thereby cleaving itself from the resin. The solution was filtered from the resin and evaporated under vacuum to give an oil. The residue was treated with liquid HF as in example I, whereby the crude, unprotected peptide was obtained. The peptide was treated and purified as in Example I.

Compound 49:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX

( sequence identity no. : 63) ( X = L- 2. 4- diaminobutyryl lactam)

Physical data: m.p. 161-181'C [o]D" -48.38 (c 0.25, H2O) FAB (C176H300N56<O>51): [M+H]<+>4016.8

AAA: Asx, 2,1{2); Glx, 6.3(6); See, 1.7(2); His, 3.3(3);

Gly, 1.1(1); Thr, 1.0(1); Ala, 2.1(2); Arg, 2.9(3);

Choice, 0.9(1); Ile, 0.9(1); Leu, 8.0(8); Light, 3.8(4).

Example V

An aqueous solution of the homoserine lactone analog of Example II was treated with porcine liver esterase (Sigma Chemical Company, St. Louis, MO). The hydrolysis of the lactone to the C-terminal homoserine was monitored by analytical HPLC. After the hydrolysis was judged to be complete, the material was purified by preparative HPLC as in Example

IN.

Compound 37:

AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX- OH

( SEQ ID NO : 51) ( X = homoserine)

Physical data: m.p. (not determined) [oc]D<25>(not determined) FAB (<C>176<H>301<N>55053): [M+H]<+>4035.1

AAA: Asx, 2.1(2); Glx, 5.9(6); Looking, 2.0(2); His, 3.1(3);

Gly, 0.8(1); hSer,'0.8(1); Thr, 1.0(1); Ala, 1.0(1);

Arg, 3.0(3); Choice, 1.3(1); Ile, 1.0(1); Leu, 8.1(8);

Light, 3.8(4).

Example VI

Following Example I, the protected peptide resin BocAVS(Bzl)E(OBz)H(Bom)QLLHD(OBzl)R(Ts)GR(Ts)S(Bzl)IQD-(OBz)LR(Ts)R( Ts)E(OBz)LLe(OBzl)R(Ts)LLK(Fmoc)R(Ts)LH(Bom)T-(Bzl)A- O -PAM synthesized on a 0.35 mmol scale. All Na groups were protected with t-butoxycarbonyl (Boe); side chain protecting groups were as indicated. After completion of the synthesis, the peptide resin was treated with 50 mL of 20% piperidine in dimethylformamide (DMF) at room temperature for 30 min to remove the fluorenylmethoxycarbonyl) (Fmoc) protecting group on lysine. The resin was washed successively with DMF, MeOH, CH 2 Cl 3 and dried to give 1.6 g of partially protected peptide. 0.8 g (0.175 mmol) of the partially protected peptide was acylated on lysine with 0.44 g (0.3 mmol) methoxydi(ethyleneoxy)acetic acid [PE6(2)CHZC00H] in the presence of 0.16 g (0. 3 mmol) benzotriazolyloxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBop) and 0.067 g (0.525 mmol) diisopropylethylamine (DIEA) in 20 ml DMF at room temperature for 5 hours. After 5 hours, the resin was filtered and washed successively with DMF, MeOH and CH 2 Cl 2 . The acylation step was repeated twice until a negative ninhydrin result was obtained on the resin. The final peptide was cleaved from the resin with removal of the side chain protecting groups and purification as in Example I; 100 mg of compound 19 was obtained.

Connection 19

AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA- OH

(sequence identity no.: 18)

CHjO (CHiCHjO)jCH,C=0

Physical data: mp. 145-195'C [α]D<25>-44.60° (c 0.2, H2O) FAB (C183<H>316N64<0>54): [M+H]<+>4276.2

AAA: Asp, 2.1(2); Glu, 5.0(5); See, 1.6(2); His, 2.9(3);

Gly, 0.9(1); Thr, 1.9(2); Arg, 7.1(7); Choice, 1.1(1);

Ile, 1.0(1); Leu, 8.0(8); Light, 0.9(1).

Example VII

Peptide was synthesized, cleaved and purified in the same manner as in Example IV, except that 2-methoxypoly(ethyleneoxy)acetic acid [PEG(5000)CH 2 CO 2 H] was used as the acylating agent. 0.8 g (0.175 mmol) of the partially protected peptide gave 300 mg of pure compound 20.

Connection 20

AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA- OH

sequence identity no.: 19)

CH,0 (CH,CH:0) „0CH3C=0

Physical data: m.p. 105°C [a]D<25>-22.95° (c 0.11,

50% aqueous HOAc)

AAA: Asp, 2.0(2); Glu, 4.8(5); See, 1.6(2); His, 2.6(3);

Gly, 1.1(1); Thr, 1.1(1); Arg, 7.3(7); Choice, 0.8(1);

Ile, 0.9(1); Leu, 8.3(8); Light, 1.1(1); Ala, 1.8(2).

Example VIII

Synthesis of the hPTHrp(l-34)-analogue

A synthetic gene encoding the hPTHrp(1-34) analog, compound 4 (SEQ ID NO: 9), was designed with the nucleotide sequence and enzyme restriction sites shown in Figure 1. The required oligodeoxynucleotides were prepared with a DNA synthesizer (Milligen/Biosearch) using the phosphoramidite method of Sinha et al., Nucleic Acid Research 12, 4539-4557 (1984). After deprotection, the crude oligonucleotides were purified by gel electrophoresis on preparative 15% polyacrylamide gels. The oligonucleotides were localized by UV, removed from the gel, desalted over Waters C18 "Sep-pak" cartridges and lyophilized.

Polymerase chain reaction (PCR) amplification was performed on a Perkin-Elmer Cetus thermal cycler with 25 cycles of: 94°C for 1 minute, 50°C for 2 minutes, and 72°C for 3 minutes, using reagents including Taq-poly -merase, from the "GeneAmp" DNA amplification kit (Perkin-Elmer Cetus).

Two overlapping oligonucleotides, one 88-mer (2 µg), PTH3, (SEQ ID NO: 31):

was prepared as the template DNA sequence for the hPTHrp(1-34) analog gene. Using the two flanking primers, PTHPCR1: CCTCTAGATC TCCGCGGCGC TAG (SEQ ID NO: 33) and PTHPCR2: CCTCGAAGCT TATGCATCAT TATC (SEQ ID NO: 34), the entire gene was amplified by PCR. The amplified DNA products were purified by gel electrophoresis on 4% "NuSieve" agarose gel. The band containing the synthetic hPTHrp(l-34) analog, approximately 150 bases long, was excised from the gel and approximately 200 ng of DNA isolated by "Elu-Quik" glass gel DNA extraction (Schleicher & Schuell, Keene,

NH).

Example IX

Molecular cloning of a hPTHrp( 1- 34U- analog gene

To subclone the hPTHrp(1-34) analog gene according to Example VI, 200 ng of the amplified DNA was isolated and cut using the restriction enzymes Hind III and Sac II. As shown in Figure 2, DNA was ligated to 2 µg of TrpLE 18 Prot (Ile3, Pro5) plasmid which had been previously digested with Hind III and Sac II.

The resulting plasmid, TrpLE 18 hPTHrp(1-34)1, containing one copy of the hPTHrp(1-34) analog gene, was then transformed into competent E. coli HB 101 cells (CLONTECH, Palo Alto, CA ). Transformants were subjected to PCR analysis to verify insertion. Transformed cell colonies were picked and boiled in 200 µl water for 5 min; 2 µl was subjected to PCR with two primers flanking the insert. The PCR product was then analyzed on 1% agarose gel to confirm the presence of one copy of the hPTHrp(1-34) gene sequence. The TrpLE 18 hPTHrp(1-34)1 construct was then verified by DNA sequencing on an automated DNA sequencer (Applied Biosystems model 373A, Foster City, CA) using the supplier's "Dye Deoxy Terminator" sequencing kit.

Example X

Construction of a Trp LE 18-weight or containing multiple copies of the hPTHrp(1-34) analog

Unique Nhe I and Xba I restriction sites are located near the beginning and end of the hPTHrp(1-34) analog sequence. These two sites, which recognize different sequences but yield identical single-stranded cohesive ends, enable the construction of the multiple copy hPTHrp(1-34) genes within the Trp LE 18 vector.

The strategy for the construction of successive hPTHrp(1-34) repeated sequences is outlined in Figure 5. In separate reactions, 5 µg of plasmid Trp LE 18 hPTHrp(1-34)1 containing a single copy of the gene was cleaved with Bam HI + Nhe I and Xba I + Bam HI. From each digestion approx. 300 ng of the fragment containing the hPTHrp(1-34) analog gene, isolated. These two fragments were mixed and ligated, creating the Trp LE 18 hPTHrp(1-34)2 plasmid. This plasmid was used to transform competent E. coli HB 101 cells. size sorting of the transformed PCR products on 1% agarose gel was used to determine the presence of two copies of the hPTH(1-34) gene insertion. TrpLE 18 hPTHrp(1-34)2 containing two copies of the gene was then confirmed by DNA sequencing. The correct fusion of the two hPTHrp(1-34) genes results in the elimination of Nhe I and Xba I sites at the junction. This makes the remaining Xba I and Nhe I sites flanking the tandem genes unique.

By repeating this procedure, the final plasmid Trp LE 18 hPTHrp(1-34)4 containing four copies of the hPTHrp(1-34) gene was constructed, as shown in Figure 4. The sequence of Trp LE 18 hPTHrpt1-34)4 was found to be correct by DNA sequence analysis.

Example XI

Expression and purification of Trp LE 18 hPTHrpf1- 34) 4

Induction of Trp LE 18 hPTHrp( 1- 34) 4

A starter culture of 50 ml LB culture medium, J.H. 3 Miller, "Experiments in Molecular Genetics", p. 431 (1972), containing 50 µg/ml ampicillin and 100 µg/ml tryptophan, was inoculated with E. coli cells containing Trp LE 18 hPTHrp(l-34)4- plasmid, and grown overnight at 37°C with vigorous shaking until an Asso value of about 6. Two liters of the LB culture medium for production were pre-warmed to 37°C and inoculated with 20 ml of the starter culture, whereby got an A550 value of approx.

0.06. The culture was then grown with vigorous shaking until an AS50 value of between 0.6 and 0.8, after which 2 ml of a 10 mg/ml

solution of indole acrylic acid (IAA) was added. Cultivation continued with good aeration for approx. 16 hours until a final A550 value of approx. 6 (usually between 4 and 10). The cells were concentrated by centrifugation and resuspended in 500 ml of 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA buffer solution (Tris buffer).

The suspension was sonicated using a Heat<0>Systems-Ultrasonics, Inc. model 220F sonicator (equipped with a 1.9 cm funnel) operated at 50% of full capacity to avoid overheating.

To determine the extent of induction, the whole cells were analyzed by SDS-PAGE. The gene products derived from the Trp<5>LE 18 hPTHrp(1-34)4 construct were seen as a major band of the predicted molecular weight of approximately 17,000. This constitutes as much as 10% of the total cellular protein.

Isolation of the fusion protein

<0>The cell lysate was centrifuged for 15 minutes at approx.

3600 x g to pellet the Trp LE 18 hPTHrp(1-34)4 fusion protein; the supernatant was discarded. The pellet was resuspended in 200 ml Tris buffer (typically 40-80 A^/ml).

5 Example XII

Processing of the fusion protein and purification of homo-Serlactone-hPTHrp(1-34)-peptide

Cleavage of the methionine residues flanking the multiple fusion protein hPTHrp(1-34) with CNBr releases the desired homo-Serlactone hPTHrp(1-34) polypeptide which was purified as described above.

CNBr treatment of fusion protein

The washed pellet of TrpLE 18 hPTHrp(1-34)4 fusion protein was resuspended by gentle agitation in 60 ml of 70% formic acid (approximately 20 mg/ml total protein; typically, material from 1000 A550 units of cells is dissolved in 3 ml). A few drops of octanol were added and N 2 bubbled through the solution for 20 minutes before adding 5.5 g of CNBr. This reaction was allowed to proceed for 6 hours at 25°C before an equal volume of 50:50 MeOH:H 2 O was mixed with the sample and then removed by rotary evaporation. After 2 to 4 repetitions of this procedure, the bulk of the formic acid and CNBr were substantially removed. The sample was then evaporated to dryness, redissolved in 200 ml of water and lyophilized for storage.

Purification of homo- Serlactone- hPTHrp( 1- 34)

The CNBr-cleaved supernatant was dialyzed against 50 mM ^2^4, pH 6.5, for 24 hours with several shifts. During dialysis, the pH was maintained at 6.5. After dialysis, the precipitates were removed by high-speed centrifugation. The supernatant was cleared through a Gelman 0.45 µm filter device ("Acrodisc 4184").

Cat in onbvtterchromato f i

Initial purification was performed by cation exchange chromatography on a "Bio-Gel TSK-SP-5PW" HPLC column (21.5 x 150 mm). Chromatographic conditions for a flow rate of 8 ml/min and a yield of approximately 12 mg of highly purified homo-Serlactone-hPTHrp(1-34) peptide were:

1. Column equilibration in 50 mM KH 2 PO 4 , pH 6.5.

2. Add 10 ml of clarified supernatant (approximately 1.5 L of culture fluid or 2.4 g of inclusion). 3. Wash the column with 50 mM KH2PO4, pH 6.5, containing 50 mM NaCl until the baseline is stabilized. 4. Elute the column with 50 mM KH 2 PO 4 , pH 6.5, containing 90 mM NaCl. Collect fractions for approx.

45 minutes.

5. Analyze the 90 mM NaCl fractions for homo-Serlactone-hPTHrp(1-34) content by C18-HPLC prior to pooling and storage.

Reverse phase HPLC chromatography

A reverse phase "Poros" R/M 4.6 x 100 mm column (Perseptive Biosystems, Cambridge, MA) was used for the final purification step. The chromatographic conditions were as follows:

Mobile phase A: 0.1% trifluoroacetic acid (TFA)/water

B: 0.1% trifluoroacetic acid (TFA)/CH 3 CN

Retention time for the homo-Serlactone hPTHrp(1-34), compound 4, was approximately 2.943 minutes. The purified peptide was approximately 98% pure as determined by mass spectroscopy.

Example XIII

The compounds of this invention were evaluated for their effect on bone mass in ovariectomized rats, generally in accordance with the methods of Gunness-Hey and Hock, Metab. Bone Dis. 5:177 181 (1984).

Adult female Sprague-Dawley rats were acclimated, weight-grouped (n = 9, 10, or 12) and subjected to bilateral ovariectomy (OVX) or sham surgery. Dosing was started 17 days after surgery and continued for 20 days. Test compound was administered subcutaneously once per day in 2% rat serum/physiological saline as vehicle.

After 20 days of dosing, the rats were euthanized and the right femurs were removed. The femurs were cut in half, and the lower half of the femurs (DHF) were further divided into trabecular bone (TB) and cortical bone (CB) by drilling out the trabeculae. Calcium was extracted and measured using the "Calcette" calcium analyzer and expressed as mean bone Ca in mg/DHF/100 g body weight.

The two-sample t-test was used to compare OVX and blank groups. One-way ANOVA was used to compare OVX groups, followed by Fisher's LSD multiple comparison to compare each treatment group with vehicle.

Ovariectomy induced essentially total bone loss, primarily from trabecular bone. Total bone calcium was 47-54% lower than for blind operated controls.

bPTH(l-34) and hPTHrp(l-34) at 80 ug/kg/day produced statistically significant increases in total bone calcium for treated OVX rats, varying from 53-95% and 18-40%, respectively; however, there was no significant increase in cortical bone calcium.

Compounds of this invention, dosed at 80 ug/kg/day, increased total bone calcium by 66 to 138% and trabecular calcium by 87 to 128%. Cortical bone calcium, trabecular thickness and bone volume were also significantly increased compared to untreated OVX controls.

In this analysis, the following compounds were tested:

In similar investigations, ovariectomized rats were dosed for 5, 10 and 20 days at 40 ug/kg/day with the following results:

Example XIV

Toxicity

In Example XI above, no toxic effects were observed with the compound according to the invention.

Claims (12)

1. Synthetic polypeptide analogue of parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrp) or a salt thereof, characterized in that the amino acid residues (22-31) in PTH or PTHrp are modified so that they form an amphipathic ct-helix, where the sequence of the residues (22 -31) is selected from: Xaa<22> and Xaa<2S> are independently Glu, Glu(OCH3), His or Phe; Xaa<23> is Leu or Phe; Xaa<26> is Lys or His; Xaa<28> and Xaa<31> are independently Leu or Ile; Xaa<29> is Ala, Arg or Glu; and Xaa<30> is Lys or Glu (SEQ ID NO: 85); wherein in a compound where Xaa<22>is Phe; Xaa<23> is Phe; Xaa<25> is His; Xaa<26> is Lys or His; Xaa<28> is Ile; Xaa<29> is Ala; and Xaa30 is Glu; is Xaa31Leu; Xaa<22> and Xaa<25> are independently Glu, Glu(OCH3), His or Phe; Xaa<23> is Leu or Phe; Xaa<29> is Glu, Lys or Lys (COCH2PEGX) and PEGX is a poly-(ethylene glycol methyl ether) radical with a molecular weight of 100 to 10,000 (SEQ ID NO: 86); 2. Polypeptide according to claim 1, characterized in that it has the formula: Xaa is Glu or Arg (SEQ ID NO: 26). 3. Polypeptide according to claim 1, characterized in that it has the formula: Xaa is Glu, Lys or Lys (COCH2PEGX) and PEGX is a poly-(ethylene glycol methyl ether) radical with a molecular weight of 100 to 10,000 (SEQ ID NO: 27). 4. Polypeptide according to claim 1, characterized in that it has the formula: Xaa<1>Xaa2 Xaa3 Xaa<4>Xaa<5>Xaa6 Xaa7 Leu His Xaa10Xaa11 Gly Xaa<13>Ser Ile Gin Xaa<17>Leu Xaa" Xaa<20>Xaa<21>Xaa< 22-31>Xaa<32>Xaa<33>Xaa<34>Xaa<35>Xaa<36>Xaa<37>Xaa<38>Term, where: Xaa<1> is absent or is Ala; Xaa2 is absent or is Val; Xaa3 is absent or is Ser; Xaa<4> is absent or is Glu or Glu(0CH3); Xaa<5> is absent or is His or Ala; Xaa6 is absent or is Gin; Xaa<7> is absent or is Leu,- Xaa<10> and Xaa<17> are independently Asp or Asp(0CH3); Xaa<11> is Lys, Arg or Leu; Xaa<13> is Lys, Arg, Tyr, Cys, Leu, Cys (CH2C0NH (CH2) 2NH (biotinyl)), Lys(7-dimethylamino-2-oxo-2H-1-benzopyran-4-acetyl) or Lys (d i hydro c i nnamoy 1) Xaa<20> is Arg or Leu; Xaa<19> and Xaa<2X> are independently Lys, Ala or Arg; Xaa<22>"<31> is selected from the sequences of claim 1; Xaa<32> is His, Pro or Lys; Xaa<33> is absent or is Pro, Thr, Glu or Ala; Xaa<34> is absent or is Pro, Arg, Met, Ala, hSer, hSerlactone, Tyr, Leu or 1,4-diaminobutyryl lactam; Xaa<35> is absent or is Pro, Glu, Ser, Ala or Gly; Xaa<36> is absent or is Ala, Arg or Ile; Xaa<37> is absent or is Arg, Trp or 3-(2-naphthyl)-L-alanine; Xaa<38>is absent or is Ala or hSer, or Xaa<38>"<42>is Thr Arg Ser Ala Trp; and Term is OR or NR2wherein each R is independently H, (C1-C4) alkyl or phenyl (C1-C4)alkyl; and pharmaceutically acceptable salts thereof. 5. Polypeptide according to claim 4, characterized in that Xaa<22>"<31> is the sequence according to claim la), and where Xaa<11> and Xaa<13> are both Lys, and Xaa19 and Xaa<21> are both Arg. 6. Polypeptide according to claim 5, characterized in that it is: 7. Polypeptide according to claim 5, characterized in that it is: 8. Polypeptide according to claim 5, characterized in that it is: 9. Polypeptide according to claim 5, characterized in that it is: 10. Pharmaceutical preparation, characterized in that it comprises an effective bone mass-increasing amount of a polypeptide according to claims 1-9 or a salt thereof, in mixture with a pharmaceutically acceptable carrier. 11. Process for solid-phase synthesis of a synthetic polypeptide analogue of parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrp) or a salt thereof, where the amino acid residues (22-31) in PTH or PTHrp are modified so that they form an amphipathic α-helix, the sequence until the residues (22-31) are selected from Xaa<22> and Xaa<25> are independently Glu, Glu(OCH3), His or Phe; Xaa<23> is Leu or Phe; Xaa<26> is Lys or His; Xaa28 and Xaa<31> are independently Leu or Ile; Xaa<29> is Ala, Arg or Glu; and Xaa<30> is Lys or Glu (SEQ ID NO: 85); wherein in a compound where Xaa<22>is Phe; Xaa23 is Phe; Xaa<25> is His; Xaa<26> is Lys or His; Xaa<28> is Ile; Xaa29 is Ala; and Xaa<30> is Glu; is Xaa<31>Leu; Xaa<22> and Xaa<25> are independently Glu, Glu(OCH3), His or Phe; Xaa<23> is Leu or Phe; Xaa<29> is Glu, Lys or Lys (COCH2PEGX) and PEGX is a poly-(ethylene glycol methyl ether) radical with a molecular weight of 100 to 10,000 (SEQ ID NO: 86); characterized in that protected amino acids are linked sequentially on a suitable resin carrier, the side chain and the N<*1-> protecting groups are removed and the polypeptide is cleaved from the resin carrier. 12. Use of a compound according to claims 1-9 or a pharmaceutically acceptable salt thereof, for the preparation of a drug for the treatment of mammalian conditions characterized by reductions in bone mass, in particular for the treatment of osteoporosis.