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Glucagon-like peptide-2 (GLP-2) agonists have therapeutic potential in clinical indications in which the integrity or absorptive function of the intestinal mucosa is compromised, such as in short bowel syndrome (SBS). Native hGLP-2, a 33-amino acid peptide secreted from the small intestine, contributes to nutritional absorption but has a very short half-life because of enzymatic cleavage and renal clearance and thus is of limited therapeutic value. The GLP-2 analog teduglutide (Revestive/Gattex; Shire Inc.) has been approved for use in SBS since 2012 but has a once-daily injection regimen. Pharmacokinetic (PK) and pharmacodynamic studies confirm that apraglutide, a novel GLP-2 analog, has very low clearance, long elimination half-life, and high plasma protein binding compared with GLP-2 analogs teduglutide and glepaglutide. Apraglutide and teduglutide retain potency and selectivity at the GLP-2 receptor comparable to native hGLP-2, whereas glepaglutide was less potent and less selective. In rat intravenous PK studies, hGLP-2, teduglutide, glepaglutide, and apraglutide had clearances of 25, 9.9, 2.8, and 0.27 ml/kg per minute, respectively, and elimination half-lives of 6.4, 19, 16, and 159 minutes, respectively. The unique PK profile of apraglutide administered via intravenous and subcutaneous routes was confirmed in monkey and minipig and translated into significantly greater in vivo pharmacodynamic activity, measured as small intestinal growth in rats. Apraglutide showed greater intestinotrophic activity than the other peptides when administered at less-frequent dosing intervals because of its prolonged half-life. We postulate that apraglutide offers several advantages over existing GLP-2 analogs and is an excellent candidate for the treatment of gastrointestinal diseases, such as SBS. SIGNIFICANCE STATEMENT: Apraglutide is a potent and selective GLP-2 agonist with an extremely low clearance and prolonged elimination half-life, which differentiates it from teduglutide (the only approved GLP-2 agonist). The enhanced pharmacokinetics of apraglutide will benefit patients by enabling a reduced dosing frequency and removing the need for daily injections., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Glucagon-like peptide-2 receptor agonists have therapeutic potential for the treatment of intestinal diseases. The native hGLP-2, a 33 amino acid gastrointestinal peptide, is not a suitable clinical candidate, due to its very short half-life in humans. In search of GLP-2 receptor agonists with better pharmacokinetic characteristics, a series of GLP-2 analogues containing Gly substitution at position 2, norleucine in position 10, and hydrophobic substitutions in positions 11 and/or 16 was designed and synthesized. In vitro receptor potency at the human GLP-2, selectivity vs the human GLP-1 and GCG receptors, and PK profile in rats were determined for the new analogues. A number of compounds more potent at the hGLP-2R than the native hormone, showing excellent receptor selectivity and very low systemic clearance (CL) were discovered. Analogues 69 ([Gly(2),Nle(10),D-Thi(11),Phe(16)]hGLP-2-(1-30)-NH2), 72 ([Gly(2),Nle(10),D-Phe(11),Leu(16)]hGLP-2-(1-33)-OH), 73 ([Gly(2),Nle(10),D-Phe(11),Leu(16)]hGLP-2-(1-33)-NH2), 81 ([Gly(2),Nle(10),D-Phe(11),Leu(16)]hGLP-2-(1-33)-NHEt), and 85 ([Gly(2),Nle(10),D-Phe(11),Leu(16)]hGLP-2-(1-33)-NH-((CH2)2O)4-(CH2)2-CONH2) displayed the desired profiles (EC50 (hGLP-2R) < 100 pM, CL in rat <0.3 mL/min/kg, selective vs hGLP-1R and hGCGR). Compound 73 (FE 203799) was selected as a candidate for clinical development.

Kappa-(kappa) opioid receptors are widely distributed in the periphery and activation results in antinociception; however supraspinal acting kappa-agonists result in unwanted side effects. Two novel, all d-amino acid, tetrapeptide kappa-opioid receptor agonists, FE 200665 and FE 200666, were identified and compared to brain penetrating (enadoline) and peripherally selective (asimadoline) kappa-agonists as potential analgesics lacking unwanted central nervous system (CNS) side effects. In vitro characterization was performed using radioligand binding and GTP gamma S binding. Antinociception was evaluated in both mice and rats. Rotarod tests were performed to determine motor impairment effects of the kappa-agonists. FE 200665 and FE 200666 showed high affinity for human kappa-opioid receptor 1 (Ki of 0.24 nM and 0.08 nM, respectively) and selectivity for human kappa-opioid receptor 1 (human kappa-opioid receptor 1/human mu-opioid receptor/human delta-opioid receptor selectivity ratios of 1/16,900/84,600 and 1/88,600/>1,250,000, respectively). Both compounds demonstrated agonist activity in the human kappa-opioid receptor 1 [35S]GTP gamma S binding assay (EC50 of 0.08 nM and 0.03 nM) and resulted in dose-related antinociception in the mouse writhing test (A50: 0.007 and 0.013 mg/kg, i.v., respectively). Markedly higher doses of FE 200665 and FE 200666 were required to induce centrally-mediated effects in the rotarod assay (548- and 182-fold higher doses, respectively), and antinociception determined in the mouse tail-flick assay (>1429- and 430-fold fold higher doses, respectively) after peripheral administration supporting a peripheral site of action. The potency ratios between central and peripheral activity suggest a therapeutic window significantly higher than previous kappa-agonists. Furthermore, FE 200665 has entered into clinical trials with great promise as a novel analgesic lacking unwanted side effects seen with current therapeutics.

Using solid-phase methodology we have synthesised peptides based on the 8-14 or 6-14 human and rat angiotensinogen sequences, containing the following different isosteric units at the P1-P1' cleavage site: Leu-psi[CH2NH]Leu; Leu-psi[CH(OH)CH2]Val; Leu-psi[CH(OH)CH2]Leu and Leu-psi[CH(NH2)CH2]Val. In vitro, peptide Piv-His-Pro-Phe-His-Leu-psi[CH(OH)CH2]Leu-Tyr-Tyr-Ser-NH2(XXI) is the most potent inhibitor of rat plasma renin reported having an IC50 of 0.21 nM; it is a much weaker inhibitor of human renin (IC50 45 nM). Peptide Boc-His-Pro-Phe-His-Leu-psi[CH(OH)CH2] Leu-Val-Ile-His-NH2 (XX) was a highly effective inhibitor of rat renin in vivo. When infused (1 mg/kg/h) into two-kidney, one-clip chronic renal hypertensive rats, it lowered blood pressure and suppressed both plasma renin and angiotensin II. When given as a bolus (1 mg/kg) there was a divergence between the rapid rebound of renin levels and blood pressure, which remained suppressed. These results indicate that potent in vivo inhibitors of rat renin could be useful not only in examining the role of circulating renin but also in elucidating the equally important involvement of extracirculatory renin pools.

A series of renin inhibitors containing the dipeptide transition state mimics (2R,4S,5S)-5-amino-4-hydroxy-2-methyl-6-cyclohexyl hexanoic acid (Cha-psi[CH(OH)CH2]Ala) and (2R,4S,5S)-5-amino-4-hydroxy-2-isopropyl-6-cyclohexyl hexanoic acid (Cha-psi[CH(OH)CH2]Val) were prepared. A structure-activity study, using pseudopeptide (Boc-Phe-His-Leu-psi[CH(OH)CH2]Val-Ile-His-OH) as our lead structure, led to a new series of inhibitors, which correspond to tripeptides and contain no natural amino acids. For example, R,S-Bpma-Ape-Cha-psi[CH(OH)CH2]Ala-NH2 (IC50 = 1.26 nM against human plasma renin at pH 6.0; molecular weight = 564) has only two thirds of the molecular weight but twice the potency of our original lead. This new class of low molecular weight renin inhibitor displays excellent specificity toward human renin versus the related aspartic proteinase pepsin and angiotensin-1-converting enzyme. Examples are given of selected inhibitors showing encouraging evidence for intestinal absorption after intracolonic and oral administration in male Sprague-Dawley rats.

Solid-phase methodology has been used to synthesize a series of peptides based on the N-terminal sequence of human angiotensinogen in which statine (Sta) or the novel analogues (3S,4S)-3,4-diamino- or (3R,4S)-3,4-diamino-6-methylheptanoic acid (Ads or R-Ads) and (3S,4S)-4-amino-3-aminomethyl- or (3R,4S)-4-amino-3-aminomethyl-6-methylheptanoic acid (Amd or R-Amd) replace either residue 10 or both residues 10-11 at the P1-P1' cleavage site. The synthesis of these novel analogues of statine together with biological results on the inhibition of human and rat renin by peptides derived from them is reported. The absolute stereochemistry of the (3S,4S) Ads was determined by an X-ray crystallographic analysis of its N gamma-Boc, B beta-Z, R(+)-1-methyl benzamide derivative. Peptide Boc-His-Pro-Phe-His-Sta-Val-Ile-His-NH2 (VI) is the best inhibitor of human renin containing Sta at position 10. However, peptides containing Ads and Amd gave better rat renin inhibitors than the corresponding Sta-containing peptides. Peptides Boc-His-Pro-Phe-His-Ads-Val-Ile-His-NH2 (VII) having Ads at position 10 had an IC50 of 12 nM against rat renin. Although Sta has come to be accepted as an isosteric replacement for a dipeptide unit rather than for a single amino acid residue, in our series of inhibitors Sta is more effective when replacing only the amino acid at position 10 in the natural angiotensinogen sequence. None of the peptides gave any effect in vivo in a hypertensive rat model.

Based on a tetrapeptide fragment [Pro387-Ser390] of HK we have developed a series of potent low molecular weight (5-600 Da) inhibitors of PK which are stable to the enzyme. These inhibitors show good selectivity for PK versus tissue kallikrein, thrombin and plasmin. Such inhibitors will help define the role of PK and kinins in human physiology and pathophysiology. They may also find clinical use in the treatment of diseases where kinins are important mediators.

1. Tissue kallikrein (TK) cleaves low molecular weight kininogen (LK) at two sites to release kallidin: site I (between Arg389 and Ser390) is a typical cleavage point for a trypsin-like enzyme whereas site II (between Met379 and Lys380) is unusual and unique to TK. In order to learn more about the structural requirements and mechanism of cleavage at site II, we studied the hydrolysis by TK of several synthetic LK fragments varying in length between 4 and 22 residues and containing either site II only or both sites I and II. 2. Blocking site I cleavage in LK fragments by substituting DArg for LArg at position 389 or omitting site I from the sequence still allowed cleavage to proceed at site II. Replacement or deletion of selected amino acid residues in these fragments demonstrated that the presence of Arg381 was essential for site II cleavage to occur whereas Pro383, Phe385 and Ser386 could be replaced with Ala without affecting binding or cleavage by TK. Ki values towards TK were determined for all LK fragments in order to compare their binding affinities to the enzyme. Short peptides containing site II only exhibited high Ki values (> or = 100 microM) whereas longer fragments containing both sites I and II had Ki values of 2-7 microM. 3. In order to bring sites I and II into close proximity spatially and thus facilitating efficient cleavage in the enzyme-substrate complex, we prepared several cyclic analogs of the longer LK fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

We have utilized the relative structural simplicity of several short, cyclic, highly active somatostatin analogs in the search for competitive antagonists of somatostatin. During an attempted synthesis of cyclo(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr), catalytic hydrogenation of the protected peptide intermediate unexpectedly gave cyclo [7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)] in which the benzyl protecting group on Thr could not be removed even upon prolonged treatment under standard conditions. Injection of this new peptide into the rat completely blocked the inhibitory effects of exogenous somatostatin on GH, insulin, and glucagon release. Indeed, in fasted rats, basal hepatic portal insulin and glucagon levels were significantly increased after analog treatment. Plasma GH levels in Nembutal-anesthetized and stimulated rats were also increased after injection of the analog. These results provide strong evidence that endogenous somatostatin exerts local tonic control of pituitary and pancreatic secretions. The availability of a somatostatin anatagonist should be of considerable value in elucidating the roles of somatostatin in these and many other physiological processes.

The effects of CRF, ACTH 1-24, alpha-MSH, and an ACTH 4-49 analog, at doses of 0, 0.1, 1, and 10 mg/kg, were tested on temperature, ptosis, and sedation in mice pretreated 18 hr previously with reserpine. IP injection of CRF at doses of 1 and 10 mg/kg significantly potentiated the reserpine-induced hypothermia while ACTH 1-24 at the same two doses had the opposite effect of significantly reversing the hypothermia as compared to diluent. The highest dose of alpha-MSH exerted a similar action to that of ACTH 1-24, but none of the doses of the ACTH 4-9 analog changed body temperature. beta-endorphin also failed to cause a reliable effect even though naloxone blocked the action of CRF on body temperature. The results suggest that CRF, like other hypothalamic peptides, can exert extra-pituitary actions after peripheral administration.

The presence of synthetic ovine corticotropin-releasing factor leads to a rapid and marked stimulation of adenosine 3', 5'-monophosphate accumulation in an enriched population of rat pituitary corticotrophs in primary culture. The increase, observed as early as 60 seconds after the addition of corticotropin-releasing factor, suggests that changes in the intracellular concentration of the cyclic nucleotide coincide with or precede the secretion of adrenocorticotropic hormone in response to corticotropin-releasing factor.

Human pancreatic growth hormone releasing factor (1-29)-amide [hpGRF (1-29)-NH2] and the following analogs: [D-Tyr-1]-hpGRF(1-29)-NH2, [D-Ala-2]-hpGRF(1-29)-NH2, [D-Asp-3]-hpGRF(1-29)-NH2, and [N-Ac-Tyr-1]-hpGRF (1-29)-NH2 were synthesized using solid phase methodology and tested for their ability to stimulate growth hormone (GH) secretion in the rat and the pig in vivo. [D-Ala-2]-hpGRF (1-29)-NH2 was approximately 50 times more potent than the parent molecule in eliciting GH secretion in the rat. The other analogs were less active, but all were more potent than the 1-29 amide in the rat. [D-Tyr-1]-hpGRF(1-29)-NH2 was 10 times more potent, [D-Asp-3]-hpGRF(1-29)-NH2 7 times more potent, and the acetylated molecule approximately 12 times more potent than hpGRF(1-29)-NH2.

The 41-residue sequence of recently identified ovine corticotropin-releasing factor (CRF) was assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptide was purified by gel filtration and reverse-phase, medium pressure, followed by high-performance liquid chromatography (HPLC). In addition to the usual criteria, the homogeneity of the final material, obtained in 7% yield, was assessed by the isolation and examination of cyanogen bromide cleavage and tryptic digestion fragments by HPLC and amino acid analysis. The synthetic 41 amino acid CRF stimulated the release of corticotropin (ACTH) in three in vitro systems: isolated rat pituitary quarters, monolayer cultures of dispersed pituitary cells, and superfused pituitary cells on a column, the responses being related to the log-dose of CRF in the range of 0.05-125 ng/ml. The synthetic peptide also augmented in vivo release of ACTH in rats pretreated with chlorpromazine, morphine, and Nembutal, as assessed by the measurement of serum corticosterone. The data indicates chemical purity and high biological activity of synthetic material.

Two related glycoproteins, fetuin in species of the order Artiodactyla (cattle, sheep, pig) and alpha 2HS glycoprotein in the human [(1987) Cell Tissue Res. 248, 33-41] have a very specific distribution in the developing brain. We have isolated and determined the first 15 N-terminal residues of a similarly distributed glycoprotein in the developing brain of the tammar wallaby (Macropus eugenii). The degree of homology is the same between wallaby glycoprotein and alpha 2HS glycoprotein as between fetuin and alpha 2HS glycoprotein (46%). Antibodies made to synthetic peptides of fetuin were used to identify the wallaby glycoprotein. A polyclonal antibody to the purified glycoprotein was used for immunocytochemical identification of brain cells positive for this protein.

Infusion of H.261, the inhibitor of human renin in the baboon, lowered blood angiotensin I, plasma angiotensin II, and arterial pressure suggesting that in the sodium-depleted state angiotensin II contributes to the maintenance of arterial pressure. In a second experiment dose-response infusions of angiotensin II were given in conscious sodium-depleted dogs before and during infusion of the renin inhibitor H.77. These suggested that the contribution of angiotensin II to the maintenance of arterial pressure in this state was made mainly by a circulating peptide. Preliminary results in normal humans show that infusion of H.142 intravenously lowered angiotensin I, angiotensin II, and arterial pressure.

Hydroxy-ethylene dipeptide analogues (Leu[CH(OH)-CH2]Leu and Leu[CH(OH)-CH2]Val) of human substrate peptides are potent in vitro inhibitors of rat renin with IC50 values as low as 0.8 nmol/l. When given to renal hypertensive rats they lower blood pressure and suppress both plasma renin and angiotensin II. There was a divergence between the rapid rebound of renin and blood pressure which remained suppressed.

Using solid-phase methodology and preparative medium- and high-performance reverse-phase liquid chromatography, we have synthesized glucagon and its Arg12 analogue in approximately 5% yields. The synthetic glucagon was fully active relative to natural material, and the Arg12 peptide exhibited 50% activity. Since perhaps the most critical part of the glucagon-family peptides is the N-terminal hexapeptide region, both batches of resin were split during synthesis in order to prepare two series of analogues based on glucagon and [Arg12]glucagon with changes in the His-Ser-Gln-Gly-Thr-Phe sequence. The following new analogues were tested for their effects on blood glucose levels in normal male rats relative to glucagon and gave the following activities: [Ac-His1,Arg12]glucagon, 46%; [3-Me-His1,Arg12]glucagon, 30%; [Phe1,Arg12 )glucagon, 31%; [Des-His1,Arg12]glucagon, 4%; [D-Ala2,Arg12]glucagon, 44%; [D-p-Cl-Phe1,D-Ala4,Arg12]glucagon, 9%; [D-Phe4]glucagon, 655%; [Ala2]glucagon, 9%. These data indicate that the amino or imidazole nitrogens of the histidine residue are not essential for biological activity. However, an aromatic group in position 1 may be important, since the Phe1 analogue is almost as active as glucagon in our bioassay. The superagonist activity with [D-Phe4]glucagon, which was synthesized to test the hypothesis that a beta-bend conformation occurs at this position in glucagon by analogy with luteinizing hormone-releasing hormone and other Gly-containing peptides, indicates that this is indeed the case and has important implications for the receptor-recognition requirements of the glucagon-secretin-vasoactive intestinal peptide family of peptides.

Synthetic human pancreatic growth hormone-releasing factor containing 40 amino acids ([hpGRF (1-40)]-OH) significantly stimulated plasma growth hormone (GH) levels in both sodium pentobarbital and urethane anesthetized rats. Synthetic secretin, gastric inhibitory polypeptide (GIP), and glucagon significantly decreased plasma GH levels while synthetic vasoactive intestinal peptide (VIP) had no effect. Secretin and GIP also altered the in vivo plasma GH response to [hpGRF(1-40)]-OH. Whether this effect is the result of an interaction at the pituitary level or is due to an extra-pituitary effect of secretin and GIP awaits further study.

In previous reports illustrating the effects of conformational restriction of the N-terminal region of human pancreatic growth hormone releasing factor, we demonstrated that D-amino acid substitutions in either of positions 1, 2, or 3 resulted in greatly increased growth hormone releasing activity both in vivo and in vitro. The most active compound, [D-Ala-2]GRF(1-29)NH2, was 51 times more active than the parent 29 amino acid peptide in the sodium pentobarbital anesthetized rat. These observations have now been extended to analogues containing multiple D-amino acid replacements in these three positions. Once again, peptides with superagonist potencies ranging from 1200% to 3800% were obtained after solid-phase synthesis and purification by medium-pressure reverse-phase liquid chromatography. In addition, it was found that [D-Asn-8]- and [D-Ala-4]GRF(1-29)NH2 were, respectively, 2.43 and 1.1 times more active than GRF(1-29)NH2 itself. In contrast, [D-Phe-6] and [D-Thr-7] analogues were virtually inactive. Chou-Fasman structural predictions suggest that the first three residues of the peptide assume no fixed type of conformation but that a reverse turn could be present between residues 6 and 10. Attempts are made to rationalize the biological results with these calculations. The effects of other side chains on the D-amino acid in position 2 were also investigated. Both the Ac-[D-Phe-2]- and Ac-[D-Arg-2]peptides had very low activity. Several of the inactive peptides were tested as possible antagonists of GRF; however, none was able to block the stimulatory effects of GRF(1-29)NH2 after combined administration.

In this study, we determined the ability of four N-terminally modified derivatives of glucagon, [3-Me-His1,Arg12]-, [Phe1,Arg12]-, [D-Ala4,Arg12]-, and [D-Phe4]glucagon, to compete with 125I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, [3-Me-His1,Arg12]glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. [Phe1,Arg12]glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. [D-Ala4,Arg12]glucagon and [D-Phe4]glucagon, analogues designed to examine the possible importance of a beta-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. [D-Ala4,Arg12]glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the [D-Phe4] derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal beta-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists.(ABSTRACT TRUNCATED AT 250 WORDS)