compound to reduce levels of parathyroid hormone, composition containing said compound and use of it

专利摘要:
THERAPEUTIC AGENTS FOR REDUCING PARATHYROID HORMONE LEVELS. Compounds that have activity to reduce parathyroid hormone levels are described. In one embodiment, the compounds are composed of a contiguous sequence of subunits, X1 - X2 - X3 -X4 -X5 -X6 -X7, where the X1 subunit comprises a thiol-containing portion and the charge distribution in the Xz-X7 subunits provides the desired activity. Methods of using the compounds for the treatment of hyperparathyroidism, bone disease and / or hypercalcemic disorders are also described and, in particular, methods for reducing serum serum PTH and calcium are provided. The compounds can be used to treat individuals who have, for example, primary, secondary or tertiary hyperparathyroidism; hypercalcemia of malignancy; metastatic bone disease; or osteoporosis.
公开号:BR112012002143B1
申请号:R112012002143-5
申请日:2010-07-29
公开日:2020-12-08
发明作者:Felix Karim；Amos Baruch；Derek MacLean；Kanad Das；Qun Yin
申请人:Kai Pharmaceuticals, Inc；
IPC主号:

专利说明:

This claim claims the benefit of US Provisional Order No. 61 / 229,695, filed on July 29, 2009, and US Provisional Order No. 61 / 255,816, filed on October 28, 2009, and US Provisional Order No. 61 / 313,635, filed on March 12, 2010. Each of these orders is incorporated by reference in its entirety. REFERENCE TO THE LISTING OF SEQUENCES, TABLES OR COMPUTER PROGRAMS
A list of strings is being submitted electronically through EFS in the form of a text file, created on July 29, 2010 and called “632008017WO00seqHst.txt” (85,400 bytes), the content of which is incorporated by reference in its entirety. TECHNICAL FIELD
The current object of study is related to compounds with activity to decrease the levels of parathyroid hormone (PTH), to the pharmaceutical compositions that comprise the compounds and to the use of these compounds and compositions in treatment methods, which include, without limitation, the treatment hypercalcemia or hyperparathyroidism or modulation of PTH levels in vivo. BACKGROUND OF THE INVENTION
Calcium homeostasis is the mechanism by which the body maintains adequate calcium levels. The process is highly regulated, and involves a complex interaction between absorption, transport, storage in bones, deposition in other tissues, and calcium excretion. PTH is a regulator of circulating calcium levels, and works to increase the concentration of calcium in the blood by increasing the release of calcium from the bone through the process of bone resorption; increased calcium reabsorption by renal tubules; and increased absorption of calcium in the intestine by increasing the production of 1.25- (OH) 2 vitamin D, the active form of vitamin D. PTH also stimulates the excretion of phosphorus by the kidney and increases release through the bone.
PTH secretion is regulated by the calcium sensor receptor (CaSR), a receptor coupled to the G protein expressed by various types of cells on the surface of parathyroid cells, which detects small fluctuations in the concentration of extracellular calcium ion (Ca2 +) and accounts for alteration of PTH secretion. Activation of CaSR by Ca2 + inhibits PTH secretion in seconds to minutes by inhibiting vesicular transport, and this process can be modulated by phosphorylation by protein kinase C (PKC) of the receptor. CaSR is also expressed in osteoblasts and in the kidney, where it regulates renal Ca2 + excretion.
In addition, PTH regulates phosphorus homeostasis, PTH stimulates the parathyroid hormone receptor 1 (PTHR1) in both apical membranes (brush border membrane) and basolateral cells in the gastrointestinal tract. PTHR1 stimulation leads to an increase in urinary phosphate (Pi) excretion as a result of the reduction by internalization of the renal Na + / phosphate (NaPi-IIa) co-transporter in the brush border membrane.
PTH is also involved in the regulation of osteoblasts and osteoclasts in bone. PTH increases circulating Ca by increasing bone resorption and renal calcium reabsorption. PTH stimulates osteoblasts to produce RANK ligand (RANKL), which binds to the RANK receptor and activates osteoclasts, leading to an increase in bone resorption and an increase in serum Ca. Osteoprotegerin (OPG) is a receptor-bait for RANKL that blocks bone resorption. Osteoporosis is caused by an imbalance between bone resorption processes by osteoclasts and bone formation by osteoblasts.
The human body contains approximately 1 kg of calcium, 99% of which reside in the bone. Under normal conditions, the circulating calcium (Ca2 +) ion is kept strictly at a level of about 9 to 10 mg / dl (ie, 2.25-2.5 mmol / 1; approximately 600 mg). Approximately 1 g of elemental calcium (Ca2 +) is taken daily. Of that amount, approximately 200 mg / day is absorbed, and 800 mg / day is excreted. In addition, approximately 500 mg / day is released by bone resorption or is deposited in the bone. About 10 g of Ca2 + is filtered through the kidney a day, with about 200 mg appearing in the urine, and the rest being reabsorbed.
Hypercalcemia is a high level of calcium in the blood. Acute hypercalcemia can result in gastrointestinal symptoms (anorexia, nausea, vomiting); renal (polyuria, polydipsia), neuromuscular (depression, confusion, stupor, coma) and cardiac (bradycardia, first-degree atrioventricular). Chronic hypercalcemia is also associated with gastrointestinal symptoms (dyspepsia, constipation, pancreatitis); renal (nephrolithiasis, nephrocalcinosis), neuromuscular (weakness) and cardiac (blockage of hypertension, sensitivity to fingerprints). Abnormal cardiac rhythms may appear, and ECG findings from a short QT interval and a flooded T wave suggest hypercalcemia. Hypercalcemia can be asymptomatic, with symptoms occurring more commonly at high calcium levels (12.0 mg / dl or 3 mmol / 1). Severe hypercalcemia (above 15-16 mg / dl or 3.75-4 mmol / 1) is considered a medical emergency: at these levels, coma and cardiac arrest can occur.
Hypercalcemia is often caused by hyperparathyroidism, leading to excessive bone resorption and elevated serum calcium levels. In primary sporadic hyperparathyroidism, PTH is overproduced by a single parathyroid adenoma; less commonly, multiple adenomas or diffuse parathyroid gland hyperplasia may be the cause. The increased secretion of PTH leads to a net increase in bone resorption, with the release of Ca2 + and phosphate (Pi). PTH also increases renal Ca2 + reabsorption and inhibits phosphate (Pi) reabsorption, resulting in a net increase in serum calcium and a decrease in phosphate.
Secondary hyperparathyroidism occurs when a decrease in circulating levels of the Ca2 + level stimulates PTH secretion. One cause of secondary hyperparathyroidism is chronic kidney failure (also called chronic kidney disease or CKD) which occurs, for example, in polycystic kidney disease or chronic pyelonephritis, or chronic kidney failure, for example, which occurs in patients on hemodialysis (also called disease end-stage renal disease or ESRD). Excessive PTH can be produced in response to hypocalcemia resulting from low calcium intake, gastrointestinal disorders, renal failure, vitamin D deficiency and renal hypercalciuria. Tertiary hyperparathyroidism can occur after a long period of secondary hyperparathyroidism and hypercalcemia.
Malignancy is a common cause of hypercalcemia not mediated by PTH. Malignant hypercalcemia is an uncommon but severe complication of cancer, which affects between 10% and 20% of cancer patients, and can occur with both solid tumors and leukemia. The condition has an abrupt onset and a very poor prognosis, with a median survival of only six weeks. Growth factors (GF) regulate the production of parathyroid hormone-related protein (PTHrP) in tumor cells. Tumor cells can be stimulated by autocrine GF to increase PTHrP production, leading to increased bone resorption. Metastatic tumor cells to the bones can also secrete PTHrP, which can produce bone resorption and release additional GF which, in turn, act in a paracrine manner to further increase the production of PTHrP.
Consequently, compounds with activity to, for example, modulate PTH levels and / or calcium levels in vivo are desired. BRIEF SUMMARY OF THE INVENTION
In one aspect, a compound is provided which comprises the formula: X1 - x2 - x3 - x4 - x5 - x6 - x7 where Xx is a subunit comprising a thiol containing group; X5 is a cationic subunit; X6 is a non-cationic subunit; X7 is a cationic subunit; and at least one, preferably two, of X2, X3 and X4 are independently a cationic subunit; and in which the compound has activity to decrease the concentration of parathyroid hormone. In one embodiment, the decrease in the concentration of parathyroid hormone is a decrease in the blood or plasma concentration of parathyroid hormone in an individual treated with the compound, in relation to the blood or plasma concentration of parathyroid hormone in the individual prior to treatment. In another embodiment, the decrease in parathyroid hormone concentration is achieved in the absence of a histamine response.
In another modality X3 and X4 are non-cationic, while X1; X5, Xs and X7 are cationic.
In one embodiment, the X3 subunit is an amino acid residue that contains thiol, in another embodiment, the thiol group of the Xi subunit is an organic portion that contains thiol.
In another embodiment, when the Xx subunit is an amino acid residue containing thiol, it is selected from the group consisting of L-cysteine, D-cysteine, glutathione, n-acetylated cysteine, homocysteine and pegylated cysteine.
In yet another embodiment, the thiol-containing organic portion is selected from thiol-alkyl, or thioacyl moieties such as, for example, 3-mercaptopropyl or 3-mercaptopropionyl, mercaptopropionic acid, mercaptoacetic acid, thiobenzyl or thiopropyl. In yet another modality, the organic portion containing thiol is mercaptopropionic acid.
In yet another embodiment, the X3 subunit is chemically modified to comprise an acetyl group, a benzoyl group, a butyl group or another amino acid such as, for example, acetylated beta-alanine.
In yet another embodiment, when the X4 subunit comprises a thiol moiety, the Xi subunit is joined by a covalent bond to a second thiol moiety.
In another modality, the formula X3 - X2 - X3 - X4 - X5 - X6 - X7 is composed of a contiguous sequence of amino acid residues (here designated (Xaal) - (Xaa2) - (Xaa3) - (Xaa4) - (Xaa5 ) - (Xaa6) (Xaa7) - SEQ ID NO: 1) or a sequence of subunits of organic compound (residues that are not amino acids).
In another embodiment, the contiguous sequence of amino acid residues is a contiguous sequence of L-amino acid residues, a contiguous sequence of D-amino acid residues, a contiguous sequence of a mixture of L-amino acid residues and D-amino acid residues , or a mixture of amino acid residues and unnatural amino acid residues.
In another embodiment, the contiguous sequence of amino acid residues is linked to a compound to facilitate transport across a cell membrane. In another embodiment, the contiguous sequence of amino acid residues is linked to a compound that increases the release of the sequence in or through one or more layers of tissue.
In another embodiment, the contiguous sequence of amino acid residues is contained within a sequence of amino acid residues of 8-50 amino acid residues, 8- 40 amino acid residues, 8-30 amino acid residues or 8-20 amino acid residues of length. In yet another embodiment, the contiguous sequence of amino acid residues is contained within an amino acid residue sequence of 8-19 amino acid residues, 8-18 amino acid residues, 8-17 amino acid residues, 8-16 amino acid residues , 8-15 amino acid residues, 8-14 amino acid residues, 8-13 amino acid residues, 8-12 amino acid residues, 8-11 amino acid residues, 8-10 amino acid residues, or 8-9 amino acid residues of lenght.
In another embodiment, the X3 subunit is a cationic amino acid residue.
In another embodiment, the X2 subunit is a non-cationic amino acid residue, and in another modality, the X4 subunit is a non-cationic amino acid residue. In one embodiment, the non-cationic amino acid residue is a D-amino acid.
In another modality, X3 and X4 are cationic D-amino acid residues.
In another embodiment, the X5 subunit is a D-amino acid residue.
In another aspect, the contiguous sequence in any of the described compounds is covalently attached via the thiol containing group in the Xi subunit to a second contiguous sequence. For example, the second contiguous sequence may be identical to the contiguous sequence (to form a dimer), or it may be non-identical, as would be the case when attached to a portion that facilitates the transfer of the contiguous sequence across a cell membrane.
In another aspect, a conjugate formed by the peptide carrrar (SEQ ID. NO: 2) is provided, in which the peptide is conjugated at its N-terminal residue to a Cys residue.
In one embodiment, the peptide is chemically modified at the N-terminus, the C-terminus, or both.
In another embodiment, the N-terminus of the peptide is chemically modified by acetylation and the C-terminus is chemically modified by amidation.
In another embodiment, the conjugate is Ac-c (C) arr-NH2 (SEQ ID. NO: 3).
In another aspect, a method of treating secondary hyperparathyroidism (SHPT) is contemplated in an individual, in which a compound as described herein is provided to the individual, in various modalities, the individual may be suffering from chronic kidney disease or another condition.
In another aspect, a method of decreasing parathyroid hormone in an individual is contemplated, in which a compound as described herein is provided to the individual.
In another aspect, a treatment regimen is provided, the regimen comprising providing a compound according to any of those described herein, in combination with a second agent.
In one embodiment, the second therapeutic agent is vitamin D, a vitamin D analogue or cinacalcet hydrochloride.
In any of the aspects or modalities described herein, any one or more of the strings are contemplated to be individually excluded or removed from the scope of the claims. In certain embodiments, the peptides identified by any one or more of the IDS. SEQ. Nos: 162-182, individually or in any combination, are excluded from the claimed compounds, compositions and methods. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph of the parathyroid hormone level, as a percentage of the pre-dose baseline value, as a function of time, in hours, in rats with acute renal failure (model 1K1C), in which the rats were dosed with Accrrrr- NH2 (SEQ ID NO: 4, diamonds), Ac-crrrrr-NH2 (SEQ ID NO: 5, closed squares), Ac-crrrrrr-NH2 (SEQ ID NO: 6 , triangles), Ac-crrrrrrr-NH2 (SEQ ID NO: 7, open squares), or saline control (x symbols); FIG. 2A is a graph of the concentration of IPi, in nM, as a function of the compound concentration of Ac-carrrar-NH2 (SEQ ID. N °: 26, squares) and Ac-arrrar-NH2 (SEQ ID. N °: 29, triangles), as a measure of the compound's ability to activate human CaSR in an in vitro cell assay when human CaSR is expressed as a stable transfected HEK-293 cell line; FIG. 2B shows the reduction in PTH concentration after in vivo administration of peptides identified as ID. SEQ. N °: 26 (Ac-carrrar-NH2) (squares) and as ID. SEQ. No. 29 (Ac-arrrar-NH2) (diamonds), where the peptides were administered as an IV bolus to normal Sprague-Dawley rats at doses of 9 mg / kg for ID. SEQ. No. 29 and 0.5 mg / kg for ID. SEQ. No. 26. An intravenous (IV) saline bolus was used as a control (dashed line). Plasma PTH levels were assessed before dosing and 1, 2, 3 and 4 hours after dosing. The results are presented as group mean + standard deviation (SD), and PTH is shown as a percentage of the pre-dose baseline value; FIG. 3 is a bar graph comparing the release of histamine after bolus IV administration of various compounds in normal Sprague-Dawley rats, in which the compounds Ac-crrrr-NH2 (SEQ ID. NO: 4), Ac- crrrrrr-NH2 (SEQ ID NO: 5), Ac-crrrrrr-NH2 (SEQ ID NO: 6) and Ac-crrrrrrrr-NH2 (SEQ ID NO: 41) were dosed in an equimolar dose per bolus IV of 2.1 pmol / kg, and plasma histamine was measured before dosing (pre-dose), 5, 15 and 30 minutes after dosing; FIG. 4 is a bar graph comparing the release of histamine after IV bolus administration of two compounds in normal Sprague-Dawley rats, in which the compounds Ac-c (C) arr-NH2 (SEQ ID NO: 3 , crossed cross bars) and Ac-crrrrrr-NH2 (SEQ ID NO: 6, open bars) were measured at 3 mg / kg, and plasma histamine was measured before dosing (time zero) and 5, 15 and 30 minutes after dosing; FIG. 5 is a graph of the parathyroid hormone level, as a percentage of the pre-dose baseline value, as a function of time, in hours, in normal rats dosed with 0.5 mg / kg per IV bolus of Ac-crrrrrr-NH2 (ID SEQ NO: 6, diamonds), Ac-carrrrr-NH2 (SEQ ID NO: 8, squares), Ac-crarrrr-NH2 (SEQ ID NO: 9, triangles), Ac-crrrarr-NH2 (SEQ ID NO: 10, symbols x), Ac-crrrrr-NH2 (SEQ ID NO: 11, symbols *), Ac-crrrr-NH2 (SEQ ID. N °: 12, circles) or Ac-crrrrra-NH2 (SEQ ID. N °: 13, symbols +); FIGS. 6A-6B are graphs of the parathyroid hormone level, as a percentage of the pre-dose baseline value, as a function of time, in hours, in healthy rats dosed with 0.5 mg / kg per IV bolus of Ac-carrrar-NH2 ( SEQ ID NO: 26, open diamonds), Ac-crrarar-NH2 (SEQ ID NO: 25, open squares), Ac-caarrrr-NH2 (SEQ ID NO: 22, triangles), Ac-crraarr-NH2 (SEQ ID NO: 17, closed squares), Ac-c (C) arrr-NH2 (SEQ ID NO: 3, diamonds Fig. 6B), Ac -craarrr-NH2 (SEQ ID. N °: 24, x symbols in Fig. 6A); Ac-c (C) rrarar-NH2 (SEQ ID NO: 28, symbols x, Fig. 6B); FIG. 7 shows the decrease in the levels of parathyroid hormone in the blood as a function of time, for the compound Ac-c (C) arrrar-NH2 (SEQ ID NO: 3) administered as an IV bolus to Sprague-Dawley rats normal doses of 1 mg / kg (diamonds), 0.5 mg / kg (squares), 0.3 mg / kg (triangles), and 0.1 mg / kg (x symbols). An intravenous (IV) saline bolus (circles) was used as a control. Plasma PTH levels were assessed before dosing and at 1, 2, 3 and 4 hours after dosing; FIG. 8 is a graph of the parathyroid hormone level, as a percentage of the pre-dose baseline value, as a function of time, in hours, in rats with acute renal failure (model 1K1C), in rats with model 1K1C acute kidney failure, in that the rats were dosed by means of bolus IV with the compound Ac-c (C) arr-NH2 (SEQ ID. NO: 3) in doses of 3 mg / kg (diamonds), 1 mg / kg (triangles) ), 0.5 mg / kg (squares) and 0.3 mg / kg (x symbols), or saline (squares); the dashed line in Fig. 8 indicating the pre-dosage baseline PTH level; FIG. 9 is a graph of the parathyroid hormone level, as a percentage of the pre-dose baseline value, as a function of time, in hours, in rats dosed intravenously with saline (symbols x) or with the compounds Ac-crrrrrr-NH2 (SEQ ID NO: 6, open diamonds), and Ac-carrrar-NH2 (SEQ ID NO: 26, open squares) at 1 mg / kg by means of an IV infusion in 30 minutes , in which plasma PTH levels were assessed before dosing, 16 hours and 24 hours after dosing; FIG. 10 is a graph of the parathyroid hormone level, as a percentage of the pre-dose baseline value, as a function of time, in hours, in rats with acute renal failure (model 1K1C), in which the rats were dosed by means of an IV bolus. with the compounds Ac-c (C) arr-NH2 (SEQ ID NO: 3, squares, symbols *) and Ac-c (Ac-C) arr-NH2 (SEQ ID NO: 14 6, triangles, diamonds) in doses of 0.3 mg / kg (squares, triangles) and 0.5 mg / kg (*, diamonds); FIG. 11 is a graph of the parathyroid hormone level, as a percentage of the pre-dose baseline value, as a function of time, in hours, in rats treated by transdermal release facilitated by Accrrrrrr-NH2 micropores (SEQ ID. No.: 6, two animals, squares and triangles) or with saline solution by means of transdermal release (diamonds); FIG. 12 is a graph of the parathyroid hormone level, as a percentage of the pre-dose baseline value, as a function of time, in hours, in rats treated by transdermal release facilitated by micropores of Ac-C (C) arrr-NH2 ( SEQ ID NO: 3); FIG. 13 is a graph of average PTH (as a percentage of the baseline level) during and after an IV infusion in 6 hours of Ac-c (C) arr-NH2 (SEQ ID: NO: 3) in normal Sprague-Dawley rats , in which the compound was infused at rates of 1 pg / kg / h (squares), 3 pg / kg / h (circles) and 10 pg / kg / h (triangles); FIG. 14A shows PTH (as a percentage of the baseline level) during and after an IV infusion in 6 hours of Ac- (C) arrr-NH2 (SEQ ID NO: 3) in model 1K1C in an acute renal failure rat , in which the rats were infused intravenously at rates close to 30 pg / kg / h (diamonds) and 100 pg / kg / h (squares); FIG. 14B is a bar graph showing serum calcium, in mg / dl, for the 1K1C model mice treated as in FIG. 14A.
The present object of study can be more easily understood by reference to the following detailed description of the preferred embodiments and the examples included therein. DETAILED DESCRIPTION I. Definitions
Within this application, unless otherwise specified, definitions of terms and illustration of the techniques of that application can be found in any of several well-known references such as, for example: Sambrook, J., et al., "Molecular Cloning: A Laboratory Manual ", Cold Spring Harbor Laboratory Press (1989); Goeddel, D., ed., Gene Expression Technology, "Methods in Enzymology", 185,
Academic Press, San Diego, CA (1991); "Guide to Protein Purification" in Deutshcer, M.P., ed., "Methods in Enzymology", Academic Press, San Diego, CA (1989); Innis, et al., "PCR Protocols: A Guide to Methods and Applications", Academic Press, San Diego, CA (1990); Freshney, R.I., "Culture of Animal Cells: A Manual of Basic Technique", 2nd Edition, Alan Liss, Inc. New York, NY (1987); Murray, EJ., Ed., "Gene Transfer and Expression Protocols", pages 109-128, The Humana Press Inc., Clifton, NJ and Lewin, B., "Genes VI", Oxford University Press, New York (1997) .
As used herein, the singular forms "urn", "uma", "o" and "a" include references in the plural, unless otherwise indicated. For example, "a" modulator peptide includes one or more modulator peptides.
As used herein, a compound has "activity to decrease the level of parathyroid hormone" or "PTH-reducing activity" when the compound, after administration to an individual, reduces the plasma parathyroid hormone (PTH) in relation to plasma concentration of PTH before administration of the compound. In one embodiment, the decrease in the PTH level is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% less than one hour after administration of the compound that the PTH level before compound administration.
As used herein, the term "absence of a histamine response" or "devoid of a histamine response" means a dose of a compound that produces an increase of less than 15 times, 14 times, 13 times, 12 times, 11 times , 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times or 3 times in histamine, measured in an in vitro assay as described here, in which the increase in the number of times is determined based on histamine levels before incubation with the compound and after incubation for 15 minutes with the compound.
As used herein, the term "amino acid" refers to natural and unnatural amino acids. The twenty naturally occurring amino acids (L-isomers) are designated by the three-letter code with the prefix "L-" (except for glycine which is achiral) or by the capital letter code: alanine ("L-Ala" or "A"), arginine ("L-Arg" or "R"), asparagine ("L-Asn" or "N"), aspartic acid ("L-Asp" or "D"), cysteine ("L- Cys H or "C"), glutamine ("L-Gln" or "Q"), glutamic acid ("L-Glu" or "E"), glycine ("Gly" or "G"), histidine ("L -His "or H"), isoleucine ("L-Ile" or "I"), leucine ("L-Leu" or "L"), lysine ("L-Lys n or" K "), methionine (" L-Met "or" M "), phenylalanine (" L-Phe "or" F "), proline (" L-Pro "or" P "), serine (" L-Ser "or" S "), threonine ("L-Thr or" T "), tryptophan (" L-Trp "or" W "), tyrosine (" L-Tyr "or" Y ") and valine (" L-Val "or" V "). L-norleucine and L-norvaline can be represented as (NLeu) and (NVal), respectively. The nineteen naturally occurring amino acids that are chiral have a corresponding D-isomer that is designated by the three-letter code prefixed with "D-" or the lowercase letter code: alanine ("D- Ala" or "a") , arginine ("D-Arg" or r), asparagine ("D-Asn" or "a"), aspartic acid ("D-Asp" or "d"), cysteine ("D-Cys" or "c" ), glutamine ("D-Gln" or "q"), glutamic acid ("D-Glu" or "e"), histidine ("D-His" or "h"), isoleucine ("D-Ile" or "i"), leucine ("D-Leu" or "1"), lysine ("D-Lys" or "k"), methionine ("D-Met" or "m"), phenylalanine ("D-Phe "or" f "), proline (" D-Pro "or" p "), serine (" D-Ser "or" s "), threonine (" D-Thr "or" t "), tryptophan (" D -Trp "or" w "), tyrosine (" D-Tyr "or" y ") and valine (" D-Val "or" v "). D-norleucine and D-norvaline can be represented as (dNLeu) and (dNVal), respectively. Although "amino acid residue" is often used in reference to a monomeric subunit of a peptide, polypeptide or protein, and "amino acid" is often used in reference to a free molecule, the use of these terms in the art overlaps and varies. The terms "amino acid" and "amino acid residue" are used interchangeably and can refer to a free molecule or a monomeric subunit of a peptide, polypeptide or protein, depending on the context.
To determine the percentage of "homology" or percentage of "identity" of two amino acid sequences, the sequences are aligned for the purpose of optimal comparison (for example, gaps can be introduced in the sequence of one polypeptide for optimal alignment with the other polypeptide) . Amino acid residues at corresponding amino acid positions are then compared. When a position in one sequence is occupied by the same amino acid residue as the corresponding position in the other sequence, then the molecules are identical in that position. As used herein, the term "homology" of amino acid or nucleic acid is equivalent to the term "identity" of amino acid or nucleic acid. Consequently, the percentage of sequence identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage of sequence identity = numbers of identical positions / total numbers of positions x 100). The percentage of sequence identity between two polypeptide sequences can be determined using the "Vector NTI" program suite (Invitrogen Corporation, 5791 Van Alien Way, Carlsbad, CA 920 08). A gap gap penalty of 10 and a gap extension penalty of 0.1 are used to determine the percent identity of two polypeptides. All other parameters are adjusted to the default settings.
The term "cationic amino acid" means an amino acid residue that has a positive net charge at physiological pH (7.4), as is the case, for example, in amino acid residues in which the side chain, or "group R", contains an amine functional group or another functional group that can accept a proton to become positively charged at physiological pH, for example, a guanidine or imidazole moiety. Cationic amino acid residues include arginine, lysine, histidine, 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), ornithine and homoarginine.
The term "cationic subunit" means a subunit that has a positive net charge at physiological pH (7.4).
As used herein, the term "conservative amino acid substitutions" are substitutions that do not result in a significant change in the activity or tertiary structure of a selected polypeptide or protein. These substitutions typically involve replacing a selected amino acid residue with a different amino acid residue that has similar physicochemical properties. The grouping of amino acids and amino acid residues by physicochemical properties is known to those skilled in the art. For example, among naturally occurring amino acids, families of amino acid residues that have similar side chains have been defined in the art, and include basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid , glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine , tryptophan), beta branching side chains (for example, threonine, valine, isoleucine) and aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, histidine).
As used herein, the term "chemical cross-linking" refers to the covalent bond of two or more molecules.
A peptide or peptide fragment is "derived from" a parent peptide or polypeptide if it has an amino acid sequence that is identical or homologous to at least one contiguous sequence of five amino acid residues, more preferably eight amino acid residues, of the peptide or polypeptide relative.
As used herein, the term "hyperparathyroidism" refers to primary, secondary and tertiary hyperparathyroidism, unless otherwise indicated.
The term "intradermal" means that, in the treatment methods described herein, a therapeutically effective amount of a calcimimetic compound is applied to the skin to release the compound to the layers of skin below the stratum corneum and thereby obtain a desired therapeutic effect.
As used herein, an "isolated" or "purified" polypeptide or biologically active portion thereof is free of a part of the cellular material, when produced by recombinant DNA techniques, or chemical precursors or other chemicals, when chemically synthesized. The language "substantially free of cellular material" includes polypeptide preparations in which the polypeptide is separated from some of the cellular components of the cells in which it is naturally or recombinantly produced. When the polypeptide or biologically active portion thereof is produced recombinantly, it is also preferably substantially free of culture medium, that is, culture medium represents less than about 20%, more preferably less than about 10% and, more preferably still, less than about 5% of the volume of the polypeptide preparation. The language "substantially free of chemical precursors or other chemicals" includes polypeptide preparations in which the polypeptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of a polypeptide that has less than about 30% (by dry weight) of chemical precursors or other chemicals, preferably less than about 20% chemical precursors or other chemicals, more preferably less than about 15% chemical precursors or other chemicals, even more preferably less than about 10% chemical precursors or other chemicals and , more preferably still, less than about 5% chemical precursors or other chemicals. In preferred embodiments, isolated polypeptides, or biologically active portions thereof, do not contain contaminating polypeptides from the same organism from which the domain polypeptide is derived.
As used herein, the term "macromolecule" refers to a molecule, for example, a peptide, polypeptide, protein or nucleic acid, which typically has a molecular weight greater than about 900 Daltons.
The term "non-cationic amino acid" means an amino acid residue that has no charge or a negative net charge at physiological pH (7.4), as is the case, for example, in the amino acid residues in which the side chain, or " group R ", is neutral (polar neutral and non-polar neutral) and acidic. Non-cationic amino acids include those residues with an R group that is an alkyl or aromatic hydrocarbon moiety (for example, valine, alanine, leucine, isoleucine, phenylalanine); a neutral polar R group (asparagine, cysteine, glutamine, serine, threonine, tryptophan, tyrosine); or a neutral non-polar R group (glycine, methionine, proline, valine, isoleucine). Non-cationic amino acids with an acid group R include aspartic acid and glutamic acid.
The term "polymer" refers to a linear chain of two or more identical or non-identical subunits joined by covalent bonds.
As used herein, the terms "peptide" and "polypeptide" refer to any polymer consisting of a chain of amino acid residues linked by peptide bonds, regardless of their size. Although the term "protein" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, the use of these terms in the art overlaps and varies. Thus, for simplicity, the term "peptide" will be used here, although in some cases the technique may refer to the same polymer as a "polypeptide". Unless otherwise indicated, the sequence for a peptide is given in order from the amino terminus to the carboxy terminus.
The terms "thiol-containing group" or "thiol-containing moiety", as used herein, refer to a functional group that comprises a sulfur-hydrogen bond (- SH), and which is capable of reacting with another thiol under physiological conditions to form a disulfide bond. A thiol that is capable of forming a disulfide bond with another thiol is referred to herein as a "reactive thiol". In a preferred embodiment, the thiol-containing group is less than 6 atoms away from the compound's framework. In a more preferred embodiment, the thiol-containing group has the structure (-SH- CH2-CH2-C (O) -O-) -.
As used herein, the term "small molecule" refers to a molecule other than a macromolecule, for example, an organic molecule and, typically, has a molecular weight of less than 1,000 Daltons.
As used herein, the term "individual" refers to a human individual or an animal individual.
The term "subunit" means a monomeric unit that is joined to more than one other monomeric unit to form a polymeric compound, where a subunit is the shortest repeating pattern of elements in the polymeric compound. Exemplary subunits are amino acids that, when linked, form a polymeric compound, such as those referred to in the art as a peptide, polypeptide or protein.
As used herein, the term "therapeutically effective amount" is an amount necessary to produce a desired therapeutic effect. For example, in methods for reducing serum calcium in hypercalcemic individuals, a therapeutically effective amount is the amount needed to reduce serum calcium levels by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or 25%. Calcium can be measured as total calcium or as ionized calcium. Just as another example, in methods for reducing PTH in vivo, a therapeutically effective amount is the amount needed to reduce PTH levels by at least 1%, 2%, 3%, 4%, 5%, 6%, 7 %, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or 25%.
As used herein, the term "transdermal" means, in the treatment methods described herein, that a therapeutically effective amount of a calcimimetic agent is applied to the skin to release the compound into the systemic circulation and thereby obtain a desired therapeutic effect.
Unless otherwise indicated, all documents cited herein are incorporated by reference in their entirety. II. Compounds
In one aspect, a compound is provided which comprises the sequence of subunits Xi - X2 - X3 - X4 - X5 - Xe - X7, where Xi is a subunit comprising a thiol group; X5 is a cationic subunit; X6 is a non-cationic subunit; X7 is a cationic subunit; and at least two of X2, X3 and X4 are independently a cationic subunit. The compounds have activity to decrease levels of parathyroid hormone (PTH) and / or decrease levels of calcium in an individual's blood. The term "decrease in parathyroid hormone levels", as will be illustrated below, means a reduction in the plasma or blood concentration of PTH in an individual in relation to the plasma or blood concentration of PTH before treatment with the compound. In one embodiment, the compound achieves a reduction in plasma PTH concentration by at least 50% within one hour after dosing, compared to plasma PTH before dosing. The compounds are exemplified by peptides, although those skilled in the art will note that non-peptide compounds that have the desired activity can be designed based on the structure-activity relationship studies described here.
As used herein, the term "parathyroid hormone" or "PTH" is an 84 amino acid peptide produced by the parathyroid gland and its breakdown products. In addition to full-length PTH (which consists of residues 1-84 and is sometimes referred to as "intact" or "bioactive" PTH), several fragments of PTH generated by proteolysis and other metabolism pathways are present in the blood. The 1-34 region of the amino terminal of the intact PTH molecule is biologically active. This region of the molecule contains the sequence of amino acids that allows PTH to bind to parathyroid hormone receptors in target tissues. It is believed that the middle and carboxy terminal 35-84 of the intact PTH molecule is biologically inert, but has immunological reactivity. PTH region 7-84 is believed to have effects that are opposite to those in PTH region 1-84. Various tests have been developed to measure PTH levels, including various degradation products, and are reviewed by Souberbielle et al, Kidney International, 11: 93-100 (2010), which is incorporated by reference. In one embodiment, a compound that has activity to decrease the level of PTH, as defined herein, is verified using a validated method of quantifying PTH that detects the intact bioactive form of PTH (1-84), and commercially available kits are known in the technique (for example, see Example 3 of that order).
In a first study, compounds containing 4 to 7 cationic subunits (eg, arginine) were generated and tested for their ability to reduce PTH, compared to baseline PTH values and animals treated with saline. Specifically, a 1K1C model of acute renal failure has been established for use in the characterization of PTH-reducing activity in a renal dysfunction environment. The 1K1C model is described in Example IA, and the compounds synthesized for testing included (i) Ac-crrrr-NH2 (SEQ ID NO: 4), (i) Ac-crrrr-NH2 (SEQ ID. N °: 5), (iií) Ac-crrrrrr-NH2 (SEQ ID NO: 6), (ív) Ac-crrrrrrr-NH2 (SEQ ID NO: 7) and (v) control saline solution.
As described in Example 1B, each of the compounds identified as ID. SEQ. No. 4, ID. SEQ. No. 5, ID. SEQ. No. 6 and ID. SEQ. N °: 7 was administered by an IV infusion in 30 minutes to animals of the 1K1C model. Fig. 1 shows the reduction in plasma PTH levels as a percentage of the pre-dosage (baseline) level. All four compounds dosed at 3 mg / kg produced a significant drop in plasma PTH, but differences in the potency and duration of PTH reduction suggest a relationship between positive net charge and PTH reduction activity. For example, the compound Ac-crrrrrr-NH2 (SEQ ID NO: 6; triangles) with six cationic subunits (arginine) had increased efficacy, as well as the duration of action, compared with the compounds Ac-crrrr-NH2 (SEQ ID NO: 4; diamonds) and Ac-crrrrr-NH2 (SEQ ID NO: 5; squares), which contain four and five cationic subunits (arginine), respectively. Surprisingly, the compound Ac-crrrrrr-NH2 (SEQ ID NO: 6; triangles) with six cationic subunits (arginine) had an increased duration of action, compared to the compound Ac-crrrrrrr-NH2 (SEQ ID. N °: 7, open squares) with seven cationic residues (arginine), suggesting that the activity or potency of the compounds is not correlated simply with the increase in the cationic charge of the compound. That is, the compound Ac-crrrrrrr-NH2 (SEQ ID NO: 7) with seven cationic subunits (arginine residues) produced an initial drop in PTH similar to compounds with less cationic residues, but over the 24 hours after dosing it was less effective than Ac-crrrrrr-NH2 (SEQ ID NO: 6) and Ac-crrrrr-NH2 (SEQ ID NO: 5). These last two compounds produced an average PTH reduction of approximately 40% and 60% at the 24 hour time point, respectively. Both the extent of PTH reduction and the duration of PTH are important criteria for obtaining optimal therapeutic benefit for patients in need of treatment. It should be noted that the compounds in that study were administered at the same dose of mg / kg, but, due to differences in molecular weight, a different number of moles of each compound was actually dosed. Therefore, Ac-crrrrrr-NH2 (SEQ ID NO: 6) was significantly more potent than Ac-crrrr-NH2 (SEQ ID NO: 4) and Ac-crrrrr-NH2 (ID. DE SEQ. No.: 5) per mole.
Additional studies were done to explore the structure-activity relationship of the compounds. The compound Ac-crrrrrr-NH2 (SEQ ID NO: 6) was modified by sequentially replacing an arginine residue with an alanine residue at each of the X2-X7 subunit positions. The compounds were characterized in a human calcium sensor receptor (CaSR) in vitro assay, described in Example 2, in which HEK-293 cells expressing the human calcium sensor receptor were used to measure the activity of exemplary compounds. Without being bound by a theory, it is believed that the mechanism by which the compounds described reduce PTH in vivo is through activation of CaSR, which is expressed in the parathyroid gland and controls the secretion of PTH. Activation of CaSR leads to an increase in intracellular calcium and inositol-3-phosphate (IP3) and the subsequent accumulation of inositol-phosphate-1 (IPi). Consequently, in this in vitro assay, the 50% effective concentration of compound was determined to reduce the generation of IPi by 50% (EC50). The same compounds were also tested in vivo to determine their PTH reducing activity, as described in Example 3. The results are shown in Table 1. The numbers in the column titled "% PTH AUC (1-4 hours) control of saline "in Table 1 define activity as a reduction in the area under the curve (AUC) of PTH over 4 hours as a percentage of PTH AUC derived from saline-treated control rats. For example, an AUC (compound treated) / AUC (saline control) * 100 that is equal to 0 would be indicative of a highly active PTH reduction compound that completely suppresses PTH (to an undetectable level) for 4 hours after a single IV administration of normal rats anesthetized with isofluorane (IF). In contrast, an AUC (compound-treated) / AUC (saline control) * 100 value that is equal to or greater than 100 would be indicative of an inactive compound. Table 1

1 Bold font indicates D-alanine substitutions of cationic amino acids (D-arginine in SEQ ID. NO: 6). 2 * Reduction of PTH after IV administration of 0.5 mg / kg in normal rats anesthetized with isofluorane - PTH was measured at 1, 2, 3 and 4 hours post-administration and the cumulative AUC was calculated. PTH data were calculated according to the following formula: AUC treated with compound / - ^ - UCcontrol solution
In Table 1, the compounds Ac-crrrrrr-NH2 (SEQ ID NO: 6), Ac-carrrrr-NH2 (SEQ ID NO: 8) and Ac-crrarrr-NH2 (SEQ ID. No. 10) were very potent, as evidenced by the decrease in the percentage of PTH to below the detection limit or basically zero, as measured in vivo after a single IV administration in normal rats. The substitution of the cationic residue (arginine) in positions 2, 3, 4 or 7 of Ac-crrrrrr-NH2 (SEQ ID NO: 6) resulted in a loss of approximately twice in potency in vitro. The substitution in position 5 to produce the compound Accrrrarr-NH2 (SEQ ID. NO: 11) produced a 5-10 fold reduction in in vitro potency, although a reduction in the percentage of PTH AUC in vivo of 45% would be active enough for clinical therapy. Surprisingly, replacing the cationic arginine residue at position 6 with the uncharged residue (alanine) actually increased the potency. The data illustrate that cationic and uncharged residues in different positions are not the same and there are changes in activity as a result of changes in the structure of the compound.
To further evaluate the effect of the change in activity due to the change in the structure of the compound, another series of analogues of Ac-crrrrrr-NH2 (SEQ ID NO: 6) was generated containing double amino acid substitutions, in which two cationic residues (arginine) were replaced by unloaded residues (alanine), and tested for potency. The data are shown in Table 2. It should be noted that this series of compounds has the same cationic net charge as the ID. SEQ. N °: 4 (four cationic residues), although surprisingly some are very active (SEQ ID. N °: 26) with very low% of PTH AUC from saline control, while others are inactive (for example, ID SEQ N °: 14).
Unexpectedly, this suggests that the position of charges, as well as the total cationic charge, may influence the potency of compounds to reduce PTH. The data shown in Table 2 are consistent with the data shown in Table 1, suggesting that the cationic residues of ID. SEQ. N °: 6 are essential in positions 5 and 7, but 10 are not necessary in position 6, for PTH reduction activity.

structural factors that influence activity. In one embodiment, the compound is Ac-craarrr-NH2 (SEQ ID NO: 22) and, in another embodiment, the compound is Ac-craarrr-NH2 (SEQ ID NO: 24).
Additional studies of the structure-activity relationship were made using the in vitro cell assay on HEK-293 cells that express the human calcium sensing receptor, as described in Example 4. The ability of the Ac-carrrar-NH2 peptides (SEQ ID. N °: 26) and Ac-arrrar-NH2 (SEQ ID. N °: 29) to activate human CaSR was verified by measuring the accumulation of inositol monophosphate (IPi), which reflects the production of IP3. The production of IP3 is a second messenger of important cell signaling and its production is a direct consequence of downstream CaSR activation. The accumulation of IPX after production of IP3 can be obtained by treating the cells used in the assay with lithium chloride (LiCl2), which inhibits the enzyme that converts IPX to inositol. In the studies described in Example 4, IPX accumulation was measured in the presence of the exemplary compounds Ac-carrrar-NH a (SEQ ID NO: 26) and Ac-arrrar-NH2 (SEQ ID NO: 29). The results are shown in Fig. 2A.
The IPX concentration is recorded as nM along the Y axis and the compound concentrations of the ID. SEQ. No. 26 or ID. SEQ. N °: 29 are recorded as M along the X axis. The absence of the N-terminal D-cysteine residue of the ID. SEQ. N °: 29 dramatically reduced the compound's ability to activate CaSR when compared to ID. SEQ. N °: 26. That is, the elimination of the N-terminal cysteine residue significantly reduced the potency of the compound, as the peptides Ac-carrrar-NH (SEQ ID. N °: 26) and Ac-arrrar -NH2 (SEQ ID. N °: 29) differ only by the presence or absence of the N-terminal D-cysteine.
The contribution of the thiol-containing group to the Xi subunit of the compound (for example, in certain embodiments in which the compound is a peptide at the N-terminal residue) was also investigated in an in vivo study. The PTH-reducing activity of the peptides identified as ID. SEQ. N °: 26 (Ac-carrrar-NH2) and as ID. SEQ. N °: 29 (Accrar-NH2) was assessed in vivo according to the procedures in Example 4. Plasma PTH levels were assessed before dosing and at 1, 2, 3 and 4 hours after dosing. The results are shown in Fig. 2B. As noted, a dose of 0.5 mg / kg of the peptide Ac-carrrar-NH2 (SEQ ID NO: 26) (squares) reduced the blood PTH concentration to an undetectable level for up to 4 hours after dosing. In contrast, the peptide devoid of an N-terminal residue with a thiol-containing group, Acrrar-NH2 (SEQ ID NO: 29), diamonds, did not reduce the PTH concentration, even in one dose substantially higher (ie 9 mg / kg).
The structure-activity relationship of the group containing thiol in the X2 subunit of the compound was further analyzed by preparing compounds with different X2 subunits. The compounds, shown in Table 3, were tested in vivo in normal mice for activity to reduce PTH.Table 3 In vivo activity of exemplary compounds
Bold font indicates respective replacement of thiol-containing residue in Ac-crrrrrr-NH2 (SEQ ID. N °: 6). ** PTH reduction after 0.5 mg / kg IV administration in 5 normal rats anesthetized with isofluorane - PTH was measured at 1, 2, 3 and 4 hours post-administration and the cumulative AUC was calculated. PTH data were calculated according to the following formula: AUC treated with compound / AUC saline control * 100.
The data in Table 3 illustrates that the thiol-containing XT subunit can be varied. Compounds with the following in the N-terminal residue were tested: D-cysteine (cys), D-penicillamine (dPen), d-homocysteine (dHcy) and mercaptopropionic acid (Mpa). In addition, a natural or unnatural amino acid, for example, beta-alanine, can be conjugated to the residue containing the N-terminal thiol. The data illustrates that cationic compounds such as Ac-crrrrrr-NH2 (SEQ ID. No. 6), which contain different thiol-containing groups in the Xi subunit, effectively reduce PTH in vivo. Replacing the N-terminal cysteine residue with methionine, which does not contain a thiol group, resulted in a compound with very little PTH reduction activity in vivo (data not shown).
Based on the studies above, compounds from the contiguous sequence of subunits Xi - X2 - X3 - X4 - X5 - X6 - X7, where Xi is a subunit comprising a group containing thiol, have activity to decrease parathyroid hormone levels . In one embodiment, the group containing thiol in the Xx subunit is selected from the group consisting of amino acid residues that contain thiol and organic portions that contain thiol; in another embodiment, the thiol-containing group is able to react with another thiol group under physiological pH and temperature. In certain embodiments in which the thiol-containing residue is an amino acid residue, the Xi subunit can be any one of cysteine, glutathione, mercaptopropionic acid, n-acetylated cysteine and PEGylated cysteine. In embodiments in which the thiol-containing group is a subunit that is not an amino acid residue, for example, a small organic molecule with a thiol-containing group, the Xi subunit may be a thiol-alkyl, or thioacyl moieties such as example, residues of 3-mercaptopropyl or 3-mercaptopropionyl. In one embodiment, the thiol is not homocysteine.
Consequently, and in another embodiment, the compounds described herein have "clinical activity to decrease the level of parathyroid hormone", which means that the compound, after administration to an individual, reduces the plasma parathyroid hormone, as measured by the area under the cumulative PTH curve (PTH AUC) over 4 hours post-administration, compared with the PTH AUC of a control subject treated with the corresponding vehicle. Plasma PTH concentrations are measured using, for example, a commercially available ELISA kit that detects intact bioactive PTH 1-84 (see Example 3 for a specific kit). The compound with clinical activity to decrease the level of parathyroid hormone reduces PTH AUC by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, compared with the PTH AUC of a control subject treated with the corresponding vehicle.
The above studies, and others described below, illustrate additional embodiments of the compounds described herein, where the Xx subunit in some embodiments can be modified chemically, for example, by chemical modification, to include an acetyl group, a benzoyl group, a benzyl group , a butyl group, a natural or unnatural amino acid, for example, acetylated beta-alanine, or is joined by a covalent bond to another thiol moiety. Therapeutic peptide substances can be vulnerable to attack by peptidases. Exopeptidases are typically non-specific enzymes that cleave amino acid residues from the amino or carboxy termini of a peptide or protein. Endopeptidases, which cleave within a sequence of amino acids, can also be non-specific; however, endopeptidases often recognize particular amino sequences (recognition sites) and cleave the peptide at or near these sites. Consequently, modifications to the compound are contemplated to protect it from proteolytic degradation.
A method of protecting a peptide from proteolytic degradation involves chemical modification, or capping, of the amino and / or carboxy terminals of the peptides. As used herein, the terms "chemically modified" or "covered" are used interchangeably to refer to the introduction of a blocking group to one end or to both ends of the compound by means of a covalent modification. Suitable blocking groups serve to cover the peptide terminals, without decreasing the biological activity of the peptides. Any residue positioned at the amino or carboxy termini, or both, of the described compounds, including the subunits that contain thiol, can be modified chemically.
In a preferred embodiment, the amino terminus of the compound is chemically modified by acetylation, to provide an N-acetyl peptide (which can be represented as "Ac-" in a structure or formula presented here). In a preferred embodiment, the carboxy terminus of the described peptides is modified chemically by amidation to provide a primary carboxamide at the C terminus (which can be represented as "-NH2" in a peptide sequence, structure or formula presented here). In a preferred embodiment, both the amino and the carboxy terminals are chemically modified by acetylation and amidation, respectively. However, other coverage groups are possible. For example, the amino terminus can be covered by acylation with groups such as an acetyl group, a benzoyl group or with natural or unnatural amino acids such as, for example, beta-alanine covered with an acetyl group, or by alkylation with groups such as, for example, a benzyl group or a butyl group, or by sulfonylation to form sulfonamides. Similarly, the carboxy terminus can be esterified, or converted to a secondary amide, and acyl sulfonamide, or the like. In some embodiments, the amino terminus or the carboxy terminus may comprise a site for adhesion of a portion of polyethylene glycol (PEG), that is, the amino or carboxy terminus can be chemically modified by reaction with a suitably functionalized PEG.
The protection of endopeptidase peptides typically involves the identification and elimination of an endopeptidase recognition site from a peptide. Protease recognition sites are well known to those skilled in the art. In this way, it is possible to identify a potential endoprotease recognition site and then eliminate that site by altering the amino acid sequence within the recognition site. Residues in the recognition sequence can be moved or removed to destroy the recognition site. Preferably, a conservative substitution is made with one or more of the amino acids that comprise an identified protease recognition site. A. Additional structure-activity relationship studies
Additional structure-activity studies were carried out to further evaluate the effect of the properties of each subunit on the compound on its therapeutic activity. These studies will now be described with reference to Example 5.
A series of compounds that have an L-amino acid residue that replaces a D-amino acid residue have been prepared based on the PTH reduction framework Ac-c (C) arrrar-NH2 (SEQ ID. NO: 3) . The compounds were administered to the subjects and plasma PTH levels were assessed before dosing and 1, 2, 3 and 4 hours after dosing, as described in Example 5, and the AUC was calculated as the sum of the concentration values of PTH at time points of 1, 2, 3 and 4 hours, normalized by the AUC for 10 saline control at the same time points, multiplied by 100. The results are shown in Table 4.
* Reduction of PTH after IV administration of 0.5 mg / kg in normal rats anesthetized with isofluorane. ** PTH was measured at 1, 2, 3 and 4 hours post-administration and cumulative AUC was calculated. PTH data were calculated according to the following formula: AUC treated with compound / UC UC saline control * 100.
The exemplary compounds shown in Table 4 have been chemically modified at both the N-terminus and the C-terminus, as indicated by the designations Ac and NH2. The sequence of seven carrrar subunits (SEQ ID. NO: 3), where all subunits were residues of D-amino acids, was modified by replacing one subunit at a time with an L-amino acid. The X4 subunit was a D-Cys residue (or L-Cys residue in SEQ ID NO: 34) conjugated via a disulfide bond to an L-Cys residue, as indicated by the designation in parentheses (C). The in vivo PTH reduction data in Table 4 shows that the chirality of Arg and Ala affects the activity of the compounds. In one embodiment, a compound of the sequence X! -X2-X3- X4 - X5 - X6 - X7 is contemplated, in which at least the subunits identified as X4 and X7 are subunits of D-amino acid residue. In another embodiment, the subunits identified as X4, X5, X6 and X7 are subunits of D-amino acid residue. In a preferred embodiment, the subunits identified as X3, X4, X5, X6 and X7 are subunits of D-amino acid residue. In most preferred embodiments, the subunits identified as X2, X3, X4, X5, X6 and X7 are subunits of D-amino acid residue, and all subunits Xlz X2, X3, X4, Xe, X6 and X7 are residue subunits of D-amino acid.
In other studies, it was also found that replacing a peptide that has all L-amino acids with all D-amino acids did not reduce the in vitro activity of the tested peptides; in fact, it appears that peptides composed entirely of D-amino acids increase the potency for CaSR activation. It was also shown that some of the cationic residues (arginine), in specific positions in relation to the cysteine residue, could be replaced with unloaded residues (alanine) with minimal effect on the activity against CaSR.
To further characterize the relationship between structure and activity against CaSR, several cationic peptides with different numbers (4 to 8) of arginine residues (all containing an N-terminal cysteine) were tested using the HEK-293 cell in-vitro assay. A direct correlation was found between the number of cationic subunits and the potency of the compound, in which the potency is evidenced by the ability to activate CaSR. The reduction in the number of cationic subunits (eg, arginine) from 5 to 4 resulted in the greatest change in potency (> 10 times), suggesting that there may be a turning point in activity between compounds that have these liquid charges, and that a cationic subunit in the X5 subunit is preferred for activity. Consequently, compounds of structure X3 - X2 - X3 - X4 - X5 - Xs - X7 are contemplated, where X5 is a cationic subunit. In certain embodiments, X3 is a subunit that comprises a thiol group that is capable of reacting with another thiol group under physiological conditions (a "reactive thiol", meaning a thiol that reacts with another thiol (for example, cysteine with cysteine) under physiological conditions of pH 7.4 and body temperature).
Unexpectedly, Ac-crrrrrr-NH2 (SEQ ID NO: 6) with six cationic residues, when evaluated in vivo, exhibited greater and more prolonged activity than Ac-crrrrrrrr-NH2 (SEQ ID NO: 41), which has eight cationic residues. This is in contrast to the observation that the ID. SEQ. N °: 41 was more potent in activating CaSR in this in vitro cell assay. Without sticking to a theory, it is believed that the superior performance of Ac-crrrrrr-NH2 (SEQ ID: No. 6) in may be due to the better pharmacokinetic properties of Ac-crrrrrr-NH2 (SEQ ID NO: 6), as Ac-crrrrrrrr-NH2 (SEQ ID NO: 41) is believed to be collected in cells due to its characteristic of penetrating the cell and, thus, removed from the vicinity of the active portion of the CaSR.
To further explore the structure-activity relationship of Ac-crrrrrr-NH2 (SEQ ID NO: 6), some of the cationic residues (arginine) have been replaced with uncharged residues (alanine). It was found that the replacement of the cationic residues (arginine) at the positions of the X2 and X4 subunit resulted in a compound (SEQ ID. N °: 15) that had significantly reduced in vitro potency in CaSR activation. On the contrary, the substitution of the cationic residues (arginine) at the subunit positions X2 and X6 resulted in a compound (SEQ ID. N °: 26) that retained much of the potency observed with Ac-crrrrrr-NH2 (SEQ ID. No.: 6). These results suggest that the position of the charged residues in the compound contributes to the potency and, in some modalities, can surpass the contribution of the total positive charge of the peptide, and it also appears that cationic residues (arginine) in certain positions, for example, position subunit X5, contribute disproportionately to the power.
It was found that the presence of a cysteine at the N-terminal markedly increases the potency of the peptides to activate CaSR. CaSR is a receptor coupled to the G 7-transmembrane protein with a large extracellular domain that functions as a homodimeric receptor. There are 18 cysteine residues in the extracellular domain, some of which were shown, by polymorphism or mutational analysis, to be important for receptor activity. Of particular importance are cysteines 129 and 131 of the Loop 2 region of the extracellular domain. Cysteines 129 and 131 are believed to form an intermolecular disulfide bridge between the two monomers of the receptor complex, which is in a closed or inhibited configuration. The cysteine 129 mutation activates CaSR, as do several other mutations that include a total deletion of the Loop 2 region. The increased potency provided by the N-terminal cysteine residue in the compounds described could result from a specific interaction with one or more of cysteine residues in the CaSR extracellular domain.
To further evaluate the effect of chirality of amino acid substitutions on CaSR activity in vitro, a series of Ac-crrrrrr-NH2 analogs (SEQ ID NO: 6) containing L-amino acid substitutions or of achiral amino acid (glycine) in various positions, and these were tested for potency against CaSR. The tested analogs included (i) Ac-cGrrrGr-NH2 (SEQ ID NO: 42), (ii) Ac-cArrrAr-NH2 (SEQ ID NO: 43) and (ill) Ac-CaRrRaR -NH2 (SEQ ID. N °: 44). All of the aforementioned analogs had significantly less potency than Ac-crrrrrr-NH2 (SEQ ID NO: 6), varying by a 10-fold difference for the ID. SEQ. N °: 44 (the most powerful of the three analogs) at a difference of more than 2,000 times for the ID. SEQ. N °: 43 (the least potent of the three analogs). With Ac-carrrar-NH2 (SEQ ID. N °: 26), in which cationic residues of D-amino acids (D-arginine residues) in positions 2 and 6 of the ID. SEQ. N °: 6 were replaced by uncharged residues of D-amino acids (D- arginine residues), the change in activity was much less (a difference of approximately 3 times). Thus, surprisingly, it was found that the interruption of the entire D-amino acid residue of Ac-crrrrrr-NH2 (SEQ ID NO: 6) with two or more L-amino acid residues resulted in a significant reduction in power. It was also surprising that the potency was decreased more than 80 times when the break residue was an amino acid residue without an achiral charge (glycine residue), compared when it was an L-amino acid unloaded residue (L-alanine residue) .
It was also surprising that the replacement of the two uncharged residues of D-amino acids (residues of D-alanine) of Ac-carrrar-NH2 (SEQ ID. N °: 26) with their L counterparts (SEQ ID. N °: 43) resulted in a decrease of more than 600 times in potency, while its replacement with an amino acid residue without achiral charge (glycine residue) (SEQ ID. NO: 42) resulted in a reduction of less than 8 times in power; and that the replacement of three cationic residues of D-amino acids (D-arginine residues) of Ac-carrrar-NH2 (SEQ ID. N °: 26) with their L counterparts (SEQ ID. N °: 44 ) resulted in a difference of less than 4 times in power.
The activity of several peptides and conjugates was tested against human CaSR. These studies were performed by measuring the production of IPX in HEK-293 cells that express human CaSR. EC50 values are shown in Table 5. Each peptide was tested at eight different concentrations, in duplicates, to establish a dose-response curve. The curve adjustment was performed using GraphPad Prism. In Table 5, and throughout the specification, the residues provided in capital letters are L-amino acids, while lower letters indicate D-amino acids. "Ac" indicates an acetyl cover group, "NH2" indicates an amide cover group, "Ac-bAla" is an acetylated beta-alanine, "GSH" indicates reduced glutathione, "GS" indicates oxidized glutathione, "PEG" if refers to polyethylene glycol, "PEG2" and "PEG5" refer to the 2 kDa and 5 kDa polyethylene glycol portions, respectively, and "Mpa" refers to mercaptopropionic acid. A group grouped in parentheses indicates that the group or portion is attached to the side chain of the preceding subunit or amino acid residue.




In another study of the structure-activity relationship, the contribution of non-cationic amino acids to the potency of the peptides was assessed by preparing a series of peptides with various residues of D-amino acids or glycine (Table 6) or with sterically hindered unnatural amino acids ( Table 7), substituted at various positions in the peptide Ac-carrrar-NH2 (SEQ ID NO: 26) and in the peptide Ac-crrarar-NH2 (SEQ ID NO: 153). The peptides were administered as an IV bolus to normal Sprague-Dawley rats at a dose of 0.5 mg / kg. An intravenous (IV) saline bolus was used as a control. Plasma PTH levels were assessed before dosing and 1, 2, 3 and 4 hours after dosing. The results are shown in the tables below, and indicate that: (1) a small amino acid, such as alanine, glycine or serine, is preferred at position 6 in the peptide Ac-carrrar-NH2 (SEQ ID. NO: 26), and (2) alanine at position 2 in Accarrrar-NH2 (SEQ ID. N °: 26) is much more permissive to substitutions and can be substituted with natural hydrophobic amino acids (for example, D-Val , D-Leu), aromatic (for example, D-Phe), or polar (for example, D-Ser, D-Gln), as well as with bulky unnatural hydrophobic amino acids (for example, dNle, dNva), but not with those acids, and that (3) the alanine residue at position 4 of the peptide Ac-crrarar-NH2 (SEQ ID NO: 25) is also very permissive to substitutions and can accommodate 5 most types of natural amino acids (as well as with bulky hydrophobic unnatural amino acids (eg, dNle, dNva), but it is not permissive to amino acids that affect secondary conformation, specifically glycine or proline or amino acid acidic side chain.




conjugation groups that contain thiol. GS = oxidized glutathione; dHcy = D-homocysteine; Mpa = mercaptopropionic acid; PEG = polyethylene glycol. 2 * Reduction of PTH after IV administration of 0.5 mg / kg in normal rats anesthetized with isofluorane - PTH was measured at 1, 2, 3 and 4 hours post-administration and the cumulative AUC was calculated. PTH data were calculated according to the following formula: AUC treated with compound / AUCCO saline control * 100. 3 ** The compound was dosed at 10 mg / kg (approximately the molarity equivalent to 0.5 mg / kg of a non-PEGylated peptide). 4 *** the compound was dosed at 00 mg / kg (approximately the molarity equivalent to 0.5 mg / kg of a non-PEGylated peptide). B. Histamine response and structure-activity relationship studies
Polycationic compounds have been reported in the literature as triggering the release of histamine from active biogenic amine. See Church et al, J. Immunol., 128 (5): 2,116-2,121 (1982); Lagunoff et al, Ann. Rev. Pharmacol. Toxicol., 23: 331-51 (1983). Histamine release is believed to be the result of mast cell and basophil activation that occurs in a Gai-dependent manner. See Aridor et al., J. Cell Biol., 111 (3): 909-17 (1990). The reduction or elimination of this physiological reaction is desirable, inter alia, to increase the therapeutic margin of cationic peptide calcimimetics for the treatment of SHPT.
Studies have been carried out to evaluate the histamine release induced after the in vivo administration of the compounds described herein. In a first study, described in Example 6, bolus or IV infusion dosing in normal Sprague-Dawley rats was used to assess histamine release associated with various compounds. To evaluate the effect of positive net charge on the release of histamine associated with a compound, peptides containing 4 to 7 cationic residues (arginine) were generated and tested for their ability to trigger the release of histamine in vivo, according to the procedure described in Example 6. The peptides tested included: (i) Ac-crrrr-NH2 (SEQ ID NO: 4), (li) Ac-crrrrr-NH2 (SEQ ID NO: 5), (ill) Ac-crrrrrr-NH2 (SEQ ID NO: 6) and (ív) Ac-crrrrrrrr-NH2 (SEQ ID NO: 41).
As shown in Fig. 3, when an equivalent number of moles of each peptide was administered by bolus IV to normal mice, the ID. SEQ. No.: 41 (8 arginine residues) exhibited the greatest induction of histamine. Other compounds with less Arg residues, including ID. SEQ. No. 6 (6 arginine residues), ID. SEQ. N °: 5 (5 arginine residues), and ID. SEQ. N °: 4 (4 arginine residues), also produced a peak in histamine level, but to a lesser extent compared to ID. SEQ. No.: 41. The ID. SEQ. No. 6, ID. SEQ. No. 5 and ID. SEQ. N °: 4 generated milder responses in their histamine release activity (approximately 2-3 times above the baseline level). 0 ID. SEQ. No. 5 and the ID. SEQ. N °: 4 were, however, less potent than the ID. SEQ. N °: 6, regarding the reduction of plasma PTH.
As the PTH reduction activity of Ac-crrrrrr-NH2 (SEQ ID. NO: 6) was accompanied by the absence of a histamine response, additional assessments were made based on Ac-crrrrrr-NH2 (ID. DE SEQ No.: 6) in order to assess whether it was possible to further decrease the histamine response, without sacrificing PTH reduction activity. As will be demonstrated in the data below, the replacement of cationic residues (arginine) in Ac-crrrrrr-NH2 (SEQ ID NO: 6) with non-cationic residues (alanine) was performed to produce a series of analogues with a charge reduced overall net and reduced charge density. Of these analogs, both Ac-cararrr-NH2 (SEQ ID NO: 15) and Ac-carrrar-NH2 (SEQ ID NO: 26) were associated with the absence of a histamine response when administered to rats per cake IV. Significantly, these two peptides retained their potent calcimimetic properties and were capable of reduced PTH secretion in both normal and renal dysfunction mice.
The compound Ac-crrrrrr-NH2 is identified as ID. SEQ. N °: 6 (2.1 pmol / kg = 2.3 mg / kg) triggered an observable histamine response about 2-3 times from baseline compared to 6-9 times with ID. SEQ. No. 41 when dosed by bolus IV (given over less than 1 minute) in normal rats. The release of histamine triggered by Ac-crrrrrr-NH2 (SEQ ID NO: 6) peaked at 5 minutes after dosing and returned to baseline levels 15 minutes later (Fig. 3). An additional reduction in the number of loaded subunits for 5 and 4 arginine residues per peptide (SEQ ID NO: 5 and SEQ ID NO: 4, respectively) further reduced the histamine response when compared to the longer oligo-arginine peptides; however, a 2-3-fold increase in histamine over baseline was still observed 5 minutes after IV bolus dosing (Fig. 3). These results suggest a relationship between the net load of the peptide and the release of associated histamine. It is also noted that peptides rich in arginine with less than 7 arginines are very limited in their ability to enter cells, suggesting that penetration into the cell is not necessary to trigger the release of histamine.
The histamine release associated with PTH reducing compounds Ac-crrrrrr-NH2 (SEQ ID NO: 6) and Ac-c (C) arrr-NH2 (SEQ ID NO: 3) was evaluated in vivo. The compound Ac-c (C) arrrar-NH2 (SEQ ID NO: 3) has the following structure:

This conjugated structure is represented here as Ac-C (C) arr-NH2 (SEQ ID NO: 273), in which the L-Cys residue attached to the residue containing thiol in the Xi subunit of the compound (here, a D-Cys residue) through a Cys-Cys disulfide bond, is placed in parentheses in the formula. This notation is used throughout the specification to designate that the portion in parentheses is linked to a second group that contains thiol. Regarding Ac-crrrrrr-NH2 (SEQ ID NO: 6), the compound Ac-c (C) arrrar-NH2 (SEQ ID NO: 3) has two substituted cationic residues (arginine) with unloaded residues (alanine) in the subunit positions X2 and X6. In addition, the D-Cys residue at the Xi position is conjugated to an L-Cys residue.
These two compounds were administered to rats (Sprague-Dawley) anesthetized with isofluorane at 3 mg / kg per intravenous (IV) bolus (given over less than 1 minute). Blood was drawn before dosing and at 5, 15 and 30 minutes after dosing. The histamine concentration was measured, and the change in the number of times in the blood histamine concentration in relation to the pre-dose blood histamine concentration is shown in Fig. 4. The compound Accrrrrrr-NH2 (SEQ ID. N °: 6, open bars) induced a histamine response, observed at the data point 5 minutes after dosing, in which a 7-fold increase in histamine level was observed. The compound Ac-c (C) arr-NH2 (SEQ ID NO: 3, crossed cross bars) did not induce apparent histamine response, as observed by the data points at 5, 10 and 15 minutes post-dosing, in which the histamine level was not increased in relation to the pre-dose histamine level (time zero).
To further assess the relationship between compound structure and histamine release, a series of compounds was prepared and evaluated for their ability to trigger histamine induction in an in-vitro assay using rat peritoneal mast cells. In this assay, the compounds are incubated at 10 pM for 15 minutes at 37 ° C with cells isolated from peritoneal lavage of SD rats. After incubation, cell medium is collected and histamine is determined. The data are shown in Table 9.



* Method shown in Example 7.
Consequently, and as can be seen in the light of the PTH data and the histamine data described above, in one embodiment, a compound is contemplated that has activity to decrease the level of PTH in an individual in the absence of a histamine response. . In certain embodiments, the absence of a histamine response means a dose of the compound that produces an increase of less than 10 times, more preferably 8 times, still more 10 preferably 5 times, and even more preferably 3 times in histamine, measured in vitro. an assay as described herein, in which the increase in the number of times is determined based on histamine levels before incubation with the compound and after incubation for 15 minutes with compound. In a specific embodiment, the histamine response is determined in an in vitrous assay using rat peritoneal mast cells isolated from peritoneal lavage of normal Sprague-Dawley rats, and where the increase in the number of times is determined based on histamine levels before incubation with the compound and after incubation for 15 minutes with compound. In the studies conducted here, in vitro evaluation of histamine release was performed using peritoneal rat mast cells isolated by peritoneal lavage using ice cold HESS + 25 mM HEPES pH 7.4 containing heparin (5 p / ml). The cells were washed twice in stimulation buffer (HBSS + 25 mM HEPES pH 7.4) and incubated with 10 pM of compound in stimulation buffer (HBSS + 25 mM HEPES pH 7.4) for 15 minutes in a 96-well plate (10s / well) at 37 ° C. The cell supernatant was analyzed for histamine using the EIA histamine kit (Cayman # 589651).
In another modality, a compound that has activity to decrease the level of PTH in an individual in the absence of a clinical histamine response is contemplated. As used herein, the term absence of a "clinical histamine response" means that a therapeutically effective amount of a compound as described herein is administered to the subject without producing a clinically adverse increase in plasma or blood histamine, as measured 5-10 minutes after completion of dosing or throughout the treatment period. For example, when a compound needed to produce a desired therapeutic effect is administered to an individual per bolus (as used herein, "bolus" means administered over a minute or less) it produces an increase in plasma or blood histamine 5-10 minutes after finishing the dosage which is less than 15 times, 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, 2 times above the pre-dose levels.
As can be seen from the studies described above, in one embodiment, the compound comprises a sequence of 3 to 35 amino acid residues, in which several positively charged subunits of amino acid residues are present in the sequence. In some embodiments, the compounds described comprise 5 to 25 subunits and, in a preferred embodiment, each subunit is an amino acid residue. In other embodiments, the described compounds comprise 6 to 12 subunits. In yet other embodiments, the described compounds comprise 3 to 9 amino acid subunits. In alternative embodiments, the described compounds comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 subunits.
The subunits of the compounds described are, in one embodiment, selected independently of natural or unnatural amino acids, or their analogs, and may have an L or D configuration (except for glycine that is achiral). Glycine, aliphatic residues alanine, valine, leucine, or isoleucine, proline, hydroxyl serine and threonine residues, aspartic acid and glutamic acid residues, asparagine, amide residues, and glutamine, basic lysine and arginine residues, histidine , aromatic residues phenylalanine, tyrosine and tryptophan, and residues containing sulfur methionine and cysteine, are all contemplated for use in the compounds described. The number of positively charged subunits and their density can affect the potency of the compound to reduce PTH. In some embodiments, the positively charged subunits are separated by one or more other subunits ("separation subunits"). In one embodiment, the separation subunits are alanine residues. In some embodiments, the chirality of the separation subunit affects the potency of the compound.
Positively charged amino acid residues of the described compounds can be a specific natural or non-natural residue, or analog thereof, which has the L or D configuration (for example, L-arginine) which is repeated in the sequence, or can be composed of several residues natural or unnatural, or analogues thereof, which have the L or D configuration. In some embodiments, the compound is a peptide containing 3 to 20 positively charged amino acid residues, 6 to 12 positively charged amino acid residues, 3 to 9 positively charged amino acid residues. In some embodiments, the peptides comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 positively charged amino acid residues.
In some embodiments, positively charged amino acid residues are selected independently of natural amino acids. In some embodiments, positively charged amino acid residues are selected independently of natural and / or unnatural amino acids. In some embodiments, positively charged amino acid residues are selected independently from the group consisting of arginine, lysine, histidine, 2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab), ornithine and homoarginine. In a preferred embodiment, the positively charged amino acid residues are arginine residues.
In some embodiments, the compound is a peptide and is a single continuous peptide chain or strand. In other embodiments, the compound is a peptide that is branched. In still other embodiments, the peptide is conjugated to one or more thiol-containing moieties (each "thiol-containing conjugation group" or "conjugation group"). In a preferred and merely illustrative embodiment, the peptide compound is conjugated to a Cys conjugation group, by means of a disulfide bond (-S-S-) (for example, -Cys- Cys-). As used herein, the term "compound" is intended to encompass both these peptides and conjugates.
The compounds typically comprise one or more thiol moieties, preferably one or more thiol reactive moieties. Subunits that have a thiol group include non-amino acid compounds that have a thiol group and amino acids with a thiol group. The thiol group of the thiol-containing subunit can be in a conjugated form (for example, by means of a disulfide bond to a conjugation group) or in an unconjugated form (i.e., as a reduced thiol). In a preferred embodiment, when the thiol group is in an unconjugated form or in a conjugated form, it is able to form a disulfide bond with a group containing thiol. The thiol-containing residue can be located at any position along the peptide chain, including the amino terminus, the carboxy terminus, or some other position. In a preferred embodiment, the thiol-containing residue or subunit may be located at the amino terminus. In other embodiments, the thiol-containing residue or subunit may be located at the carboxy terminus or within the peptide sequence.
Some representative examples of thiol-containing residues include, without limitation, cysteine, mercaptopropionic acid, homocysteine and penicillamine. When the thiol containing residue contains a chiral center, it may be present in the L or D configuration. In a preferred embodiment, the thiol containing residue is cysteine.
In some embodiments, the crossover between the thiol containing subunit at position Xi in the compound and the thiol containing conjugation group may be cleavable and / or interchangeable with other thiol containing conjugation groups such as, for example, cysteine (e.g. by reducing the disulfide bond) in vivo to generate a biologically active form of the compound. In this way, a conjugate can function as a prodrug for the compound. A conjugation group can also be used to modify the physicochemical, pharmacokinetic and / or pharmacodynamic properties of the compounds described (for example, conjugation via a disulfide bond to a large PEGylated portion to increase pharmacokinetics).
In some embodiments, the compound is a peptide composed of the amino acid sequence (Xaai) - (Xaa2) - (Xaa3) - (Xaa4) - (Xaa5) - (Xaa5) - (Xaa7) (SEQ ID. NO: 155), where (Xaal) is an amino acid residue that contains thiol. (Xaa2) is a non-cationic amino acid residue, (Xaa3) is any amino acid residue, (Xaa4) is any amino acid residue, (Xaa5) is a cationic amino acid residue, (Xaa6) is a non-cationic residue and (Xaa7 ) is any amino acid residue. The peptide can be modified at the N-terminus, the C-terminus, or both. In a preferred embodiment, the peptide is modified at both the N-terminus and the C-terminus by acetylation and amidation, respectively.
In some embodiments, a peptide comprises the amino acid sequence (D-Cys) - (Xaa2) - (Xaa3) - (Xaa3) - (Xaa5) - (XaaS) - (Xaa7) (SEQ ID: 156 ), where (Xaa2) is a non-cationic amino acid residue, (Xaa3) is any amino acid residue, (Xaa4) is any amino acid residue, (Xaa5) is selected from the group consisting of D- Arg, L-Arg , D-Lys and L-Lys, (Xaa6) is a non-cationic residue and (Xaa7) is any amino acid residue. The peptide may have an N-terminus, a C-terminus, or both. In a preferred embodiment, the peptide has both an N-terminus and a C-terminus.
In some embodiments, a peptide comprises the amino acid sequence (D-Cys) - (Xaa2) - (Xaa3) - (Xaa4) - (Xaa5) - (Xaa6) - (Xaa7) (SEQ ID. NO: 157 ), where (Xaa2), (Xaa3) and (Xaa4) are, independently, any amino acid residue (but, in a preferred embodiment, are selected independently from the group consisting of D-Ala, D-Val, D-Leu , D-NorVal and D-NorLeu), (Xaa5) and (Xaa7) are, independently, any cationic amino acid residue (but, in a preferred embodiment, are selected independently from the group consisting of D-Arg, L-Arg, D- Lys and L-Lys), (Xaa6) is a non-cationic amino acid residue (in a preferred embodiment, selected from the group consisting of D-Ala, D-Val, D-Leu, D-NorVal and D-NorLeu ). The peptide may have an N-terminus, a C-terminus, or both. In a preferred embodiment, the peptide has both an N-terminus and a C-terminus.
In some embodiments, a peptide comprises the amino acid sequence (D-Cys) - (Xaa2) - (Xaa3) - (Xaa4) - (Xaa5) - (Xaa6) - (Xaa7) (SEQ ID: 158 ), where (Xaa2) is a non-cationic amino acid residue, (Xaa3) is any amino acid residue, (Xaa4) is any amino acid residue, (Xaa5) is selected from the group consisting of D- Arg, L-Arg , D-Lys and L-Lys, (Xaa5) is a non-cationic residue and (Xaa7) is any amino acid residue. The peptide can have an N-terminal cover, a C-terminal cover, or both. In a preferred embodiment, the peptide has both an N-terminus and a C-terminus.
In some embodiments, a peptide comprises the amino acid sequence (D-Cys) - (D-Ala) - (Xaa3) - (Xaa4) - (D- Arg) - (D-Ala) - (Xaa7) (ID. DE SEQ. No.: 159), where (Xaa3) is any cationic amino acid residue, (Xaa4) is any cationic amino acid residue and (Xaa7) is any cationic amino acid residue. The peptide can have an N-terminal cover, a C-terminal cover, or both. In a preferred embodiment, the peptide has both an N-terminus and a C-terminus.
In some embodiments, a peptide comprises the amino acid sequence (D-Cys) - (Xaa2) - (Xaa3) - (D-Ala) - (D-Arg) - (D-Ala) - (Xaa7) (ID. DE SEQ. No.: 160), where (Xaa2), (Xaa3) and (Xaa7) are, independently, any cationic amino acid residue. The peptide can have an N-terminal cover, a C-terminal cover, or both. In a preferred embodiment, the peptide has both an N-terminus and a C-terminus.
Another embodiment is a calcimimetic peptide that comprises a sequence of amino acids linked by peptide bonds, where the sequence comprises 5 to 10 amino acid residues, and where the sequence comprises an amino terminus, a carboxy terminus, at least one residue containing thiol , and from 3 to 9 positively charged residues. In one embodiment, the (at least one) thiol-containing residue is a cysteine residue. In another aspect, the cysteine residue is positioned at the amino terminus of the peptide. In certain embodiments, the cysteine residue is an L-Cys residue, a D-Cys residue or an L- or D-homoCys residue. In other embodiments, the amino acid residues of the peptide are D-amino acids or L-amino acids.
Also included within the scope of the claimed compounds are peptidomimetic molecules that comprise approximately seven subunits, in which at least one subunit contains a thiol portion, preferably a reactive thiol portion, and other subunits are several non-cationic subunits, and from 1 to 4 charged subunits positively. These peptidomimetic molecules can comprise non-peptide bonds between two or more of the subunits. The various characteristics of the compounds discussed above generally apply to the peptidomimetic molecule. For example, as discussed above, the subunits used to construct the molecules can be naturally occurring amino acids, or residues with unnatural side chains, the terminals of the modules can be covered or not covered in the manner discussed above. Similarly, the amino acid residues of the molecule can be L- or D-amino acid residues. Also as discussed above, the thiol-containing residues can be in a reduced or oxidized form with any of the thiol-containing moieties discussed above.
Many peptidomimetic frameworks and methods for their synthesis have been developed (Babine, RE; Bender, SL, Chem. Rev., 97: 1.359, 1997; Hanessian, S .; et al., Tetrahedron, 53: 12.789, 1997; Fletcher, MD Cambeli, MC, Chem. Rev., 98: 763, 1998); "Peptidomimetics Protocols"; Kazmierski W.M., Ed.; "Methods in Molecular Medicine Series", Vol. 23; Humana Press, Inc .; Totowa, NJ. (1999). Conjugated
In some embodiments, the compound is chemically interconnected to a thiol-containing conjugation group via a disulfide bond between the compound's thiol and a conjugation group's thiol. The thiol-containing conjugation group can be a small molecule, for example, cysteine, or a macromolecule, for example, a polypeptide that contains a cysteine residue. Examples of suitable thiol-containing conjugation groups include cysteine, glutathione, thioalkyl, portions such as, for example, thiobenzyl, mercaptopropionic acid, N-acetylated cysteine, cysteamide, N-acetylcysteamide, homocysteine, penicillamine and poly (ethylene glycol) modified thiols. (PEG) (called "PEGylated") such as, for example, PEGylated cysteine or a duplication of the compound (i.e., to form a disulfide bonded homodimer). In a preferred embodiment, the thiol-containing conjugation group is cysteine. Other cysteine counterparts are also contemplated for use as thiol-containing conjugation groups, alone or comprised in a large conjugation group. Similarly, stereoisomers of cysteine, homocysteine and cysteamide are suitable for use as thiol-containing moieties. Conjugation groups can be used to increase chemical stability and, therefore, the validity of a pharmaceutical product. In certain embodiments, the thiol-containing conjugation group and the peptide are the same (i.e., the conjugate is a dimer), which unexpectedly showed very good chemical stability compared to a heterologous conjugation group, for example, cysteine. Without attaching to a theory, presumably when the thiol-containing conjugation group and the peptide are the same, any disproportionation (for example, shuffling of the conjugation group) will reconstitute the original dimer compound. In contrast, the disproportionation of a compound to a heterologous conjugation group such as, for example, cysteine, can lead to the formation of homodimers of the peptide plus cystine (cysteine homodimer - cysteine) plus residual parent compound. A homodimer of the peptide (that is, the conjugation group and the peptide are the same) would be converted into a cysteine conjugated form of the peptide in vivo due to the high concentration of reduced cysteine in the systemic circulation.
In some embodiments, the teachings include a disulfide conjugate of a thiol-containing conjugation group and a peptide comprising the amino acid sequence (Xaal) - (Xaa2) - (Xaa3) - (Xaa4) - (Xaa5) - (XaaS ) - (Xaa7) (SEQ ID. N °: 155), where (Xaai) is an amino acid residue with a portion containing thiol, (Xaa2) is a non-cationic amino acid residue, (Xaa3) is any amino acid residue, (Xaa4) is any amino acid residue, (Xaa5) is a cationic amino acid residue, (Xaa6) is a non-cationic residue and (Xaa7) is any amino acid residue. The peptide can have an N-terminal cover, a C-terminal cover, or both. In a preferred embodiment, the peptide has both an N-terminus and a C-terminus. In a preferred embodiment, the thiol-containing conjugation group is selected from the group consisting of D-Cys, L-Cys, a peptide that contains D-Cys and a peptide that contains L-Cys. When the thiol-containing conjugated group is an amino acid or a peptide, it can have an N-terminus, a C-terminus, or both. In a preferred embodiment, the thiol-containing conjugate group has both an N-terminus and a C-terminus. In some embodiments, the thiol-containing conjugation group itself is a peptide comprising the ID amino acid sequence. SEQ. N °: 155. In some embodiments, the thiol-containing conjugation group and the peptide are the same (that is, the conjugate is a dimer).
In some embodiments, the teachings include a conjugate from a conjugation group that contains thiol and a peptide that comprises the amino acid sequence (D-Cys) - (Xaa2) - (Xaa3) - (Xaa3) - (Xaa5) - (Xaa6 ) - (Xaa7) (SEQ ID. N °: 156), where (Xaa2) is a non-cationic amino acid residue, (Xaa3) is any amino acid residue, (Xaa4) is any amino acid residue, (Xaa5 ) is selected from the group consisting of D-Arg, L-Arg, D-Lys and L-Lys, (Xaa6) is a non-cationic residue and (Xaa7) is any amino acid residue. The peptide can have an N-terminal cover, a C-terminal cover, or both. In a preferred embodiment, the peptide has both an N-terminus and a C-terminus. In a preferred embodiment, the thiol-containing conjugation group is selected from the group consisting of D-Cys, L-Cys, a peptide that contains D-Cys and a peptide that contains L-Cys. When the thiol-containing conjugated group is an amino acid or a peptide, it can have an N-terminus, a C-terminus, or both. In a preferred embodiment, the thiol-containing conjugate group has both an N-terminus and a C-terminus. In some embodiments, the thiol-containing conjugation group itself is a peptide comprising the ID amino acid sequence. SEQ. No. 156. In some embodiments, the thiol-containing conjugation group and the peptide are the same (that is, the conjugate is a dimer).
In some embodiments, the teachings include a conjugate from a conjugation group that contains thiol and a peptide that comprises the amino acid sequence (L-Cys) - (Xaa2) - (Xaa3) - (Xaa4) - (Xaa5) - (XaaS ) - (Xaa7) (SEQ ID. NO: 183), where (Xaa2) is a non-cationic amino acid residue, (Xaa3) is any amino acid residue, (Xaa4) is any amino acid residue, (Xaa5 ) is selected from the group consisting of D-Arg, L-Arg, D-Lys and L-Lys, (Xaa6) is a non-cationic residue and (Xaa7) is any amino acid residue. The peptide may have an N-terminus, a C-terminus, or both. In a preferred embodiment, the peptide has both an N-terminus and a C-terminus. In a preferred embodiment, the thiol-containing conjugation group is selected from the group consisting of D-Cys, L-Cys, a peptide that contains D-Cys and a peptide that contains L-Cys. When the thiol-containing conjugate group is an amino acid or a peptide, it can have an N-terminal cover, a C-terminal cover, or both, in a preferred embodiment, the thiol-containing conjugate group has both an N-terminal cover as for a C-terminal cover. In some embodiments, the thiol-containing conjugation group itself is a peptide comprising the ID amino acid sequence. SEQ. N °: 183. In some embodiments, the thiol-containing conjugation group and the peptide are the same (that is, the conjugate is a dimer).
In some embodiments, the teachings include a conjugate from a conjugation group that contains thiol and a peptide that comprises the amino acid sequence (D-Cys) - (D-Ala) - (Xaa3) - (Xaa4) - (D-Arg ) - (D-Ala) - (Xaa7) (SEQ ID. NO: 161), where (Xaa3) is any amino acid residue, (Xaa4) is any amino acid residue and (Xaa7) is any residue amino acid. The peptide can have an N-terminal cover, a C-terminal cover, or both. In a preferred embodiment, the peptide has both an N-terminus and a C-terminus. And a preferred embodiment, the thiol-containing conjugation group is selected from the group consisting of D-Cys, L-Cys, a peptide that contains D-Cys and a peptide that contains L-Cys. When the thiol-containing conjugated group is an amino acid or a peptide, it can have an N-terminus, a C-terminus, or both. In a preferred embodiment, the thiol-containing conjugate group has both an N-terminus and a C-terminus. In some embodiments, the thiol-containing conjugation group itself is a peptide comprising the ID amino acid sequence. SEQ. N °: 161. In some embodiments, the thiol-containing conjugation group and the peptide are the same (that is, the conjugate is a dimer). III. Usage methods
In one aspect, methods for preventing, treating or ameliorating hyperparathyroidism, bone disease and / or other hypercalcemia disorders are contemplated by administering the compounds described herein. As illustrated above, the compounds have activity to decrease the levels of PTH and / or calcium in a target tissue or tissues, or in an individual. In certain embodiments, the described compounds are able to decrease the levels of PTH and / or calcium when a therapeutically effective amount of the compound is administered to an individual in need of such treatment. The methods of use will now be described with reference to Examples 3 and 8-11.
With reference again to Example 3 and, as discussed above with respect to Table 1, the series of compounds in which a cationic residue (arginine) was sequentially replaced with a non-cationic residue (alanine) was administered to the rats. Fig. 5 shows the time profile of each compound's ability to reduce blood PTH and the duration of action of the various compounds. In Fig. 5, the compounds Ac-crrrrrr-NH2 (SEQ ID NO: 6, diamonds), Ac-carrrrr-NH2 (SEQ ID NO: 8, squares) and Ac-crrarrr-NH2 (SEQ ID NO: 10, symbols x) and Ac-crrrrar-NH2 (SEQ ID NO: 12, circles) were potent in vivo, as evidenced by the decrease in the percentage of PTH from the pre-baseline level -dose until practically zero and a potency duration is provided, in which the blood PTH concentration remained decreased for at least four hours. The compounds Ac-crarrrr-NH2 (SEQ ID NO: 9, triangles), Ac-crrrarr-NH2 (SEQ ID NO: 11, symbols *) and Ac-crrrr-NH2 (ID. DE SEQ N °: 13, symbols +) decreased the percentage of PTH from the baseline level for about 2-3 hours and therefore the blood PTH concentration started to increase. The substitution of the cationic residue (arginine) at the subunit 5 or 7 positions of Ac-crrrrrr-NH2 (SEQ ID NO: 6) had an impact on the duration of PTH reduction activity.
The PTH reduction profile for a series of compounds that contain double amino acid substitutions was also evaluated. Selected compounds shown in Table 2 above were administered to normal mice by IV bolus at a dose of 0.5 mg / kg, and the reduction in PTH in relation to the pre-dose blood PTH level was assessed. The data are shown in Figs. 6A-6B, in which the compounds are identified as follows: Ac-carrrar-NH2 (SEQ ID NO: 26, open diamonds), Ac-crrarar-NH2 (SEQ ID NO: 25, open squares), Ac-caarrrr-NH2 (SEQ ID NO: 22, triangles), Ac-crraarr-NH2 (SEQ ID NO: 17, circles), Ac-c (C) NH2 (SEQ ID NO: 3, diamonds Fig. 6B), Ac-c (C) rrar-NH2 (SEQ ID NO: 28, symbols x, Fig. 6B).
Another study was done to further assess the potency of the compound Ac-c (C) arrrar-NH2 (SEQ ID. NO: 3). The compound was administered intravenously to normal mice, as detailed in Example 2, in doses of 1 mg / kg, 0.5 mg / kg, 0.3 mg / kg and 0.1 mg / kg. Plasma PTH levels were assessed before dosing and for 4 hours thereafter. Fig. 7 shows the results, in which the blood PTH concentration is shown as a percentage of the pre-dose baseline value. A dose-related reduction in PTH was observed after a single administration per IV bolus, with the highest dose of 1 mg / kg (diamonds) having obtained the greatest reduction in PTH, followed by the dose of 0.5 mg / kg (squares) , 0.3 mg / kg (triangles) and 0.1 mg / kg (x symbols). The saline control is shown by the circle symbols. As noted, the peptide, when administered in a therapeutically effective dose, achieves a reduction in PTH of more than 50%, in relation to the concentration of PTH before dosing ("baseline"). Specifically, the peptide, when administered in doses above 0.1 mg / kg, reduced the PTH concentration to less than 90% of the baseline PTH concentration 1 hour after IV administration. Those doses of the peptide identified as ID. SEQ. N °: 3 also obtained an area under the curve (AUC) of less than 50%, the AUC calculated as the sum of the PTH concentration values at the 1, 2, 3 and 4 hour time points, normalized by the AUC for saline control at the same time points, multiplied by 100.
The same compound was also tested in individuals (rats) with renal failure. In this study, the 1K1C model of acute renal failure was used to evaluate the compound Ac-c (C) arrrar-NH2 (SEQ ID NO: 3) to characterize its PTH reduction activity in an environment of renal dysfunction . The model is described in Example IA. The compound was administered intravenously as a bolus to animals with renal impairment (rats) at doses of 3 mg / kg (n = 2), 1 mg / kg (n = 5), 0.5 mg / kg (n = 6) and 0.3 mg / kg (n = 5). A group of control animals was dosed with saline. Plasma PTH levels were assessed before dosing and for several hours thereafter. Fig. 8 shows the results, in which the animals treated with saline solution (squares) had an increased PTH concentration in relation to the starting PTH level. In various doses of ID. SEQ. N °: 3, a dose-dependent effect on the duration and extent of PTH reduction was observed. Animals treated with the lowest dose of 0.3 mg / kg (x symbols) exhibited reduced PTH at the earliest time point and an increase in PTH between 1-24 hours after dosing. The dose levels of 3 mg / kg (diamonds), 1 mg / kg (triangles) and 0.5 mg / kg (squares) provided a reduced blood PTH concentration for more than 15 hours and, for the highest dose, for more than 24 hours.
In another study to assess the effect of replacing cationic subunits with unloaded subunits, as exemplified by alanine amino acid residues, in the context of an individual with renal failure, an analog of Ac-crrrrrr-NH2 (SEQ ID. N °: 6) was generated and tested for its ability to reduce PTH in animals of the 1K1C model after a single intravenous administration of 1 mg / kg. In the Ac-carrrar-NH2 analogue (SEQ ID NO: 26) tested, the cationic subunits at positions X2 and X6 of Ac-crrrrrr-NH2 (SEQ ID NO: 6) were replaced with uncharged amino acids .
As shown in Fig. 9, Ac-carrrar-NH2 (SEQ ID NO: 26, open squares) shows activity that is equivalent to Ac-crrrrrr-NH2 (SEQ ID NO: 6, open diamonds ) at the tested dose (1 mg / kg) with a similar extended duration of action over 24 hours. The Ac-crrrrrr-NH2 analogue (SEQ ID NO: 6) with no-load subunit substitutions has been found to retain activity and may actually have a potency and duration of action in vivo greater than that of the identified compound as ID. SEQ. N °: 6. In the compound Ac-carrrar-NH2 (SEQ ID. N °: 26), the residues of D-Arg in positions X2 and X6 were replaced with residues of D-Ala in relation to the compound identified as ID . SEQ. N °: 6.
Significantly, as discussed above, administration of the compound Ac-carrrar-NH2 (SEQ ID. NO: 26) was not accompanied by histamine release, an undesirable side effect that is seen with Accrrrrrr-NH2 (ID. OF SEQ NO: 6) and other similar compounds when administered in larger doses (> 1 mg / kg) per IV bolus. The marked attenuation of histamine release with the compound identified as ID. SEQ. N °: 26 increases the therapeutic margin between the desired PTH reduction activity and the unwanted histamine induction activity after IV bolus administration. Consequently, in a preferred embodiment, compounds are provided that have activity to reduce PTH concentration in vivo in the absence of a histamine response. Consequently, in one embodiment, a compound is provided that has PTH-lowering activity, where the compound, when administered to a human or other individual, decreases the PTH level to below 50% of the pre-dose level by up to one hour after dosing. In a specific embodiment, a compound that has significant PTH-lowering activity means a compound that, when administered to a normal mouse, decreases the PTH level to below 50% of the pre-dose level within one hour after IV bolus dosing. .
In another study, detailed in Example 8, compounds are provided in the form of a conjugate, in which the thiol containing subunit at the Xi position was linked via a disulfide bond to an L-Cys residue. These compounds have the following structures:

In the annotation used here, the compound that is linked to the thiol-containing portion in the Xi subunit is identified in parentheses, in which, in these exemplary conjugates, the L-Cys compound is indicated (C) and is linked to the thiol-containing portion in the subunit Xx: Ac-c (C) arr-NH2 (SEQ ID NO: 3) and Ac-c (Ac-C) arr-NH2 (SEQ ID NO: 141).
These compounds were administered via bolus IV to animals with acute renal failure (model 1K1C) in doses of 0.3 and 0.5 mg / kg, and the results are shown in Fig. 10. The compound Ac-c (C ) arrrar-NH2 (SEQ ID. NO .: 3) is represented by squares (0.3 mg / kg, n = 5) and symbols * (0.5 mg / kg, n = 6) and the compound Ac -c (Ac-C) arr-NH2 (SEQ ID NO: 141) by triangles (0.3 mg / kg, n = 8) and diamonds (0.5 mg / kg, n = 7). This dose-response in vivo with ID. SEQ. N °: 3 exhibits a dose-dependent reduction in PTH very similar to Ac-crrrrrr-NH2 (SEQ ID: N °: 6).
In some of the in vivo studies described herein, the compounds, including compounds in conjugated form in which the thiol in the Xi subunit is cross-linked via a disulfide bond to another subunit, were administered as an IV infusion in 30 minutes. However, it should be noted that shorter infusions (e.g., <5 minutes) or IV bolus release typically produce a pharmacodynamic reduction in PTH comparable to a longer infusion of 30 minutes. Subcutaneous bolus administration 25 also proved to be an effective delivery route that generated a lower initial drop in PTH, but exhibited a sustained reduction in PTH similar to the profile seen by route IV. As shown in Fig. 11, the compound Ac-crrrrrr-NH2 (SEQ ID NO: 6) was also administered by micropore-facilitated transdermal release (eg, microporation of the stratum corneum), and demonstrated a reduction in PTH plasma for the several hours that was monitored. The compound Ac-crrrrrr-NH2 (SEQ ID. NO: 6) was also administered transdermally after microporation, resulting in a reduction in plasma PTH for several hours. The transdermal release of Ac-crrrrrr-NH2 (SEQ ID. NO: 6) provides an additional option for clinical delivery of the described compounds.
To assess the effect of the route of administration on the activity of Ac-crrrrrr-NH2 (SEQ ID NO: 6) in the context of an individual with renal failure, mice in the 1K1C model received 1 mg / kg of the peptide as a subcutaneous bobo (SC) or as an IV infusion in 30 minutes. Both routes of administration effectively reduced plasma PTH levels over 24 hours. When Ac-crrrrrr-NH2 (SEQ ID. NO: 6) was released by IV infusion, PTH levels dropped rapidly by 80-90% from baseline. At about 16 hours after dosing, PTH levels began to rise, although they were still reduced by approximately 80% from baseline. When Accrrrrrr-NH2 (SEQ ID NO: 6) was released by SC bolus, PTH levels exhibited a more moderate initial drop to approximately 40% from baseline, but exhibited a duration of reduction similar to that occurred when the peptide was released via the IV route. Twenty-four hours after dosing, PTH levels in animals dosed by either route had partially returned, although both still exhibited reduced PTH levels that were approximately 40-60% of the baseline level. The results showed that this route of administration provides a similar profile with respect to the effectiveness and duration of PTH reduction than IV administration, thereby providing an alternative route for clinical dosing (data not shown).
Consequently, in a preferred embodiment, an individual who has secondary hyperparathyroidism (SHPT) is treated using the compounds described to reduce plasma PTH and / or calcium levels. Patients with SHPT not treated with moderately severe hyperparathyroidism often have baseline circulating levels of intact PTH of> 300 pg / ml, and levels that can exceed 600 pg / ml. In a preferred embodiment, the decrease in PTH levels is measured as a decrease in intact PTH below baseline pretreatment levels. In another modality, the desired decrease in PTH is to bring plasma PTH levels to guidelines generally recognized by the "National Kidney Foundation" or other specialists in the treatment of kidney disorders and kidney failure.
In another aspect, methods are provided for the treatment of hyperparathyroidism, hypercalcemia and / or bone disease, which comprise the administration of a therapeutically effective amount of a described compound. In another embodiment, the subject can be treated with a described compound in combination with one or more other therapeutically effective agents.
In another aspect, the described compound is administered in an amount effective to reduce PTH or the effect of PTH. In some modalities, the reduction in plasma PTH is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% , 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25% or 30% below baseline pretreatment levels for at least 10 hours post-administration of the described compound. In specific modalities, the reduction in plasma PTH is at least 20% in 10 hours post-administration. In preferred embodiments, the reduction in plasma PTH is 15 to 40%, preferably 20 to 50%, more preferably 30 to 70% below baseline pretreatment levels for at least 48 hours post-administration of the described compound.
In another aspect, the described compound is administered in an amount effective to decrease serum calcium or the effect of calcium. In some modalities, the reduction in serum calcium is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% , 14%, 15%, 16%, 17%, 18%, 19%, 20% or 25% below pre-treatment levels for at least 10 hours post-administration of the polycationic peptide. In some preferred embodiments, the reduction in serum calcium is at least 5% within 10 hours post-administration. In some preferred embodiments, the reduction in serum calcium is 5 to 10%, preferably 5 to 20% below pretreatment levels for at least 48 hours post-administration of the described compound.
In another aspect, a method is provided for the treatment of hyperparathyroidism and / or hypercalcemia in a needy individual, which comprises administering a therapeutically effective amount of a described compound, in which PTH and / or calcium is reduced.
Based on the relationship between serum calcium, bone metabolism and PTH, the compounds described are believed to be beneficial for the treatment of various forms of bone disease and / or hypercalcemia, in addition to hyperparathyroidism. The described compounds may have advantages compared to current therapeutic agents, because they can be administered parenterally and may not be associated with adverse gastrointestinal effects, they are not metabolized by cytochrome P450 and may result in more effective reductions in plasma PTH and calcium.
As discussed above, the described methods can be used alone or in combination with one or more other therapeutically effective agents. Such other therapeutically effective agents include, without limitation, treatment with bisphosphonate anti-resorptive agents, for example, alendronate and risedronate; integrin blockers, for example, avp3 antagonists; conjugated estrogens used in hormone replacement therapy, such as PREMPRO ™, PREMARIN ™ and ENDOMETRION ™; selective estrogen receptor modulators (SERMs), for example, raloxifene, droloxifene, CP-336,156 (Pfizer) and iasofoxifene; cathepsin K inhibitors; vitamin D therapy; vitamin D analogs, such as ZEMPLAR ™ (paricaicitol); CALCIJEX® (calcitriol), HECTOROL® (doxercalciferol), ONE-ALPHA® (alfacalcidol) and the analogues in development from cytochromes known as CTA-018, CTAP201 and CTAP101; other calcimimetics such as Sensipar® (cinacalcet); family of inhibitors of the type II sodium-dependent phosphate transporter, SLC34 (including the two renal isoforms NaPi-IIa and NaPi-IIc, and the intestinal transporter NaPi-IIb); phosphatonins (including FGF-23, sFRP4, MEPE or FGF-7); treatment with low dose of PTH (with or without estrogen); calcitonin; RANK ligand inhibitors; antibodies against RANK ligand, osteoprotegrin; adenosine antagonists; and ATP proton pump inhibitors.
In one embodiment, a described compound is administered in a dose sufficient to decrease both PTH levels and serum calcium levels. In another embodiment, a described compound is administered in a dose sufficient to decrease PTH without significantly affecting serum calcium levels. In an additional embodiment, a described compound is administered in a dose sufficient to increase PTH, without significantly affecting serum calcium levels. Formulations
A pharmaceutical composition is provided which comprises a described compound and at least one pharmaceutically acceptable excipient or carrier. Methods of preparing such pharmaceutical compositions typically comprise the step of associating a described compound with a carrier and, optionally, one or more accessory ingredients. The described compounds and / or pharmaceutical compositions comprising them can be formulated in pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. Typically, formulations are prepared by placing a compound described with liquid vehicles, or finely divided solid vehicles, or both, and then, if necessary, shaping the product in uniform association and intimately.
The pharmaceutical compositions of the present invention suitable for parenteral administration comprise one or more compounds described in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, emulsions, or sterile powders that can be reconstituted in sterile solutions or dispersions injectables immediately before use, which may contain sugars, alcohols, amino acids, antioxidants, buffers, bacteriostats, solutes that make the formulation isotonic with the blood of the desired recipient or suspending or thickening agents.
Examples of suitable aqueous and non-aqueous vehicles that can be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as , olive oil, and injectable organic esters such as ethyl oleate. Adequate fluidity can be maintained, for example, by using coating materials such as, for example, lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.
Such pharmaceutical compositions can also contain adjuvants such as, for example, preservatives, wetting agents, emulsifying agents and dispersing agents. The prevention of the action of microorganisms on the described compounds can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include agents to control tonicity, for example, sugars, sodium chloride, and the like, in the compositions. In addition, the prolonged absorption of the injectable pharmaceutical form can be created by the inclusion of agents that delay absorption, for example, aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug by subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material that has little water solubility. The rate of absorption of the drug then depends on its rate of dissolution which, in turn, may depend on the size of the crystal and the crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oily vehicle.
For example, a described compound can be released to a human in a form of solution that is made by reconstituting a solid form of the drug with liquid. This solution can also be diluted with infusion fluid, such as water for injection, 0.9% sodium chloride injection, 5% dextrose injection and Ringer-lactate injection. It is preferred that the reconstituted and diluted solutions are used within 4-6 hours to release maximum power. Alternatively, a described compound can be delivered to a human in the form of a tablet or capsule.
Injectable forms of deposit are made by forming microencapsulated matrices of the compounds described in biodegradable polymers such as, for example, polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Injectable depot formulations can also be prepared by capturing the drug in liposomes or microemulsions that are compatible with body tissue.
When the compounds described are administered as pharmaceutical substances to humans and animals, they can be given alone or as a pharmaceutical composition that contains, for example, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier. In other embodiments, the pharmaceutical composition may contain 0.2-25%, preferably 0.5-5% or 0.5-2%, of active ingredient. Such compounds can be administered to humans and other animals for therapy by any suitable route of administration, including, for example, subcutaneous injection, subcutaneous deposit, intravenous injection, intravenous or subcutaneous infusion. These compounds can be administered quickly (in less than <1 minute) as a cake or more slowly over an extended period of time (over several minutes, hours or days). These compounds can be released daily or over several days, continuously or intermittently. In one embodiment, the compounds can be administered transdermally (for example, using a plaster, microneedles, micropores, ointment, micro-jet or nano-jet).
Regardless of the selected route of administration, the described compounds, which can be used in a suitable hydrated form, and / or pharmaceutical compositions, are formulated in pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.
The actual dosage levels of the active ingredients in the pharmaceutical compositions can be varied in order to obtain an amount of the active ingredient that is effective in obtaining the desired therapeutic response for a particular patient, composition and mode of administration, without being toxic to the patient. .
The dosage level selected will depend on several factors, including the activity of the compound described in particular employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being being employed, rate and extent of absorption, duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in medical techniques.
A doctor or veterinarian skilled in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, the doctor or veterinarian could initiate doses of the described compounds employed in the pharmaceutical composition at levels lower than that necessary in order to obtain the desired therapeutic effect and gradually increase the dosage until the desired effect is obtained.
In general, an adequate daily dose of a described compound will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. This effective dose will generally depend on the factors described above. Generally, intravenous, intramuscular, transdermal, intra-cerebroventricular and subcutaneous doses of the compounds described for a patient, when used for the indicated effects, will vary from about 1 pg to about 5 mg per kilogram of body weight per hour. In other embodiments, the dose will vary from about 5 pg to about 2.5 mg per kilogram of body weight per hour. In additional embodiments, the dose will vary from about 5 pg to about 1 mg per kilogram of body weight per hour.
If desired, the effective daily dose of a described compound can be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In one embodiment, the described compound is administered as one dose per day. In additional embodiments, the compound is administered continuously, via the intravenous route or by other routes. In other embodiments, the compound is administered less frequently than daily, for example, every 2-3 days, in conjunction with dialysis treatment, weekly or less frequently.
The individual receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as horses, cattle, pigs and sheep; and poultry and pets in general.
The described compounds can be administered as such or mixed with pharmaceutically acceptable carriers, and can also be administered in conjunction with antimicrobial agents such as, for example, penicillins, cephalosporins, aminoglycosides and glycopeptides. Thus, joint therapy includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutic effects of the one administered first do not entirely disappear when the subsequent one is administered. Routes of administration for the disclosed compounds
These compounds can be administered to humans and other animals for therapy by any suitable route of administration. As used herein, the term "route" of administration is intended to include, without limitation, subcutaneous injection, subcutaneous deposit, intravenous injection, intravenous or subcutaneous infusion, intraocular injection, intradermal injection, intramuscular injection, intraperitoneal injection, intratracheal administration, intratracheal administration -adipose, intra-articular administration, intrathecal administration, epidural administration, inhalation, intranasal administration, sublingual administration, buccal administration, rectal administration, vaginal administration, intracisternal administration and topical administration, transdermal administration or administration by means of local release (for example, catheter or endoprosthesis).
The transdermal delivery of drugs to the body is a desirable and convenient method for systemic delivery of biologically active substances to an individual and, in particular, for delivery of substances that have low oral bioavailability, such as proteins and peptides. The transdermal delivery pathway was particularly successful with small lipophilic compounds (eg less than about 1,000 Daltons), eg scopolamine and nicotine, which can penetrate the outer layer of the skin's stratum corneum, which serves as a barrier effective entry of substances into the body. Below the stratum corneum is the viable epidermis, which does not contain blood vessels, but has some nerves. Even deeper is the dermis, which contains blood vessels, lymph and nerves. Drugs that cross the stratum corneum barrier can generally spread to the capillaries in the dermis for systemic absorption and distribution.
Technological advances in transdermal delivery have focused on addressing the need in the technique for the release of hydrophilic compounds, of high molecular weight, for example, proteins and peptides, through the skin. One approach involves rupture of the stratum corneum using chemical or physical methods to reduce the barrier imposed by the stratum corneum. Skin microporation technology, which involves creating micron-sized transport pathways (micropores) in the skin (in particular, the micropores in the stratum corneum) using a minimally invasive technique, is the most recent approach. Techniques for creating micropores in the skin (stratum corneum) include microporation or thermal ablation, microneedle arrangements, phonophoresis, laser ablation and radiofrequency ablation (Prausnitz and Langer (2008) Nat. Biotechnology11: 1.261-68; Arora et al. , Int. J. Pharmaceutics, 364: 227 (2008); Nanda et al, Current Drug Delivery, 3: 233 (2006); Meidan et al American J. Therapeutics, H: 312 (2004)).
As noted above, PTH secretion is regulated by CaSR which is expressed on the cell surface of parathyroid cells. Thus, in order to activate CaSR, the agent or compound must be released into the parathyroid cell. The transdermal release of calcimimetic agents must obtain release through the stratum corneum and provide systemic exposure to reach the parathyroid cell. To date, the technique has not demonstrated whether a calcimimetic compound can be released transdermally in an amount sufficient to obtain therapeutic benefit and, in particular, in an amount sufficient to decrease PTH and / or for treatment, attenuation, reduction and / or hypercalcemia relief.
In addition to calcimimetics, vitamin D3 1,25- (OH) 2 analogs are the most commonly used treatments for patients with hyperparathyroidism associated with chronic kidney disease and end-stage kidney disease. Vitamin D analogs work by facilitating intestinal absorption of dietary calcium, and reduce PTH levels by inhibiting PTH synthesis and secretion. Although the intravenous and oral release of vitamin D has been used therapeutically, to date the technique has not demonstrated whether vitamin D analogs, for example, ZEMPLAR ™ (paricalcitol), CALCIJEX® (calcitriol), ONE-ALPHA® (alfacalcidol) and HECTOROL® (doxercalciferol), can be delivered transdermally in an amount sufficient to obtain therapeutic benefit and, in particular, in an amount sufficient to decrease parathyroid hormone (PTH). In addition, the technique has not yet demonstrated whether co-administration by transdermal release of a calcimimetic agent in combination with a vitamin D analog (as separate formulations or as a co-formulation) in sufficient quantities to obtain therapeutic benefit, and in particular , in sufficient quantities to decrease PTH and provide effective treatment for patients suffering from hyperparathyroidism.
Calcimimetic agents can be administered through the stratum corneum and / or other layers of the epidermis, for local or systemic release, to decrease parathyroid hormone (PTH) and / or for the treatment of hypercalcemia. In one embodiment, the calcimimetic agent is released through microporation. Any one of several techniques for microporation is contemplated, and several will be briefly described.
Microporation can be achieved by mechanical means and / or external driving forces, to disrupt the stratum corneum to release the calcimimetic agents described here through the skin surface and into the underlying skin layers and / or the bloodstream.
In a first modality, the microporation technique is the ablation of the stratum corneum in a specific region of the skin using a pulsed laser light of wavelength, pulse length, pulse energy, number of pulses and sufficient repetition rate to ablate the stratum corneum, without significantly damaging the underlying epidermis. The calcimimetic agent is then applied to the ablation region. Another microporation technique by laser ablation, called laser-induced stress waves (LISW), involves broadband, unipolar and compressible waves generated by pulsed lasers of high power. LISWs interact with tissues to break down lipids in the stratum corneum, creating intercellular channels transiently within the stratum corneum. These channels, or micropores, in the stratum corneum allow the calcimimetic agent to enter.
Sonophoresis or phonophoresis is another microporation technique that uses ultrasound energy. Ultrasound is a sound wave that has frequencies above 20 KHz. The ultrasound can be applied continuously or pulsed, and applied in various frequency and intensity ranges (Nanda et al, Current Drug Delivery, 3: 233 (2006)).
Another microporation technique involves the use of a microneedle arrangement. The microneedle arrangement, when applied to a region of skin in an individual, pierces the stratum corneum and does not penetrate to a depth that significantly stimulates nerves or pierces capillaries. The patient thus does not feel discomfort or pain, or feels minimal discomfort or pain with the application of the microneedle arrangement for the generation of micropores through which the calcimimetic agent is released.
Arrangements of microneedles are made up of hollow or solid microneedles, in which the calcimimetic agent can be coated on the outer surface of the needles or dispensed from inside the hollow needles. Examples of microneedle arrangements are described, for example, in Nanda et al, Current Drug Delivery, 3: 233 (2006) and Meidan et al. American J. Therapeutics, 11: 312 (2004). First generation microneedle arrays were composed of solid silicon microneedles that were coated externally with a therapeutic agent. When the microarray of needles was pressed against the skin and removed after about 10 seconds, permeation of the agent in the needles into the body was quickly achieved.
Second generation microneedle arrays consisted of solid or hollow silicon microneedles, of polycarbonate, titanium or other suitable polymer, and coated or filled with a solution of the therapeutic compound. New generations of microneedle arrangements are prepared from biodegradable polymers, in which the needle tips coated with a therapeutic agent remain in the stratum corneum and slowly dissolve.
Microneedles can be made from a variety of materials, including metals, ceramics, semiconductors, organics, polymers and composite materials. Exemplary building materials include pharmaceutical grade stainless steel, gold, titanium, nickel, iron, tin, chromium, copper, palladium, platinum, alloys of these or other metals, silicon, silicon dioxide and polymers. Representative biodegradable polymers include hydroxy acid polymers such as, for example, lactic acid and polylactide glycolic acid, polyglycolide, polylactide-co-glycolide, and copolymers with poly (ethylene glycol), polyanhydrides, poly (ortho) esters, polyurethanes, poly (acid butyric acid), poly (valeric acid) and poly (lactide-co-caprolactone). Representative non-biodegradable polymers include polycarbonate, polyester and polyacrylamides.
Microneedles can have straight or narrowed axes. In one embodiment, the diameter of the microneedle is larger at the base end of the microneedle and narrows to a point near the distal end of the base. The microneedle can also be manufactured to have an axis that includes both a straight (not narrowed) and a narrowed portion. The needles may also have no narrowed ends, meaning they may simply be cylindrical with blunt or flat ends. A hollow microneedle that has a substantially uniform diameter, but does not narrow to a point, is here called a "microtube". As used herein, the term "microneedle" includes both microtubes and narrowed needles, unless otherwise indicated.
Electroporation is another technique for creating micropores in the skin. This approach uses the application of high-voltage microsecond or millisecond electrical pulses to create transient permeable pores within the stratum corneum.
Other microporation techniques include using radio waves to create microchannels in the skin. Thermal ablation is yet another approach for obtaining the release of higher molecular weight compounds transdermally.
Applicants have found that low doses of calcimimetic agents can be administered therapeutically over an extended period of time to treat SHPT. This differs markedly from the current dose requirements of other calcimimetics (for example, cinacalcet hydrochloride hydrochloride).
The transdermal release of the compounds described herein has been demonstrated in the studies described in Examples 9-10. In a first study, the compound Ac-crrrrrr-NH2 (SEQ ID. NO: 6) was administered transdermally to rats in which a small area of skin was microporated by 5 passages of a 1.0 cm dermal cylinder mm under moderate pressure. A solution of Ac-crrrrrr-NH2 (SEQ ID NO: 6) or saline was placed in the microporated area of the skin.
Blood samples were taken over a period of 4 hours and the plasma was analyzed for PTH levels by ELISA. The results are shown in Fig. 11, in which plasma PTH is shown as a percentage of the pre-dose baseline level for the animal treated with saline (diamonds) and the two animals treated with the test compound (squares, triangles) . These data indicate that the compound Ac-crrrrrr-NH2 (SEQ ID. N °: 6) can be released systemically in sufficient amounts transdermally (in this case, transdermal release facilitated by micropores) using a dermal cylinder to reduce effectively and significantly increased PTH levels from baseline for approximately the 4 hours that were studied. It should be noted that Accrrrrrr-NH2 (SEQ ID NO: 6) has been shown to effectively reduce PTH levels from baseline in the 1K1C model in acute renal failure rat when administered by short IV infusion, as well as in normal mice (data not shown).
In another study, described in Example 10, the compound Ac-c (C) arr-NH2 (SEQ ID NO: 3) was administered by microporous transdermal delivery to normal mice using a transdermal patch. A transdermal patch system containing 10% (by weight) solution of Ac-c (C) arr-NH2 (SEQ ID NO: 3) in saline was placed over the microporated area and left in place for approximately 30 hours. Blood samples were collected from the rats periodically over 30 hours and plasma samples were analyzed for PTH levels by ELISA. The results are shown in Fig. 12. Surprisingly, these data demonstrate that micropore-facilitated transdermal release can achieve sufficient sustained release of Ac-c (C) arr-NH2 (SEQ ID NO: 3) to produce a significant and extended reduction in PTH for> 30 hours in rats with normal renal function. These data demonstrate that transdermal release facilitated by microporation of Ac-c (C) arr-NH2 (SEQ ID NO: 3) conjugated using a patch can obtain sufficient blood exposure of the peptide over the treatment period to produce a significant and sustained reduction in PTH from baseline for> 30 hours. These data demonstrate that the release of transdermal plaster daily or for a longer period would allow the treatment of patients on both dialysis and non-hemodialysis who need treatment. For example, CKD (stage 4), primary hyperparathyroidism and secondary hyperparathyroidism (SHPT) in kidney transplant patients who are not typically treated with IV drugs, but could easily be treated by a daily transdermal patch through transdermal delivery facilitated by microporation.
Another study was carried out to further evaluate the route of administration of the compounds. As described in Example 11, the compound Ac-c (C) arrrar-NH2 (SEQ ID. NO: 3) was administered by very low dose IV infusion to normal mice and to mice with renal failure to identify the smallest dose required to be administered by infusion, transdermal patch system or other means of sustained release to achieve a significant reduction in PTH. Healthy rats were infused intravenously for six hours at very low doses (1 pg / kg / h, 3 pg / kg / h and 10 pg / kg / h) of Ac-c (C) arr-NH2 (ID. DE SEQ. No.: 3). Blood samples were collected before dosing (pre) and in 2 hours, 4 hours, 6 hours (immediately before the end of the infusion; EO1) and 8 hours (2 hours post-EOl) after the start of the infusion, and the plasma was analyzed for PTH levels by ELISA. Surprisingly, the data shown in Fig. 13 demonstrate that the infusion of very low doses of Ac- (C) arrrar-NH2 (SEQ ID NO: 3) (1 pg / kg / h (squares), 3 pg / kg / h (circles) and 10 pg / kg / h (triangles) for 6 hours are effective in producing a significant reduction in PTH from the baseline level over the infusion period. These data indicate that low doses delivered continuously could be as effective as (or even more effective than) the release of much larger doses like a single cake.
The effect of reducing PTH was further evaluated in the 1K1C rat model of acute renal failure. Model 1K1C rats were infused intravenously with low doses of Ac-c (C) arrr-NH2 (SEQ ID NO: 3) (30 pg / kg / h and 100 pg / kg / h) for 6 hours . Blood samples were collected before dosing (Pre), and in 2 hours, 4 hours, 6 hours (immediately before the end of the infusion; EO1), 8 hours (2 hours post-EOl) and 24 hours after the start of the infusion , and the plasma was analyzed for PTH levels by ELISA. The data shown in Fig. 14A demonstrate that the IV infusion of low doses of Ac-c (C) arr-NH2 (SEQ ID NO: 3) significantly reduces PTH from baseline levels in the 1K1C model, a renal failure model in which baseline PTH levels can be observed to be 400 to> 1,100 pg / ml. Surprisingly, 6 hours of low dose IV infusion (diamonds, 30 pg / kg / h and squares, 100 pg / kg / h) of Ac-c (C) arr-NH2 (SEQ ID. NO: 3) able to reduce PTH from baseline for approximately 24 hours. Consistent with this dramatic reduction in PTH in the 1K1C rat model, Fig. 14B shows a bar graph that tabulates serum calcium data in this acute renal failure, and shows a corresponding reduction in serum calcium after low-dose IV infusion of Ac- c (C) arrr-NH2 (SEQ ID NO: 3). These data demonstrate that Ac-c (C) arrrar-NH2 (SEQ ID NO: 3) is a very potent calcimimetic compound that is able to reduce PTH and calcium after infusion or release of low doses (for example , by transdermal release) over a period of approximately 24 hours. These data further support the conclusion that the sustained release of a low dose of a calcimimetic agent by IV infusion or by micropore-facilitated transdermal release could be an effective treatment for patients on a daily or less frequent basis. Combination therapy
As described above, the methods of use can be used alone or in combination with other approaches for the treatment of hypercalcemia and / or bone disease. These other approaches include, but are not limited to, treatment with agents such as bisphosphonate agents, integrin blockers, hormone replacement therapy, selective estrogen receptor modulators, cathepsin K inhibitors, vitamin D therapy, vitamin D analogs such as ZEMPLAR ™ (paricyclicol), CALCIJEX® (calcitriol), ONE-ALPHA® (alfacalcidol) and HECTOROL® (doxercalciferol), anti-inflammatory agents, low-dose PTH therapy (with or without estrogen), calcimimetics, binders phosphate, calcitonin, RANK ligand inhibitors, antibodies against RANK ligand, osteoprotegrin, adenosine antagonists and ATP proton pump inhibitors.
In one embodiment, a combination therapy uses vitamin D or a vitamin D analog in combination with a calcimimetic agent. Vitamin D aids in calcium absorption and works to maintain normal blood levels of calcium and phosphorus. PTH works to increase calcium absorption in the intestine by increasing the production of vitamin D 1,25- (OH) 2, the active form of vitamin D. PTH also stimulates the excretion of phosphorus by the kidney and increases the release by the bones.
As discussed above, secondary hyperparathyroidism is characterized by an increase in parathyroid hormone (PTH) associated with inadequate levels of active vitamin D hormone. Vitamin D or a vitamin D analog can be used to reduce high levels of PTH in the treatment of secondary hyperparathyroidism. In one embodiment, the invention includes a pharmaceutical composition that comprises a calcimimetic agent and a vitamin D analog.
In one embodiment, the invention includes a pharmaceutical composition that comprises a calcimimetic agent and ZEMPLAR ™ (paricalcitol). Paricyclicol is a synthetic analogue of calcitriol, the metabolically active form of vitamin D. The recommended starting dose of Zemplar is based on baseline levels of intact parathyroid hormone (iPTH). If the baseline level of iPTH is less than or equal to 500 pg / ml, the daily dose is 1 pg and the dose "three times a week" (to be administered at most every other day) is 2 pg. If the baseline iPTH is greater than 500 pg / ml, the daily dose is 2 pg, and the "three times a week" dose (to be administered at most every other day) is 4 pg. Thereafter, the dosage should be individualized and based on serum plasma PTH levels, with monitoring of serum calcium and serum phosphorus. Paricalcitol is described in U.S. Patent No. 5,246,925 and in U.S. Patent No. 5,587,497.
In another embodiment, the invention includes a pharmaceutical composition that comprises a calcimimetic agent and CALCIJEX® (calcitriol). Calcitriol is the metabolically active form of vitamin D. The recommended initial dosage for CALCIJEX® (oral) is 0.25 p / day. This amount can be increased by 0.25 pg / day at intervals of 4 to 8 weeks. Normal or only slightly reduced calcium levels may respond to dosages of 0.25 pg every other day. For dialysis patients, the recommended starting dose for CALCIJEX® (IV) is 0.02 pg / kg (1 to 2 pg) 3 times / week, on alternate days. This amount can be increased by 0.5 to 1 pg, every 2 to 4 weeks. Calcitriol is described in U.S. Patent No. 6,051,567 and U.S. Patent No. 6,265,392 and U.S. Patent No. 6,274,169.
In one embodiment, a pharmaceutical composition is provided that comprises a calcimimetic agent and HECTOROL® (doxercalciferol). Doxercalciferol is a synthetic vitamin D analog that undergoes metabolic activation in vivo to form la, 25-dihydroxyvitamin D2, a naturally occurring biologically active form of vitamin D. The recommended starting dose of HECTOROL® is 10 pg administered three times a day. dialysis week (approximately every other day). The starting dose should be adjusted, as needed, to reduce blood iPTH in the range 150 to 300 pg / ml. The dose can be increased at intervals of 8 weeks for 2, 5 pg if iPTH has not been reduced by 50% and fails to reach the target range The maximum recommended dose of HECTOROL is 20 pg administered three times a week on dialysis for a total of 60 pg per week. Doxercalciferol is described in US Patent No. 5,602,116 and in US Patent No. 5,861,386 and in US Patent No. 5,869,473 and in US Patent No. 6,903,083.
The particular combination of therapies (therapeutic substances or procedures) to be employed in a combination regime will take into account the compatibility of the desired therapeutic substances and / or procedures and the desired therapeutic effect to be obtained. It will also be noted that the therapies employed can achieve a desired effect for the same disorder (for example, a compound of the invention can be administered concomitantly with another agent used to treat the same disorder), or they can achieve different effects (for example, control any adverse effects). As used herein, additional therapeutic agents that are normally administered to treat or prevent a particular disease or condition are known to be "appropriate for the disease or condition to be treated".
A combined treatment of the present invention, as defined herein, can be obtained by the simultaneous, sequential or separate administration of the individual components of said treatment.
The pharmaceutically acceptable compounds or compositions thereof can also be incorporated into compositions for coating implantable medical devices, bioerosible polymers, implantable pumps and suppositories. Consequently, in another aspect, a composition for coating an implantable device is contemplated which comprises a described compound, as generally described above, and a vehicle suitable for coating the implantable device. In yet another aspect, an implantable device coated with a composition comprising a compound as generally described above is included, and a vehicle suitable for coating said implantable device.
Suitable coatings and the general preparation of coated implantable devices are described in U.S. Patent Nos. 6,099,562, 5,886,026 and 5,304,121. The coatings are typically biocompatible polymeric materials such as, for example, a hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof. The coatings can optionally also be covered with a suitable top coat of fluorsilicone, polysaccharides, polyethylene glycol, phospholipids, or combinations thereof, to impart controlled release characteristics in the composition. Potential clinical markers for determining treatment effectiveness
The determination of the effectiveness of a described treatment method can be done by several methods.
Normal serum calcium levels are in the range of 8.8 mg / dl to 10.4 mg / dl (2.2 mmol / 1 to 2.6 mmol / 1). In certain cases, the effectiveness of the treatment can be determined by measuring calcium-related serum and urinary markers, including, without limitation, total and ionized serum calcium, albumin, plasma PTH, PTHrP, phosphate, vitamin Dx and magnesium.
In other cases, effectiveness can be determined by measuring bone mineral density (BMD), or by measuring biochemical markers for bone formation and / or bone resorption in serum or urine. Potential bone formation markers include, without limitation, total alkaline phosphatase, bone alkaline phosphatase, osteocalcin, subcarboxylated osteocalcin, type I pre-collagen C-terminal peptide and type I pre-collagen pro-peptide. Bone resorption potentials include, without limitation, hydroxyproline, hydroxylysine, glycosylgalactosyl hydroxyzine, galactosyl hydroxylysine, pyridinoline, deoxypyridinoline, type I collagen N-terminal cross-over telopeptide, collagen-type C-terminal telopeptide, collagen-type telopeptide. of cross-linking of type I collagen C terminal generated by MMPs, bone sialoprotein, acid phosphatase and tartrate resistant acid phosphatase.
In other cases, efficacy can be determined by a percentage reduction in PTH from a pre-dosage (baseline) level and / or by achieving a desirable level of PTH as generally accepted as being beneficial to patients (eg established guidelines by "National Kidney
Foundation "). In yet other cases, effectiveness can be determined by measuring the reduction in parathyroid gland hyperplasia associated with a hyperparathyroidism disease.
It is expected that when a treatment method described is administered to an individual in need, the treatment method will produce an effect, as measured by, for example, one or more of: total serum calcium, ionized serum calcium, total blood calcium , ionized blood calcium, albumin, plasma PTH, blood PTH, PTHrP, phosphate, vitamin D, magnesium, bone mineral density (BMD), total alkaline phosphatase, bone alkaline phosphatase, osteocalcin, subcarboxylated osteocalcin, pre-C-terminal pre-peptide -collagen type I, N-terminal pro-peptide of pre-collagen type I, hydroxyproline, hydroxyzine, glycosyl-galactosyl hydroxyzine, galactosyl hydroxyzine, pyridinoline, deoxypyridinoline, type I collagen cross-terminal telopeptide, interlace telopeptide type I collagen C-terminus, type I collagen C-terminus telopeptide generated by MMPs, bone sialoprotein, acid phosphatase and resistant acid phosphatase to tartrato. The effects include prophylactic treatment, as well as treatment of existing illness.
A biologically effective molecule can be operably linked to a described peptide with a covalent bond or a non-covalent interaction. In specific embodiments, biologically effective molecules operatively linked can alter the pharmacokinetics of the compounds described by virtue of conferring properties on the compound as part of a linked molecule. Some of the properties that biologically effective molecules can impart to the compounds described include, without limitation: releasing a compound to a distinct location within the body; concentrating the activity of a compound in a desired location in the body and reducing its effects elsewhere; reduction of side effects of treatment with a compound; changing the permeability of a compound; alteration of the bioavailability or rate of release to the body of a compound; changing the duration of the effect of treatment with a compound; alteration of chemical stability in composite vitrode; alteration of stability, half-life, clearance, absorption, distribution and / or excretion of the compound in vivo; alteration of the rate of appearance and fall of the effects of a compound; providing a permissive action by allowing a compound to have an effect.
In a further aspect, the described compound can be conjugated to polyethylene glycol (PEG). The selected PEG can be of any convenient molecular weight, and can be linear or branched, and can be optionally conjugated via a linker. The average molecular weight of PEG will preferably range from about 2 kilodaltons (kDa) to about 100 kDa, more preferably from about 5 kDa to about 40 kDa. Alternatively, the PEG portion used can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 kDa.
The described compounds can be conjugated to PEG by means of a suitable amino acid residue located at any position in the compounds. The described compounds can optionally contain an additional amino acid residue to which the PEG is conjugated, including, for example, a residue that contains additional amine, for example, lysine.
PEGylated peptides are known in the art to increase the serum half-life of conjugated peptides. Several methods are known in the art for the formation of PEGylated peptides. For example, the PEG moiety can be attached to the amino terminus, to the carboxy terminus or via a side chain of the claimed peptide, optionally through the presence of a linking group. In other embodiments, the PEG moiety may be attached to the sulfur of an amino acid that contains thiol, for example, cysteine, or it may be attached to the side chain of an amino acid that contains amine, for example, lysine.
PEG groups will generally be attached to the described compound by acylation or alkylation by means of a reactive group in the PEG portion (for example, an aldehyde, amine, oxime, hydrazine thiol, ester or carboxylic acid group) to a reactive group in the described compound (for example, an aldehyde, amine, oxime, hydrazine, ester, acid or thiol group), which may be located at the amino terminal, carboxy terminal or at a side chain position of the described compound. One approach to the preparation of PEGylation of synthetic peptides consists of combining by linking a conjugate in solution, a peptide and a portion of PEG, each housing a functional group that is mutually reactive with each other. Peptides can be easily prepared using conventional phase-in-solution or solid-phase synthesis techniques. The conjugation of the peptide and PEG is typically done in an aqueous phase and can be monitored by reverse phase HPLC. PEGylated peptides can be easily purified and characterized using standardized methodologies known to those skilled in the art.
One or more individual subunits of the described compounds can also be modified with various derivatizing agents that are known to react with specific side chains or terminal residues. For example, lysinyl residues and amino terminal residues can be reacted with succinic anhydride or other similar carboxylic acid anhydrides which reverse the charge of the lysinyl or amino residue. Other suitable reagents include, for example, imidoesters such as, for example, methyl picolinimidate; pyridoxal; pyridoxal phosphate; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione and transaminase-catalyzed reaction with glyoxalate. Arginyl residues can be modified by reaction with conventional agents such as, for example, phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin.
In addition, the described compounds can be modified to include non-cationic residues that provide immunogenic residues useful for the development of antibodies for bioanalytical ELISA measurements, as well as for assessing immunogenicity. For example, the described compounds can be modified by incorporating tyrosine and / or glycine residues. Specific modifications of tyrosyl residues are of particular interest for the introduction of spectral labels on tyrosyl residues.
Non-limiting examples include reaction with aromatic diazonium or tetranitromethane compounds. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyl tyrosyl and 3-nitro derivatives, respectively. Kits that comprise the disclosed compounds
The invention also provides kits for carrying out the therapeutic regimes of the invention. These kits comprise therapeutically effective amounts of the described compounds that have activity as a CaSR modulator, in pharmaceutically acceptable form, alone or in combination with other agents in pharmaceutically acceptable form. Preferred dosage forms include the compounds described in combination with sterile saline, dextrose solution, buffered solution, sterile water or other pharmaceutically acceptable sterile fluid. Alternatively, the composition can include an antimicrobial or bacteriostatic agent. Alternatively, the composition can be lyophilized or dried. In that case, the kit may further comprise a pharmaceutically acceptable solution, preferably sterile, to form a solution for injection purposes. In another embodiment, the kit may further comprise a needle or syringe, preferably packaged in a sterile manner, for injection of the composition. In other embodiments, the kit further comprises a means of instruction for administering the composition to an individual. The means of instruction may be a written package insert, an audio tape, an audiovisual tape, or any other means of instructing the administration of the composition to an individual.
In one embodiment, the kit comprises: (i) a first container that contains a described compound that has activity as a CaSR modulator; and (ii) means of instruction for use. In another embodiment, the kit comprises: (i) a first container that contains a compound as described herein, and (ii) a second container that contains a pharmaceutically acceptable carrier for dilution or reconstitution.
In another embodiment, the kit comprises: (i) a first container that contains a described compound that has activity as a CaSR modulator; (ii) a second container that contains an anti-cancer agent; and (iii) means of instruction for use.
In one embodiment, the anti-cancer agent is an agent selected from the group consisting of bisphosphonate agents, hormone replacement therapy agents, vitamin D therapy, vitamin D analogs, for example, ZEMPLAR ™ (paricalcitol); CALCIJEX® (calcitriol), ONE-ALPHA® (alfacalcidol) and HECTOROL® (doxercalciferol), low dose PTH (with or without estrogen) and calcitonin.
In related aspects, the invention provides manufactured articles that comprise the contents of the kits described above. For example, the invention provides a manufactured article that comprises an effective amount of a described peptide, alone or in combination with other agents, and means of instruction that indicate the use for the treatment of diseases described herein. EXAMPLES
The following examples are offered to illustrate, but not limit, the compounds and methods described herein. Various modifications can be made by those skilled in the art, without departing from the true spirit and scope of the object of study described here. Example 1 Cationic compounds with PTH reduction activity Model of renal failure
A rat model of acute renal failure (also called model 1K1C) was developed to stimulate the pathology of SHPT associated with end-stage renal disease. The model exhibits pathological features of hyperparathyroidism associated with an absence of renal function, specifically the significant elevation of plasma PTH and a reduction in serum calcium. The development of this model allowed the additional characterization of compounds described in the context of an individual with renal dysfunction and high PTH. Typical baseline levels of PTH in this model averaged approximately 450 pg / ml.
The 1K1C model of acute renal failure involves removal of a kidney, followed by exposure of the remaining kidney to 45 minutes of ischemia and 48 hours of reperfusion. Ischemia / reperfusion (I / R) damage subsequent to the remaining kidney results in significant necrosis and renal failure. Serum creatinine levels were elevated for more than 24-48 hours after I / R injury (data not shown). Also as a result of the resulting renal dysfunction, total PTH levels are dramatically increased from pre-injury I / R levels of approximately 100 pg / ml. By about 48 hours post-I / R, plasma PTH levels were elevated to approximately 450 pg / ml (an increase of approximately 5 times) and, in some cases, even reached approximately 1,200 pg / ml. This reproducible increase in serum creatinine and PTH provided a robust model that mimics the physiological observed in patients with ESRD.
Cinacalcet hydrochloride (SENSIPAR®), an approved calcimimetic agent that is used to reduce PTH for the treatment of SHPT, was tested in the 1K1C model of acute renal failure. Oral administration of cinacalcet at 30 mg / kg significantly reduced PTH by approximately 50% for up to 6 hours. This result is consistent with published preclinical data for cinacalcet (Nemeth et al., J. Pharmacol. Exp. Ther., 308 (2): 627-35 (2004)) and validates that the 1K1C model of acute renal failure it is an adequate model for assessing the activity of calcimimetics for this indication.
The protocol used in this study is as follows: male Sprague-Dawley rats were purchased from Charles River Laboratories (Hollister, CA; weight requested when purchasing 250-275 g). For studies with test articles, the animals were pre-cannulated in the femoral and jugular veins for drug administration and blood withdrawal, respectively. The animals were kept in a temperature-controlled environment with a constant cycle of 12 hours of light / 12 hours of darkness and free access to food and water at all times. All experimental procedures with animals were performed according to the IACUC guidelines.
General anesthesia was induced and maintained by intraperitoneal (IP) injection of sodium pentobarbital (5.2%, 0.4 ml / rat). For animals that received 45 minutes of renal ischemia, an additional IP injection of sodium pentobarbital (5.2%, 0.1 ml / rat) was given to maintain the anesthetic plan. Blood was collected for PTH measurements after the administration of compounds in normal rats under continuous anesthesia with isofluorane.
A clean aseptic technique was used throughout the procedure. After the rats were anesthetized, the abdomen was scraped with electric scissors before the operation and the skin was cleaned with a 70% alcohol solution.
For model development studies, the left femoral vein was cannulated with a PE-10 tube to draw blood. Both kidneys were exposed through a laparotomy. A right nephrectomy was performed after the right renal pedicle and the ureter were ligated with 2-0 double silk sutures. After confirmation of the absence of bleeding in the right pedicle, the left renal artery was carefully dissected and clamped with microvascular forceps to induce global renal ischemia. Renal ischemia was confirmed by observing an overall white-gray color change (bleaching). The abdominal incision was temporarily covered with gauze to help maintain the temperature of Organs abdominal organs. After 45 minutes, the planned period of ischemia, the forceps were removed and the flow of the left renal artery was considered restored by observing a global restoration of red color. The abdominal incision was closed in layers with 2-0 silk sutures. The animal was then recovered from anesthesia. Physiological parameters, including body temperature (36-37.5oC) and body weight, were measured throughout the procedure. Body temperature was monitored and maintained using a rectal probe-heating pad feedback system.
Approximately 48 hours after 1K1C surgery (I / R injury), the animals were dosed with various compounds to measure the effects on plasma PTH and calcium. In most cases, the test articles were administered by IV infusion (infusion time of 5, 10 or 30 minutes), although in some studies the compounds were administered by IV bolus or subcutaneous bolus (SC) injection. For drug administration and blood withdrawal, the animals were anesthetized with isofluorane.
Blood samples were collected periodically over the course of the study. Serum samples were analyzed for calcium levels and plasma samples were analyzed for PTH. Depending on the range of baseline PTH values for individual rats, all data are normalized to pre-dosing (baseline) levels. Serum creatinine was measured using a commercially available kit from BioAssay Systems (Hayward, Ca), catalog number DICT-500. The analyzes were performed according to the manufacturer's instructions. B. Testing of compounds in the renal failure model
Compounds with the following sequences were prepared for testing in the renal failure model: Ac-crrrr-NH2 (SEQ ID. No.: 4), n = 4, Ac-crrrrr-NH2 (SEQ ID. No.: 5), n = 4, Ac-crrrrrr-NH2 (SEQ ID NO: 6), n = 7, Ac-crrrrrr-NH2 (SEQ ID NO: 7), n = 4 and control saline, n = 2. The peptides were administered to the animals at a dose of 3 mg / kg by an IV infusion in 30 minutes. Prior to dosing, a blood sample was taken to determine the pre-dosage baseline plasma concentration of PTH. The results are shown in Fig. 1 as follows: Ac-crrrr-NH2 (SEQ ID NO: 4, diamonds), Ac-crrrrr-NH2 (SEQ ID NO: 5, squares), Ac-crrrrrr-NH2 (SEQ ID NO: 6, triangles) and Ac-crrrrrrr-NH2 (SEQ ID NO: 7, open squares). Example 2 In vitro cell assay in HEK-293 cells that express the human calcium sensor receptor
2 93T human embryonic kidney (HEK) cells were seeded in a T25 flask at 2 million cells per flask and were incubated at 37 ° C in 5% CO2 overnight. The next day, these cells were transfected with a human CaSR receptor using lipofectamine 2000 transfection reagent and, 24 hours post-transfection, the cells were seeded in 384-well plates at 8,000 cells / well. The assays were performed 48 hours after transfection. In some cases, EC50 values were determined by measuring the production of inositol monophosphate in HEK-293 cells, stably transfected with the human calcium sensor receptor (see Table 1).
The cell culture medium was aspirated from the wells and replaced with 28 µl of IX stimulation buffer (10 mM Hepes, 1 mM CaCl2, 0.5 mM MgCl2, 4.2 mM KC1, 14 6 mM NaCl, 5.5 mM glucose, 50 mM LiCl pH 7.4). The cells were incubated with compounds in various concentrations (1 mM or 300 pM the largest and additional serial log% dilutions) at 37 ° C for 1.5 hours, before the end of the reaction. The production of IPi was determined in cells using the Cisbio IP-One Tb kit (621 PAPEC) and according to the manufacturer's instructions. Briefly, the incubation with the compound was terminated by sequential addition of D2-labeled IPi and cryptate-labeled anti-IPi in lysis buffer and additional incubation at room temperature for 60 minutes. The plates were read at 620 nm and 668 nm with excitation of 314 nm. Non-transfected 293 cells were used as a negative control.
The fluorescence ratio at 668 nm and 620 nm was determined, and IPi concentrations were calculated from standard curves (generated with Graph Pad Prism version 4) using known concentrations of IPi standards. EC50 values were calculated based on the values of the fluorescent ratio (OD 668 nm) / (OD 620 nm) using non-linear regression curve adjustment in the Prism software.
Peptides and conjugates were prepared by solid phase chemistry on a 0.25 mmol scale on an automated ABI synthesizer. The sequential coupling of Fmoc-amino acids (4 eq, Anaspec) to the Rink-amide resin (NovaBiochem) was obtained using HBTU / DIEA activation. The assembled peptide was cleaved with a cocktail of TFA (phenol (5%), triisopropylsilane (2.5%) and water (2.5%); 10 ml per gram of resin) and isolated by precipitation with diethyl ether. After purification by HPLC Ci8, the final product was isolated in the form of TFA salt by lyophilization of appropriate fractions, and characterized by HPLC (> 95% purity) and LC-MS (confirmed PM). Example 3 In vivo administration of compounds with cationic subunits
The peptides were administered intravenously at a dose of 0.5 mg / kg in normal Sprague-Dawley rats anesthetized with isofluorane. A group of control rats was treated with saline. Blood was drawn before dosing and every hour for 4 hours. The rats were maintained under isofluorane anesthesia throughout the study. The plasma PTH concentration was measured by ELISA, which detects intact bioactive PTH 1-84 (Immutopics International catalog number 60-2700), and the area under the cumulative curve for AUC was calculated for the data points, including 1-4 hours. The percentage reduction in PTH was calculated according to the following formula: AUC treated with compound / A-UCCOI1 saline control * 100. Example 4 Structure-activity relationship studies: in vivo activity
The peptides tested here identified as ID. SEQ. N °: 26 (Ac-carrrar-NH2) and as ID. SEQ. No. 29 (Achrar-NH2) were tested in vitrous using HEK-293 cells transfected with CaSR, according to the procedure in Example 2. The peptides were also tested in vivo by administration as an IV bolus to Sprague-rats Normal Dawley at doses of 9 mg / kg for ID. SEQ. No.: 29 and 0.5 mg / kg for the ID. SEQ. No. 26. An intravenous (IV) saline bolus was used as a control. Plasma PTH levels (K2EDTA) were assessed before dosing and 1, 2, 3 and 4 hours after dosing. The rats were maintained under isofluorane anesthesia throughout the study. The results are shown in Figs. 2A-2B, presented as group mean + standard deviation (SD). In Fig. 2B, PTH is shown as a percentage of the pre-dose baseline value. Example 5 Structure-activity relationship studies: D- and L-amino acid subunits
A series of compounds that have an L-amino acid residue that replaces a D-amino acid residue have been prepared. The compounds were administered as an IV bolus to normal Sprague-Dawley rats at a dose of 0.5 mg / kg. An intravenous (IV) saline bolus was used as a control. Plasma PTH levels (K2EDTA) were assessed before dosing and 1, 2, 3 and 4 hours after dosing, and AUC was calculated as described above. The rats were maintained under isofluorane anesthesia throughout the study. The results are shown in Table 4 above. Example 6 Structure-activity relationship studies: histamine release
To assess the effect of positive net charge on histamine release associated with a compound, peptides containing 4 to 7 cationic residues (arginine) were generated and tested for their ability to trigger histamine release in vivo. The peptides tested included: (i) Ac-crrrr-NH2 (SEQ ID NO: 4), (ii) Ac-crrrr-NH2 (SEQ ID NO: 5), (ii) Ac- crrrrrr-NH2 (SEQ ID NO: 6) and (ív) Ac-crrrrrrrr-NH2 (SEQ ID NO: 41).
Male Sprague-Dawley rats were obtained (Charles River) pre-cannulated in the femoral and jugular veins for drug infusion and blood withdrawal, respectively. All IV drug treatments were performed under anesthesia (isofluorane). The animals were dosed by a 1-minute IV bolus in a total volume of 0.5 ml. Blood samples were collected at 5, 15 and 30 minutes after bolus IV to generate plasma samples (K2EDTA) for histamine analysis. For IV infusion studies in 30 minutes, the sample was collected at the end of the infusion. In some cases rats were used in the 1K1C model of acute renal ischemia.
An equal volume of saline was injected after each blood draw to replace the lost volume. Approximately 0.2 ml of blood was drawn at each time point using syringes pre-coated with EDTA to facilitate serum collection.
Histamine ELISAs were performed on diluted plasma using the "Histamine Enzyme Immunoassay" (EIA) immunoassay kit (Catalog No. A05890, SPI-BIO, Montigny le Bretonneux, France). The EIA histamine kit is a competitive derivatized amplified enzyme immunoassay that detects histamine within the range of 40 pg / ml to 5,500 pg / ml. The samples were analyzed in duplicate according to the manufacturer's protocol.
Lyophilized peptides (TFA salts) were weighed and the recorded mass was adjusted for the peptide content. Solutions were prepared by dissolving the material in normal saline to generate the desired peptide concentration. In some cases, the molarity of the peptide has been adjusted to allow comparison between peptides. The peptides were administered by bolus IV in a dose equivalent in molar terms to the ID. SEQ. No.: 41 (ie 0.7 pmol / rat) by a 1 minute IV bolus and plasma histamine was measured before dosing (pre-dose), 5, 15 and 30 minutes after dosing. Data are presented as group means (n = 2) ± SD. Histamine release is shown as a change in the number of times from pre-dose (baseline) levels. The results are shown in Fig. 3. The data are presented as group means (n = 2) ± SD. Example 7 Structure-activity relationship studies: histamine release
For in vitro evaluation of histamine release, peritoneal mast cells from isolated rats were isolated by performing peritoneal lavage using cold HBSS + 25 mM HEPES pH 7.4 containing heparin (5 p / ml). The cells were washed twice in stimulation buffer (HBSS + 25 mM HEPES pH 7.4) and incubated with 10 pM of compound in stimulation buffer (HBSS + 25 mM HEPES pH 7.4) for 15 minutes in a 96-well plate (106 / well) at 37 ° C. The cell supernatant was analyzed for histamine using the EIA histamine kit (Cayman # 589651). The data are shown in Table 10.
For in vivo evaluation of histamine release, compounds were dosed in normal anofluorane anesthetized rats at 2 mg / kg per IV bolus (administered over less than one minute). Plasma histamine was measured 5 minutes after administration of the compound (Cayman histamine EIA # 589651). The data are shown in Table 11.
The abbreviations used here and, in particular, in Tables 10-11, are summarized here.

The compound Ac-c (C) arrrar-NH2 (SEQ ID NO: 3) was prepared for comparison with the compound Ac-carrrar-NH2 (SEQ ID NO: 26). In the compound Ac-c (C) arrrar-NH2 (SEQ ID NO: 3), the thiol containing subunit at the Xi position is conjugated by means of a disulfide bond to an L-Cys residue. The two compounds were administered via bolus IV to animals with the 1K1C model of acute renal failure at doses of 0.3 and 0.5 mg / kg. Plasma PTH levels were assessed before dosing and 10 periodically for 24 hours after dosing. The results are shown in Fig. 10, in which the data shown are means of the ± SEM groups, where, as a function of time, in hours, in rats with model 1K1C of acute renal failure, the compound Ac-c (C) arrrar-NH2 (SEQ ID. NO .: 3) is represented by squares (0.3 mg / kg, n = 5) and symbols * (0.5 mg / kg, n = 6) and the compound Ac- C (Ac-C) arr-NH2 (SEQ ID. N °: 141) by triangles (0.3 mg / kg, n = 8) and diamonds (0.5 mg / kg, n = 7). Example 9 Transdermal release facilitated by micropores of calcimimetic agents
To assess the systemic release of a calcimimetic agent, Ac-crrrrrr-NH2 (SEQ ID NO: 6) was administered to hairless CD rats transdermally using a reservoir. Ac-crrrrrr-NH2 (SEQ ID. N °: 6) was applied as a 10% saline solution to an area of approximately 1 cm2 on the back of CD® rats without hairs that were microporated by 5 passes of one 1.0 mm dermal cylinder under moderate pressure. A polystyrene chamber (ID 9.5 mm) was glued over the microporated area of the skin to create a drug reservoir in which the Ac-crrrrrr-NH2 solution (SEQ ID NO: 6) or saline was applied. A 10% solution of Accrrrrrr-NH2 (SEQ ID. NO: 6) was administered to the reservoir chamber in two rats, and saline alone was administered to the reservoir chamber in a rat. The reservoirs were covered with tape to prevent evaporation. Blood samples were taken over a period of 4 hours and the plasma was analyzed for PTH levels by ELISA. The results are shown in Fig. 11. Example 10 Sustained release of calcimimetic agents by transdermal patch facilitated by micropores
To further assess the systemic release of a calcimimetic agent, Ac-c (C) arr-NH2 (SEQ ID. NO: 3) was administered transdermally to normal rats using a transdermal patch. Normal rats were treated with a microneedle arrangement and a transdermal patch system. A small area of hair on the back of Sprague-Dawley rats (approximately 350 g) was clipped using scissors and a skin area was microporated using an arrangement of 14 x 14 (approximately 1 cm2) of microneedles (approximately 0.5 mm). A transdermal patch system containing 10% (by weight) solution of Ac-C (C) arrrar-NH2 (SEQ ID. NO .: 3) in saline was placed over the microporated area and left in place for approximately 30 hours. Blood samples were collected from the rats periodically over the course of 30 hours and plasma samples were analyzed for PTH levels by ELISA. The results are shown in Fig. 12. Example 11 Infusion of calcimimetic agents
To further assess the effect of the PTH reduction of the calcimimetic compound Ac-c (C) arrrar-NH2 (SEQ ID NO: 3), it was administered by IV infusion of a very low dose to normal rats and rats with insufficiency to identify the lowest dose required to be administered by infusion, transdermal patch system or other means of sustained release to achieve a significant reduction in PTH. Males of normal Sprague-Dawley rats (250-300 g) were infused intravenously for 6 hours with Ac-c (C) arr-NH2 (SEQ ID NO: 3) at dose rates of 1 pg / kg / h, 3 pg / kg / h and 10 pg / kg / h. Blood samples were collected before dosing, in 2 hours, 4 hours, 6 hours (immediately before the end of the infusion; EO1) and 8 hours (2 hours post-EOl) and the plasma was analyzed for PTH levels by ELISA . The data are shown in Fig. 13, in which rats treated with 1 pg / kg / h (squares), 3 pg / kg / h (diamonds) and 10 pg / kg / h (triangles) for 6 hours were effective in producing a significant reduction in PTH at 5 from the baseline level over the course of the infusion.
A similar study was carried out in rats with the 1K1C model of acute renal failure. Model 1K1C rats were infused intravenously with Ac-c (C) arr-NH2 (SEQ ID NO: 273 at dose rates of 30 pg / kg / h and 100 10 pg / kg / h for 6 hours Blood samples were collected before dosing (Pre), in 2 hours, 4 hours, 6 hours (immediately before the end of the infusion; EO1), 8 hours (2 hours post-EOl) and 24 hours, and the plasma was analyzed for PTH levels by ELISA The data are shown in Fig. 14A (30 pg / kg / h, diamonds, and 100 pg / kg / h, squares) and the serum calcium for animals is shown in Fig. 14B .

权利要求:
Claims (15)
[0001]
1. A compound characterized by comprising a peptide and a conjugation group, in which the peptide comprises the amino acid sequence of the formula: X1 - X2 - X3 - X4 - X5 - X6 - X7 where: X1 is D-cysteine; X2 is D-arginine, D-alanine, D-valine, D-leucine, D-phenylalanine, D-serine, D-glutamine, D-norleucine or D-norvaline; X3 is D-arginine; X4 is D-arginine or a non-cationic amino acid, where X4 is not glycine, proline or an amino acid with an acidic side chain; X5 is D-arginine; X6 is D-alanine, D-glycine or D-serine; X7 is D-arginine; and at least two of X2, X3 and X4 are, independently, a cationic subunit; and wherein the peptide is linked by a disulfide bond at its N-terminal residue to the conjugation group.
[0002]
2. Compound according to claim 1, characterized in that the compound comprises Accrarar-NH2 (SEQ. ID. NO: 25) or Ac-carrrar-NH2 (SEQ. ID. NO: 26).
[0003]
3. A compound according to claim 1, characterized by the fact that the peptide is 8 to 11, 8 to 10 or 8 to 9 amino acids in length.
[0004]
A compound according to any one of claims 1 to 3, characterized by the fact that the conjugation group is selected from L-cysteine, D-cysteine, homocysteine, glutathione, PEGylated cysteine or the peptide sequence as defined in any one of claims 1 to 3.
[0005]
5. A compound according to claim 4, characterized by the fact that the conjugation group is cysteine, in which the cysteine is n-acetylated cysteine.
[0006]
6. Compound according to claim 4 or 5, characterized by the fact that it comprises:
[0007]
7. A compound according to claim 1, characterized by the fact that the peptide comprises a sequence of amino acids which is to be carried (SEQ ID NO: 2).
[0008]
A compound according to claim 7, characterized by the fact that the peptide is chemically modified at the N-terminus, the C-terminus, or both.
[0009]
A compound according to claim 7 or 8, characterized by the fact that the compound is Ac-c (C) arr-NH2 (SEQ. ID. NO: 3).
[0010]
A compound according to any one of claims 1 to 5, characterized by the fact that the compound is Ac-c (C) rrar-NH2 (SEQ. ID. NO: 28).
[0011]
A pharmaceutical composition characterized by comprising a compound, as defined in any one of claims 1 to 10, and at least one pharmaceutically acceptable excipient or carrier.
[0012]
Pharmaceutical composition according to claim 11, characterized in that it further comprises a second therapeutic agent which is vitamin D, a vitamin D analog or cinacalcet hydrochloride.
[0013]
13. Use of a compound, as defined in any one of claims 1 to 10, characterized by being in the preparation of a composition for treating secondary hyperparathyroidism (SHPT) or hypercalcemia disorder in an individual.
[0014]
14. Use of a compound, as defined in any one of claims 1 to 10, characterized by being in the preparation of a composition to decrease the levels of parathyroid hormone in an individual.
[0015]
Use of a compound according to claim 13 or 14, characterized in that it further comprises a second therapeutic agent which is vitamin D, a vitamin D analogue or cinacalcet hydrochloride.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-01-29| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2019-06-11| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-04-28| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

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2020-09-29| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-08| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 08/12/2020, OBSERVADAS AS CONDICOES LEGAIS. |

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2021-05-25| B16C| Correction of notification of the grant|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 29/07/2010 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

优先权:

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申请号 | 申请日 | 专利标题

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US22969509P| true| 2009-07-29|2009-07-29|

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US61/229,695|2009-07-29|

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US25581609P| true| 2009-10-28|2009-10-28|

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US61/255,816|2009-10-28|

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US31363510P| true| 2010-03-12|2010-03-12|

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US61/313,635|2010-03-12|

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PCT/US2010/043792|WO2011014707A2|2009-07-29|2010-07-29|Therapeutic agents for reducing parathyroid hormone levels|

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