PTH analogs for treating hypoparathyroidism

Abstract

Novel derivatives of parathyroid hormone are provided that have an extended duration of action compared to known natural parathyroid hormone agonist peptides while minimizing excessive effects immediately after administration. Compositions containing the novel parathyroid hormone conjugates can be used to treat hypoparathyroidism and osteoporosis.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under U.S. Provisional Patent Application No. 63 / 030004, filed on 26 May 2020, and U.S. Provisional Patent Application No. 63 / 033586, filed on 2 June 2020, the disclosures of which are expressly incorporated herein. Integration by referencing electronically submitted documents The computer-readable nucleotide / amino acid sequence listing submitted with this specification and identified below is incorporated in its entirety by reference: an 80-kilobyte ACII (text) file named "338101Final_ST25.txt" created on 24 May 2021. [Background technology]

[0002] Through the secretion of parathyroid hormone (PTH), the parathyroid glands regulate blood calcium within a very narrow range (8-10 mg / dL in adults). Calcium stored in the bones is released in response to PTH, supporting central nervous system function, vasoconstriction and muscle contraction, enzyme and hormone secretion, and blood clotting. Mechanistically, PTH increases calcium concentration by stimulating the production of a physiologically active form of vitamin D in the kidneys, while simultaneously releasing calcium and phosphate from the bones. This signals the kidneys to eliminate excess phosphate and maximize tubular reabsorption of calcium within the kidneys.

[0003] Hypoparathyroidism (hypo PT) is a rare disorder characterized by insufficient production of parathyroid hormone (PTH). Patients with hypo PT excrete excess calcium in the urine, have excess phosphate in the blood, and have abnormally low bone turnover. Until Natpara® (rhPTH) of NPS / Shire was approved by the U.S. Food and Drug Administration (FDA) in January 2015, hypoparathyroidism was one of the very few classic endocrine disorders that were not treated with hormone replacement therapy. Natpara® offers only moderate efficacy compared to standard treatment consisting of high-dose calcium and vitamin D supplementation. Nevertheless, the drug has been well-received since its commercial launch in 2015, demonstrating a significant unmet need that exists in the treatment of hypo PT.

[0004] Because hypo PT is a rare disease, drugs developed for hypo PT benefit from FDA and European Medicines Agency (EMA) orphan drug designations. Each new competitor is aimed at restoring PTH in a more physiological way than Natpara®, primarily to normalize serum calcium and reduce the long-term consequences of poorly controlled disease. The competitors differ in their approach to normalizing PTH and have considerable differences in their product profiles, including route of administration, potency, frequency of administration, and therapeutic index. The compositions disclosed herein provide weekly treatments for hypo PT patients that provide patients with specific pharmacological benefits and significant convenience by stably restoring and maintaining physiological levels of PTH throughout the week.

[0005] Hypo-parathyroidism (hypo-PT) can be divided into primary and secondary disorders. Primary disorders occur when there is an inherent defect within the parathyroid gland due to a genetic cause. Hypo-PT due to genetic causes is particularly rare and is thought to account for less than 10% of all cases. Secondary, or acquired, disorders occur when previously functioning parathyroid glands are impaired, destroyed, or removed. Secondary disorders are by far the most common, accounting for approximately 90% of all cases.

[0006] At least 75% of acquired hypo-hypo-PT cases are caused by anterior neck surgery (i.e., total thyroidectomy or radical neck dissection for head and neck malignancies) due to accidental or unavoidable removal or damage to the thyroid gland and / or its blood supply. Transient hypo-PT after thyroid surgery is relatively common, occurring in an estimated 7–46% of thyroid surgeries. Transient hypo-PT resolves within weeks or months of surgery. Chronic hypo-PT is rare, occurring in 0.9–1.6% of cases in experienced endocrine surgeons and surgical centers with a high number of cases. However, a high rate of 6.6% has been reported after thyroid surgery, highlighting the importance of expertise and experience in avoiding permanent damage resulting in hypo-PT.

[0007] Following anterior neck surgery, the second most common acquired cause of hypo PT in adults is thought to be autoimmune disease. This can affect only or multiple endocrine glands. Autoimmune-mediated diseases are estimated to cause less than 10% of acquired hypo PT. Other secondary causes may include rare invasive lesions due to metastatic disease or iron / copper excess, exposure to ionizing radiation, or be of unknown cause (idiopathic).

[0008] Estimates of the prevalence of hypo-PT in the United States range from 60,000 to 115,000. Experts suggest that the best estimate based on large health insurance claims databases is 77,000, of which 58,793 were insured. Data for other regions is extremely limited, with recorded estimates ranging from 70,000 to 267,000 for Europe, 20,000 for Japan, and 30,000 for the rest of the world.

[0009] Chronic hypothyroidism (hypo-PT) is a growing disease driven by the development of thyroid diseases, including cancer, and their treatment, most notably surgery. For example, between 1996 and 2006, the total number of thyroidectomies performed in the United States increased by 39%, from 66,864 to 92,931. The number of thyroidectomies performed annually in the United States is now estimated to be around 150,000. The number of people potentially affected by hypo-PT is increasing. Since thyroid diseases affect women more than men, over 70% of hypo-PT patients are women.

[0010] Most people with calcium levels outside the normal physiological range feel unwell. Due to the vital role of calcium in nerve and muscle function, patients with hypocalcemia may experience tingling or burning sensations in the extremities (paresthesia), muscle spasms, muscle pain, involuntary muscle contractions (tetany), dry / rough skin, difficulty concentrating, anxiety, and / or depression. Severely low levels of blood calcium can cause life-threatening laryngospasm, seizures, or arrhythmias requiring emergency treatment with IV calcium. Serious long-term consequences of hypocalcemia may include nephrocalcinosis, renal dysfunction / chronic kidney disease, calcium deposits in soft tissues, and overcalcified bones.

[0011] Hypo-PT is typically diagnosed through clinical history and laboratory examination. The diagnosis is typically characterized by low / undetectable levels of serum PTH, hypocalcemia defined as total serum calcium below the lower limit of normal, and hyperphosphatemia. Levels of active vitamin D and bone metabolism markers are also typically in the low-to-excessive range of normal, indicating increased calcium excretion. Such clinical outcomes in the context of recent cervical surgery can lead to a straightforward diagnosis of hypo-PT. However, diagnosing cases of hypo-PT can be very difficult, especially in the absence of known damage to the parathyroid glands.

[0012] There are no definitive guidelines for the management of hypo PT, so treatment is based on experience and clinical judgment. The generally acceptable primary goals of chronic treatment are to maintain serum total calcium (low to low normal range), serum phosphorus (high normal range), 24-hour urinary calcium excretion (less than 7.5 mmol / day), and calcium phosphate product (less than 4.4 mmol / L2) within acceptable ranges.

[0013] Standard treatment includes calcium, vitamin D metabolites, and sometimes thiazide diuretics. Recommended calcium supplements are calcium carbonate and calcium citrate, with the required amounts varying greatly among patients (9-fold). 1,25(OH)2D3 (calcitriol) is an active metabolite of vitamin D and helps maintain serum calcium by improving intestinal calcium absorption efficiency. Calcitriol is also administered over a wide (8-fold) dosage range. Thiazide diuretics (benzothiadiazine class drugs) may also be useful in treating hypo PT by enhancing distal tubular calcium reabsorption.

[0014] While supplementing calcium deficiency with calcium and vitamin D supplements may sound straightforward, in reality, achieving good control is extremely difficult. Most patients experience a rollercoaster of dosage levels, alternating between being too high and too low. Furthermore, calcium and vitamin D supplements do nothing to correct the underlying PTH deficiency, which would have therapeutic effects.

[0015] Patients with hypoparathyroidism (hypo-PT) experience a significant burden of the disease, with substantial negative impacts on their daily quality of life and on their caregivers, family, and friends. NPS Pharmaceuticals conducted PARADOX, an epidemiological study of 374 patients, to assess the clinical, social, and economic impacts of hypo-PT. Data were collected through a 30-minute web-based method developed with input from clinical experts, the Hypoparathyroidism Society, and patients. This method was primarily distributed via email to Hypoparathyroidism Society members, including US adults living with hypo-PT for more than six months. The results were published by Hadker et al. in Endocrine Practice in the following key points: 72% experienced more than 10 symptoms daily, with the most frequently reported being: Physical symptoms: fatigue (82%), muscle pain / cramps (78%), paresthesia (76%), tetany (70%), joint or bone pain (67%), and limb pain or weakness (53%). Emotional symptoms: anxiety (59%) and depression (53%) Cognitive symptoms: Brain fog / mental apathy (72%), inability to concentrate (65%), memory loss (61.5%), and sleep disturbances (57%). 79% required hospitalization or a visit to the emergency department. 45% reported significant impairment to their lives. 85% reported being unable to engage in household activities, and 20% experienced a (negative) change in employment status related to their illness.

[0016] Other publications also confirm the disease burden of hypo PT. A cross-sectional study by Arlt et al., published in the European Journal of Endocrinology, compared well-being and mood using validated questionnaires in 25 women with postoperative hypo PT stably managed with calcium and vitamin D treatment and 25 women with intact parathyroid function after thyroid surgery. Hypo PT patients had significantly higher overall complaint scores on Geissen's Complaints List, von Zerssen's Symptoms List, and the Symptom Checklist-90, and had elevated subscale scores for anxiety, phobic anxiety, and physical equivalence. Importantly, current conventional standard treatments for hypo PT did not restore well-being in these patients.

[0017] It is often desirable to extend the release time of an injected drug to increase its duration of action or to reduce its toxic effects. Formulations that are readily soluble in the body are usually absorbed rapidly, resulting in a sudden burst of available drug, in contrast to the more desirable, gradual release of pharmacologically active products.

[0018] While various attempts have been made to provide controlled and extended-release pharmaceutical compounds, previously disclosed technologies have not succeeded in overcoming all of the technological challenges, including achieving optimal extended release time, maximizing stability and efficacy, reducing toxicity, maximizing reproducibility in preparation, and eliminating unwanted physical, biochemical, or toxicological effects introduced by undesirable matrix materials. Therefore, there is a need for formulations that can safely and effectively extend the half-life of existing drugs and improve the therapeutic index between doses and patients. This is especially important for drugs with a narrow therapeutic index for differentiating the effective dose from the toxic dose. PTH is recognized as a drug in which even a slight excess of calcium beyond the physiological range can lead to acute and chronic adverse consequences, similar to insulin or thyroid hormones.

[0019] Mechanisms that provide extended release and enhanced therapeutic index include molecular capture at the injection site or the use of prodrug derivative forms of the drug, where the prodrug derivative is designed to delay the onset of action of the drug and extend its half-life. Delayed onset of action is advantageous in that it allows for systemic distribution of the prodrug before its activation. Therefore, administration of a prodrug can eliminate complications caused by peak activity at the time of administration and increase the therapeutic index of the parent drug.

[0020] Furthermore, receptor recognition and subsequent processing of peptide and protein agonists are the primary degradation pathways for many peptide and protein-based drugs. Thus, while the binding of a peptide drug to its receptor results in biological stimulation, it also initiates subsequent inactivation of peptide / protein-induced pharmacology through enzymatic degradation of the peptide or protein. Therefore, the use of prodrugs can also delay the duration of action of the administered drug, allowing for uniform distribution throughout the body before activation. This disclosure provides compositions and methods for safely extending the biological effects of PTH while minimizing excessive effects immediately after administration. [Overview of the project] [Means for solving the problem]

[0021] According to this disclosure, PTH peptides can be modified to prevent interaction with their corresponding receptors. More specifically, as disclosed herein, PTH peptides can be reversibly modified by the binding of a non-enzymatically self-cleaved dipeptide to the drug to form a complex that functions as either a depot composition that localizes the drug at the injection site for controlled release, and / or a prodrug that is distributed throughout the body but cannot interact with its receptor. Furthermore, such prodrug derivatives of PTH peptides can be further modified by covalent bonding of a fatty-acyl or diacyl group to the PTH peptide to enhance retention time and extend the duration of action of the underlying peptide during prodrug retention and cleavage of the prodrug portion.

[0022] Advantageously, the PTH conjugates of this disclosure safely extend the biological effects of PTH while minimizing excessive effects immediately after administration. Thus, the compositions disclosed herein provide patients with the ability to safely normalize serum calcium levels without the risk of excessive calcium elevation, which can have life-changing results. This technology should increase the ease of drug administration and lead to increased treatment compliance. As disclosed herein, the PTH analogues of this disclosure demonstrate an extended duration of action that allows for once-weekly administration to patients. However, daily administration to patients is also conceivable as a way to enable even more precise control of patients' serum calcium levels.

[0023] The compositions of the present disclosure may be administered using standard routes such as subcutaneous administration. In one embodiment, the composition is formulated for oral delivery by co-formulating the PTH conjugate of the present disclosure with an absorption enhancer capable of significantly increasing the absorption of the PTH conjugate. Sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) is a delivery agent that has been reported to enhance the permeability of molecules across a diverse spectrum, including peptides such as insulin, GLP-1, and calcitonin, and other macromolecules such as heparin. According to one embodiment, a pharmaceutical composition comprising the PTH conjugate of the present disclosure and SNAC, optionally formulated as a tablet, is provided for oral delivery.

[0024] According to one embodiment, there is provided an acylated conjugate derivative of parathyroid hormone that, when administered to a warm-blooded mammal, such as a human (Homo sapiens), has an improved therapeutic index and an extended in vivo action time. In some embodiments, the invention provides an acylated 31 amino acid PTH peptide further modified by a covalent bond of a self-cleaving dipeptide via an amide bond. In some embodiments, the invention provides an acylated 32 amino acid PTH peptide further modified by a covalent bond of a self-cleaving dipeptide via an amide bond. In some embodiments, the invention provides an acylated 33 amino acid PTH peptide further modified by a covalent bond of a self-cleaving dipeptide via an amide bond. In some embodiments, the invention provides an acylated 34 amino acid PTH peptide further modified by a covalent bond of a self-cleaving dipeptide via an amide bond. In some embodiments, the invention provides an acylated 38 amino acid PTH peptide further modified by a covalent bond of a self-cleaving dipeptide via an amide bond. More specifically, in one embodiment, the modified parathyroid hormone (PTH) is a 33, 34 or 35 amino acid peptide of SEQ ID NO: 2, 3 or 30, respectively, and the PTH peptide is optionally further modified by a covalent bond of a self-cleaving dipeptide via an amide bond at the N-terminal alpha amine of the PTH peptide.

[0025] In one embodiment, the PTH peptide is SVSEIQLMHX 10 LGX 13 HLX 16 SX 18 ERVEWLRX 26 X 27 LQDX 31 H-Z, (SEQ ID NO: 133); SVSEIQLMHX 10 LX 12 KHLX 16 X 17 X 18 ERVEWLRKKLQDVH-Z; (SEQ ID NO: 134); SVSEIQLMHX 10 LGKHLX 16SX 18 ERVEWLRKKLQDVH-Z (Sequence ID 135) and SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (Sequence ID 7); (In the formula, Z is X) 33 , X 33 F, battery 33 FX 35 , X 33 FVX 35 , X 33 FVAX 35 , X 33 FVALX 35 , X 33 FVALGX 35 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 And in some cases, Z is X 33 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 and; X 10 and X 16 These are independently Asp, Gln, or Asn; X 12 It is Gly or Aib; X 17 It is aminoisobutyric acid (Aib) or Ser; X 18 is Met, Met(O), Leu, or Nleu; X 13 , X 26 , and X 27It was independently selected from the group consisting of Arg, Glu, Asp, and Lys; X 31 is Gly or Val; X 33 and X 35 Each comprises an acylated amino acid containing a C16-C30 fatty acid or C16-C30 diacid, which is covalently linked to the side chain of an amino acid, sometimes via a spacer, and the acylated amino acid is sometimes selected from the group consisting of Lys, dLys, ornithine, Cys, and homocysteine; X 53 is Gln or Asn, depending on the case, however X 12 , X 16 and X 17 (One or fewer of these are Aib, and in some cases the C-terminal amino acid is modified to replace the carboxyl terminus with an amide.) The PTH peptide comprises an amino acid sequence selected from the group consisting of the following. In some embodiments, the PTH peptide further comprises a self-cleaving dipeptide covalently bonded to the PTH peptide via an amide bond, and optionally the self-cleaving dipeptide is covalently linked to the N-terminal alphaamine of the PTH peptide. In one embodiment, the self-cleaving dipeptide comprises a structure AB (wherein the formula, A is an amino acid containing a C16-C30 fatty acid or C16-C30 diacid, possibly covalently linked to the side chain of an amino acid via a spacer, and possibly an acylated amino acid; (B is an N-alkylated amino acid.) This includes, in certain embodiments, the PTH peptide comprises epsilon-acylated Lys, epsilon-acylated dLys, ornithine, epsilon-acylated ornithine, cysteine, S-acylated cysteine, homocysteine, or S-acylated homocysteine, and optionally the acylated amino acid is the C-terminal amino acid of the PTH peptide.

[0026] In some embodiments, the PTH peptide further comprises non-natural amino acids. In some embodiments, the PTH peptide comprises one, two, or three amino acid substitutions. In some embodiments, the amino acid substitutions are conserved substitutions. In some embodiments, the substitutions are made with non-conserved amino acids. In some embodiments, the PTH peptide of the present invention comprises one or more non-natural amino acids. Non-limiting examples of non-natural amino acids for use in the PTH peptide of the present invention include benzophenone, ketone, iodide, or phenylalanine derivatives containing azido substitutions; O-propargyltyrosine; α-aminocaprylic acid, O-methyltyrosine, O-nitrobenzylcysteine; 3-(naphthalene-2-ylamino)-2-amino-propanoic acid; para-substituted phenylalanine derivatives, e.g., p-aminophenylalanine and p-methoxyphenylalanine; meta-substituted tyrosine derivatives, e.g., 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenoside This includes stain; p-boronophenylalanine; o-nitrobenzyltyrosine; amide and carbamate-substituted lysines, e.g., 2-amino-6-((R)-tetrahydrofuran-2-carboxamide)hexanoic acid, N-ε-D-prolyl-L-lysine, and N-ε-cyclopentyloxycarbonyl-L-lysine; N-ε-acryloyl-L-lysine; N-ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]-L-lysine; azidoalanine; 2-(4'-pentenyl)alanine; alaninal; and N-ε-(1-methylcyclopro-2-encarboxamide)lysine.

[0027] In one embodiment, a PTH conjugate is provided comprising any of the PTH peptides disclosed herein and a self-cleaving dipeptide covalently bonded to the PTH peptide via an amide bond, and optionally to the N-terminal alpha-amine of the PTH peptide. In one embodiment, the conjugate is SVSEIQLMHNLX 12 X 13 HLX 16 X 17 MERVEWLRX 26X 27 LQDX 31 H-Z (SEQ ID NO: 4), SVSEIQLMHNLGX 13 HLNSMERVEWLRX 26 X 27 LQDX 31 H-Z (SEQ ID NO: 5), SVSEIQLMHNLX 12 KHLX 56 X 17 MERVEWLRKKLQDVH-Z (SEQ ID NO: 6); SVSEIQLMHX 10 LGKHLX 16 SX 18 ERVEWLRKKLQDVH-Z (SEQ ID NO: 135); and SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (SEQ ID NO: 7) (wherein, Z is X 33 F, X 53 FX 35 , X 33 FX 35 , or X 33 and optionally, Z is X 53 FX 35 or X 33 ; X 10 and X 16 are independently Asp, Gln or Asn; X 12 is aminoisobutyric acid (Aib) or Gly; X<000​​​​​​​​​​​​​​​​​​​​​27 It is selected independently of Glu and Lys; X 31 is Gly or Val; X 33 and X 35 Each contains an acylated amino acid; X 53 It is Gln, Asp, Glu, or Asn, and in some cases X 53 is Asn, Depending on the circumstances, however, X 12 , X 16 and X 17 Only one of them is Aib. It comprises a PTH peptide having an amino acid sequence selected from the group consisting of; The conjugate self-cleaving dipeptide has a general structure AB- (In the formula, A is an acylated amino acid; (B is an N-alkylated amino acid.) Includes; X 33 , X 35Each of the acylated amino acids in and A is an amino acid containing a C16-C30 fatty acid or C16-C30 diacid, which is covalently linked to the amino acid side chain, possibly via a spacer, and the self-cleaving dipeptide is linked to the PTH peptide through the formation of an amide bond between B and the primary amine of the PTH peptide, which may be located on the side chain of a lysine substitution present at position 13, 16, 19, 22, or 26 of the PTH peptide, or on the N-terminal alpha amine. In a further embodiment, the amino acid "A" of the self-cleaving dipeptide is a lysine residue acylated with a C16-C30 fatty acid or C16-C30 diacid. In one embodiment, A and B are selected to provide a chemical cleavage half-life (t1 / 2) of A and B from the PTH peptide in a standard PBS solution under physiological conditions of at least about 24 to about 240 hours, about 48 to about 168 hours, about 48 to about 120 hours, or about 70 to about 120 hours, about 80 to about 120 hours, about 90 to about 120 hours, or about 100 to about 120 hours. In one embodiment, the C-terminal amino acid of any of the PTH conjugates disclosed herein may be modified to replace the native carboxyl group with an amide.

[0028] According to one embodiment, a self-cleaving dipeptide is covalently bonded to the N-terminal alpha-amine of the PTH peptide via an amide bond, and furthermore, the PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX 33 -Z (Sequence ID 2), SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHFNX 35 (Sequence ID 15) or SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX 33 (Array of Sequence ID 2) (In the formula, X 33 and X 35 Each of these is an amino acid, and the side chain of the amino acid is acylated with a C16-C20 fatty acid or a C16-C20 diacid, and in some cases, X 33 and X 35This is independently selected from C16-C20 acylated lysine, C16-C20 acylated ornithine, C16-C20 acylated cysteine, and C16-C20 acylated homocysteine, and in some cases, X 33 and X 35 Both are C16-C20 acylated Lys; Z is selected from the group consisting of FR, FV-R, FVA-R, FVAL-R, FVALG-R, and FVALGA-R (wherein R is COOH or CONH2). The self-cleaving dipeptide has the structure:

[0029] [ka]

[0030] (In the formula, R1 is C1~C 18 A side chain selected from the group consisting of alkyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)SH, (C1-C4 alkyl)COOH, and (C1-C4 alkyl)NH2, wherein a C16-C20 fatty acid or a C16-C20 diacid is covalently linked to the side chain; R2, R4, and R8 are independently H or C1-C4 alkyl groups; R3 is a C1-C4 alkyl group, or R4 and R3, together with the atom to which they are bonded, form a 5-membered or 6-membered heterocyclic ring, for example, containing a pyrrolidine ring; R5 is NH2, however, if R4 and R3, together with the atoms to which they are bonded, form a 5-membered or 6-membered heterocyclic ring, for example, including a pyrrolidine ring, then R2 is not H. A conjugate derivative of PTH, which is a dipeptide of A, X is provided. In one embodiment, A, X 33 and X 35 The acylated amino acids

[0031] [ka]

[0032] (In the formula, n is an integer selected from 1 to 4, R 50 NH-CO(CH2) 14~20 COOH, NH-[spacer]-CO(CH2) 14~20 COOH, S(CH2) 14~20 COOH, S-[spacer]-CO(CH2) 14~20 COOH, N=N=N-[spacer]-CO(CH2) 14~20 COO, HC≡C-[spacer]-CO(CH2) 14~20 COO and CHO-[spacer]-CO(CH2) 14~20 (Selected from a group consisting of COOs) It is independently selected from amino acids having the general structure of A, X 33 and X 35 The acylated amino acid is independently selected from lysine, d-lysine, ornithine, cysteine, homocysteine, azidoalanine, 2-(4'-pentenyl)alanine, or alaninal, and the side chain of the acylated amino acid is covalently linked to a C16-C22 fatty acid or C16-C22 diacitic acid through a spacer which optionally contains an amino acid or dipeptide. In one embodiment, the spacer contains gamma glutamic acid. In one embodiment, any spacer contains two gamma glutamic acids, and optionally the two gamma glutamic acids are interposed in a functionalized PEG polymer, [COCH2(OCH2CH2)] k They are bonded to each other via HN]q (wherein k and q are integers independently selected from 1, 2, 3, 4, 5, 6, 7, or 8). In one embodiment, the spacer is -{gamma glutamate-[COCH2(OCH2CH2)2NH-COCH2(OCH2CH2)2]NH-gamma glutamate}-. In a further embodiment, the self-cleaving dipeptide is R1 is (C1~C4 alkyl)NH-CO(CH2) 14~20 COOH or (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20It is COOH; R2 and R8 are H respectively; R4 is H or CH3; R3 is CH3 and R5 is NH2, and optionally the first amino acid of the self-cleaving dipeptide is an amino acid with a D-stereochemical configuration, and the spacer is gamma glutamate, gamma glutamate-gamma glutamate dipeptide, and gamma glutamate-[COCH2(OCH2CH2)] k The structure of formula I is selected from the group consisting of HN]q-gamma-glutamic acid (wherein k is an integer selected from the range of 1 to 8 or 2 to 4, and q is 1, 2, 4, or 8). In one embodiment, k is 2 or 4 and q is 1 or 2.

[0033] According to one embodiment, a pharmaceutical composition is provided comprising one of the novel PTH conjugates disclosed herein, preferably with a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 97%, 98%, or 99%, and a pharmaceutically acceptable diluent, carrier, or excipient. Such a composition may contain the PTH conjugate disclosed herein at a concentration of at least 0.1 to 10 mg / ml or higher. In one embodiment, the pharmaceutical composition comprises an aqueous solution that is sterile and optionally stored in various packaging containers. In other embodiments, the pharmaceutical composition comprises a lyophilized powder. The pharmaceutical composition may be further packaged as part of a kit including a disposable device for administering the composition to a patient. The container or kit may be labeled for storage at ambient room temperature or refrigerated temperature.

[0034] According to one embodiment, an improved method is provided for treating hypoparathyroidism in patients requiring treatment for hypoparathyroidism. The method comprises the step of administering a therapeutically effective amount of the PTH conjugate of the present disclosure to control hypoparathyroidism. In one embodiment, the PTH peptide is acylated with a fatty acid or diacid group of sufficient size to bind to serum albumin with high affinity, and further, the PTH peptide is linked to a self-cleaving dipeptide, the amino acids of the dipeptide optionally acylated with a fatty acid or diacid group of sufficient size to bind to serum albumin with high affinity.

[0035] According to one embodiment, an improved method is provided for treating osteoporosis or osteopenia in patients requiring treatment for osteoporosis or osteopenia. The method comprises the step of administering a therapeutically effective amount of the PTH conjugate of the present disclosure to control serum calcium levels. In one embodiment, the PTH peptide is acylated with a fatty acid or diacyl group of sufficient size to bind to serum albumin with high affinity, and is further modified by binding to an autocleaved dipeptide, wherein the amino acids of the dipeptide are optionally acylated with a fatty-acyl group of sufficient size to bind to serum albumin with high affinity. [Brief explanation of the drawing]

[0036] [Figure 1] This graph shows the effect of Natpara® (SEQ ID NO: 1) on calcium urinary excretion rate in humans. It shows the time course of plasma PTH levels and calcium urinary excretion rate after subcutaneous administration of 100 μg of Natpara®. While Natpara® is effective in reversing calcium loss, this compound has an insufficient duration of action, and adequate control of serum calcium levels is limited to only one-third of the day, with excessively high and low levels for the remaining time. [Figure 2A]Figures 2A and 2B demonstrate the use of dipeptides to form prodrugs of PTH. Figure 2A is a schematic diagram of the reaction in which an amide-linked dipeptide is cleaved from a PTH conjugate to form a biologically activated PTH peptide and diketopiperazine (DKP). Figure 2B is a diagram demonstrating that the chemical cleavage is a concentration-independent, linear, zero-order reaction that does not require additional components (enzymes or catalysts). The reaction rate at a particular pH and temperature is a function of the particular dipeptide and can be adjusted from less than 30 minutes to more than 500 hours. [Figure 2B] Same as above. [Figure 3] Figures 3A and 3B demonstrate the ability of PTH analogs to stimulate the PTH receptor in cells stably transfected with PTH receptor-1 using a cAMP-producing luciferase reporter. Figure 3A provides data for SEQ ID NOs: 9, 10, 11, and 12. Figure 3B provides data for SEQ ID NOs: 9, 12, 13, and 14. The amino acid sequences of the PTH analogs are provided below. The data demonstrate that PTH analogs in which the 35-amino acid PTH peptide is covalently modified by the addition of acylated lysine (having a C18 fatty acid disaccharide chain linked to its side chain) at position 35 retain high potency at the PTH receptor (SEQ ID NO: 12), while attachment of acylated dipeptides (SEQ ID NOs: 10, 11, and 14) to PTH(1-34) reduces potency, and a combination of N-terminal acylated dipeptide attachment and carboxy-terminal amino acid acylation also results in low potency (SEQ ID NO: 13).

[0037] [Table 1] [Figure 4]This figure shows the ability of a 33-amino acid PTH analog (containing the PTH sequence of SEQ ID NO: 2) to stimulate the PTH receptor in cells stably transfected with PTH receptor-1, using a cAMP-producing luciferase reporter. SEQ ID NO: 18 is a PTH analog containing an alanine substitution at position 8 that reduces PTH activity at the PTH receptor. The data demonstrate that, similar to the 34-amino acid PTH analog, the addition of a fatty acid chain to the C-terminus of the 33-amino acid PTH analog retains the high potency of the parent compound (SEQ ID NO: 16), while the combination of dipeptide and lipo-acylation at the carboxyl terminus reduces potency (SEQ ID NO: 17).

[0038] [Table 2] [Figure 5A] Figures 5A-5C are graphs demonstrating the in vivo efficacy in mice of PTH analogs containing an acylated amino acid at the C-terminus of the PTH peptide, which dose-response increased serum calcium levels (Figure 5A) and decreased serum phosphate levels (Figure 5B) over an extended period of 72 hours. SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHK(γE-COC16H32CO2H)F-OH (SEQ ID NO: 102). Figure 5C is a graph demonstrating the in vivo efficacy in mice of additional acylated PTH analogs (PTH(1-34), K33(γE-dioxide C18)(SEQ ID NO: 102); PTH(1-33), K33(γE-dioxide C18)(SEQ ID NO: 74); and PTH(1-33), K33(γE-(miniPEG)2-γE-dioxide C18)(SEQ ID NO: 77), which increase serum calcium levels. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6]Figures 6A and 6B show the results of pharmacokinetic (PK) studies in mice to determine the onset and elimination times using PTH analogues designed for sustained duration but unable to be converted to the active drug. Each compound was administered subcutaneously to mice at a dose of 100 nmol / kg SC. Figure 6A shows the data for (dK)GSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHK(γE-(miniPEG)2-γE-COC16H32CO2H)-OH (sequence number 78); (dK)GSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHK(γE-(miniPEG)2-γE-COC18H32CO2H)-OH (sequence number 84); and (dK)(γE-(miniPEG)2-γE-COC16H32CO2H)GSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHK(γE-(miniPEG)2-γE-COC16H32CO2H)-OH (sequence number 79). Figure 6B shows the same results for sequence numbers 78, 84, and 79 as in Figure 6A, but presented in logarithmic scale. Figures 6A and 6B demonstrate the importance of fatty acyl length and the difference between the acylated PTH analog and the diacylated PTH analog of the present invention. [Figure 7A]Figures 7A and 7B show the results of pharmacokinetic (PK) studies to determine retention times in monkeys administered a single subcutaneous dose of a 25 nmol / kg prodrug PTH analogue, in which the PTH peptide is modified by alanine substitution at position 8 to produce a low-potency PTH analogue. Figure 7A is a graph demonstrating the detection levels of the prodrug PTH analogue SEQ ID NO: 94:PTH(1-33), A8, K33(γE-2xOEG-γE-dioxide C18)dK-1, N(Me)G0 and its activated form SEQ ID NO: 93:PTH(1-33), A8, K33(γE-2xOEG-γE-dioxide C18) over time after a single dose of the prodrug (SEQ ID NO: 94). Figure 7B is a graph demonstrating the detection levels of the prodrug PTH analogues SEQ ID NO: 95:PTH(1-33), A8, K33(γE-2xOEG-γE-dioxide C18), dK-1(γE-2xOEG-γE-dioxide C18), N(Me)G0 and their active forms: SEQ ID NO: 93:PTH(1-33), A8, K33(γE-2xOEG-γE-dioxide C18) over time following a single dose of the prodrug (SEQ ID NO: 95). The data demonstrates the accumulation of the active forms over time, corresponding to the decrease in the prodrug forms, resulting in a relatively consistent amount of the active forms over an extended period. The bilipacylated prodrug (SEQ ID NO: 95) achieves higher concentrations that persist for a longer period than the monolipacylated prodrug (SEQ ID NO: 94). Figure 7C shows the same drug concentration results as provided in Figure 7A, provided on a logritmetic scale. Figure 7D shows the same drug concentration results as those provided in Figure 7B, but on a logarithmic scale. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 8A]Figure 8A shows the functional response of cyclic AMP production in CHO-K1 PTHR1 cells used in the cAMPHunter Teriparitide Bioassay described in Example 11, in response to treatment with PTH(1-34) and the PTH agonist SEQ ID NO: 77 of the present invention; Figure 8B shows the non-cleavable prodrug SEQ ID NO: 79; and Figure 8C shows the functional response of cyclic AMP production in CHO-K1 PTHR1 cells used in the cAMPHunter Teriparitide Bioassay described in Example 11, in response to treatment with PTH(1-34) and the cleavable prodrug SEQ ID NO: 87. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9A] Figures 9A to 9F show the LCMS results for the PTH prodrug of the present invention, SEQ ID NO: 87, to its active drug form, SEQ ID NO: 77, in PBS buffer over an 8-day period. The peak at 7.3 minutes indicates the prodrug, and in Figure 9A, this is the only peak at day 0. In Figure 9B, at day 1, the onset of the active drug peak is present at 6.5 minutes. In Figures 9C and 9D, at days 2 and 3, respectively, less active drug is still present than the prodrug. In Figures 9E and 9F, at days 5 and 8, respectively, more active drug is present than the prodrug. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 9E] Same as above. [Figure 9F] Same as above. [Figure 10] This graph shows the LCMS assay data for Example 11, applied to zero-order reaction kinetics. The LCMS assay shows the conversion of prodrug SEQ ID NO: 87 to active drug SEQ ID NO: 77 over 192 hours, with a high linear correlation (R>0.99) and a half-life of 112 hours. [Figure 11] This figure shows the functional response of cyclic AMP production in CHO-K1 PTHR1 cells from cAMPHunter Teriparitide Bioassay after various incubation lengths from day 0 to day 8 of treatment with peptide SEQ ID NO: 87, SEQ ID NO: 77, or PTH(1-34) of the present invention, as evaluated in Figures 9A to 9F. [Figure 12] This figure shows the functional response to cyclic AMP production in CHO-K1 PTHR1 cells from cAMPHunter Teriparitide Bioassay after treatment with peptides SEQ ID NO: 77, SEQ ID NO: 109, and SEQ ID NO: 110. [Figure 13] Figures 13A and 13B show the functional response of cyclic AMP production in CHO-K1 PTHR1 cells from cAMPHunter Teriparitide Bioassay after treatment with peptides SEQ ID NO: 77, SEQ ID NO: 118, and SEQ ID NO: 120 in Figure 13A, and peptides SEQ ID NO: 77, SEQ ID NO: 122, and SEQ ID NO: 124 in Figure 13B. [Figure 14] Figures 14A and 14B are graphs of absolute serum calcium levels (Figure 14A) and relative changes in calcium levels (Figure 14B) in mice treated with either a vehicle or 20 or 40 nmol / kg of PTH analogues SEQ ID NO: 77 over a 48-hour period. [Figure 15] This figure shows the serum concentrations in rats over a period of one week after administration of the prodrug SEQ ID NO: 87 and the active drug SEQ ID NO: 77. A single subcutaneous injection of either the prodrug or the active drug was administered, and serum concentrations were measured over a one-week period. The amount of active drug induced from the prodrug was also measured (black dashed line, square symbol). [Figure 16] Figures 16A and 16B show the absolute serum calcium concentration and its changes in rats over a period of one week after subcutaneous administration of the prodrug SEQ ID NO: 87 at 30 or 60 nmol / kg. [Figure 17] Figures 17A and 17B show the absolute serum phosphate concentration and its changes in rats over a period of one week after subcutaneous administration of the prodrug SEQ ID NO: 87 at concentrations of 30 or 60 nmol / kg. [Figure 18]Figures 18A and 18B are graphs showing the changes in serum calcium levels (Figure 18A) and serum phosphate levels (Figure 18B) in Sprague-Dawley rats after daily subcutaneous injection of either the vehicle, the active drug SEQ ID NO: 77 at 20 nmol / kg, or the prodrug SEQ ID NO: 87 at 20 nmol / kg or 40 nmol / kg. [Figure 19A] Figures 19A and 19B are graphs of serum concentrations of the prodrug SEQ ID NO: 87 (Figure 19A) and the active drug SEQ ID NO: 77 (Figure 19B) in rats after seven daily subcutaneous injections starting on day 0, measured at various time points over 144 hours. A four-fold difference exists between the total concentrations of the prodrug and the active drug. [Figure 19B] Same as above. [Figure 20] Figures 20A and 20B are graphs showing calcium levels in rodents treated with 28 repeated daily doses of vehicle, 4, 8, and 12 nmol / kg prodrug SEQ ID NO: 87, starting on day 0 over a 28-day period (Figure 20A), and washout calcium levels after the final dose on day 28 (Figure 20B). [Figure 21] This figure shows the pharmacodynamic analysis by LC-MS of the conversion of the prodrug SEQ ID NO: 87 from various starting doses (4, 8, or 12 nmol / kg) to the active drug in rats after repeated administration over 28 days, as detailed in Example 5. The solid line shows the plasma concentration (nM) of the prodrug, and the corresponding dashed line shows the plasma concentration of the active drug SEQ ID NO: 77. [Figure 22] Figure 21 shows the pharmacodynamic analysis by LCMS for the shorter time period, illustrating the plasma concentration levels of the active drug (solid bars) and prodrug (bars with white dots) after discontinuation of repeated administration from Example 15, as well as the changes in plasma concentration at time points (2, 7, and 24) after the last administration. [Figure 23]This graph shows the plasma calcium concentration in surgically treated control rats (Siamese) and disease model rats, including others that underwent surgical parathyroidectomy (these were subsequently treated with either vehicle or prodrug SEQ ID NO: 87 at 10, 25, or 40 nmol / kg). Doses were given before each time measurement, which was performed at times 0, 24, 48, and 72 (the last dose), and additional measurements were taken at 144 and 72 hours after the last dose. [Figure 24] This graph shows serum calcium levels in disease model rats, including the sham control described in Example 5. The rats were administered a vehicle or various levels of the compound, 10, 25, and 40 nmol of the prodrug SEQ ID NO: 87, daily for 10 days and determined over 25 days. [Figure 25] This graph shows serum phosphate levels in disease model rats, including the sham control described in Example 5. The rats were administered a vehicle or various levels of the compound, 10, 25, and 40 nmol of the prodrug SEQ ID NO: 87, daily for 10 days and determined over 25 days. [Figure 26A] Figures 26A and 26B are graphs of serum concentrations of prodrug SEQ ID NO: 87 (Figure 26A) and active drug SEQ ID NO: 77 (Figure 26B) in monkeys after subcutaneous administration of prodrug SEQ ID NO: 87 at various concentrations (2.5, 3.75, and 5.0 nmol / kg). There is approximately a four-fold difference between the prodrug and the active drug. [Figure 26B] Same as above. [Modes for carrying out the invention]

[0039] definition In describing and claiming the present invention, the following terms will be used in accordance with the definitions set forth below.

[0040] As used herein, the term “PTH peptide” includes any peptide comprising the amino acid sequence of SEQ ID NO: 7, or any analogue of the amino acid sequence of SEQ ID NO: 7, including amino acid substitutions, additions, deletions or post-translational modifications of the peptide (e.g., methylation, acylation, alkylation, PEGylation, etc.), which, when administered to a patient, stimulate an increase in blood calcium levels.

[0041] As used herein, the term “about” means 10 percent greater than or less than a stated value or range of values, but no value or range of values ​​is intended to be limited solely to this broad definition. Each value or range of values ​​preceded by the term “about” is also intended to encompass embodiments of the stated absolute value or range of values.

[0042] As used herein, the terms “natural” or “spontaneous” define a state in which something is found in nature. “Natural amino acids” are naturally occurring amino acids obtained through natural means.

[0043] As used herein, the term “amino acid” encompasses any molecule containing both an amino group and a carboxyl group, wherein the amino group and the carboxyl group are attached to the same carbon (alpha carbon). The alpha carbon may optionally have one or two further organic substituents. Amino acids may be designated by their three-letter code, one-letter code, or, as applicable, by the name of their side chain. For example, a non-canonical amino acid containing a cyclohexane group attached to the alpha carbon is called “cyclohexane” or “cyclohexyl.” For the purposes of this disclosure, any designation of an amino acid that does not specify its stereochemistry is intended to encompass either the L-form or D-form of the amino acid, or a racemic mixture. However, in examples where an amino acid is designated by its three-letter code (e.g., Lys) or one-letter code (e.g., K), such designation is intended to specify the native L-form of the amino acid, and the D-form is specified by including a lowercase d before the three-letter or one-letter code (i.e., dLys or dK). As used herein, the designation of a particular amino acid is intended to encompass natural amino acids as well as any isotopically enriched derivatives thereof that have different molecular weights from natural amino acids but possess equivalent physical and biological properties.

[0044] As used herein, the term "hydroxyl acid" refers to an amino acid that has been modified to replace an alpha-carbon amino group with a hydroxyl group. As used herein, the term “non-coding (non-canonical) amino acid” includes any amino acid that is not an L-isomer of any of the following 20 amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr.

[0045] A "dipeptide" is the result of the bonding of an alpha amino acid or alpha hydroxy acid to another amino acid via a peptide bond. As used herein, the term “chemical cleavage,” unless otherwise specified, encompasses non-enzymatic reactions resulting in the breaking of covalent chemical bonds.

[0046] "Bioactive peptide" refers to a peptide capable of exerting biological effects in vitro and / or in vivo. As used herein, general references to peptides are intended to include peptides having modified amino-terminated and carboxyl-terminated structures. For example, an amino acid sequence specifying a standard amino acid is intended to include the standard amino acids at the N-terminus and C-terminus, as well as the corresponding C-terminal amino acid modified to include an amide group instead of the corresponding hydroxyl acid and / or terminal carboxylic acid at the N-terminus.

[0047] As used herein, an "acylated" amino acid is an amino acid containing an acyl group that is unnatural to naturally occurring amino acids, regardless of the means by which it is produced. Exemplary methods for producing acylated amino acids and acylated peptides are known in the art and include acylation of amino acids before inclusion in a peptide or peptide synthesis and subsequent chemical acylation of the peptide. In some embodiments, the acyl group causes the peptide to have one or more of the following: (i) an extended half-life in circulation, (ii) delayed onset of action, (iii) an extended duration of action, (iv) improved resistance to proteases such as DPP-IV, and (v) altered potency at the receptor for PTH.

[0048] As used herein, an alkylated amino acid is an amino acid that contains an alkyl group that is unnatural relative to a naturally occurring amino acid, regardless of the means by which it is produced. Exemplary methods for producing alkylated amino acids and alkylated peptides are known in the art and include alkylation of amino acids or peptide synthesis prior to inclusion in the peptide and subsequent chemical alkylation of the peptide.

[0049] As used herein, the term “prodrug” is defined as any compound that undergoes chemical modification before exhibiting its pharmacological effects. As used herein, “receptor” is a molecule that recognizes a specific molecule through high affinity interactions and binds to it, thereby producing a biological effect (directly or indirectly) within the cell of a host organism, or on the cell and / or tissue. “Cellular receptor” is a molecule on or inside a cell that recognizes a specific molecule and binds to it, thereby producing an effect (directly or indirectly) within the cell.

[0050] As used herein, the term “identity” refers to the similarity between two or more sequences. Identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the quotient by 100 to obtain a percentage. Thus, two copies of exactly the same sequence have 100% identity, while two sequences that have amino acid deletions, additions, or substitutions relative to each other have a lower degree of identity. Those skilled in the art will recognize that several computer programs, such as BLAST (Basic Local Alignment Search Tool, Altschul et al. (1993) J.Mol.Biol.215:403~410), using algorithms, are available to determine sequence identity.

[0051] The term "PTH peptide" is directed to peptides that have biological activity (as an agonist or antagonist) in the receptor for natural PTH and include an amino acid sequence that shares at least 70% sequence identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%) with the peptide sequence of (SEQ ID NO: 7) in an aligned portion.

[0052] As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, emulsions such as oil / water or water / oil emulsions, and various types of wetting agents. The term also includes any of the drugs approved by U.S. federal regulatory authorities for use in animals, including humans, or listed in the United States Pharmacopeia.

[0053] As used herein, the terms “phosphate-buffered saline” or “PBS” refer to an aqueous solution containing sodium chloride and sodium phosphate. While various formulations of PBS are known to those skilled in the art, for the purposes of this disclosure, the phrase “standard PBS” refers to a solution having a final concentration of 137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl, and a pH of 7.2–7.4.

[0054] As used herein, the term “pharmaceutically acceptable salt” refers to a salt of a compound that retains the biological activity of the parent compound and is not biologically or otherwise undesirable. Many of the compounds disclosed herein can form acid and / or base salts by the presence of an amino group and / or a carboxyl group or similar groups.

[0055] As used herein, the term “treat” includes the prevention of a particular disorder or condition, or the alleviation of symptoms associated with a particular disorder or condition, and / or the prevention or elimination of such symptoms.

[0056] As used herein, the “effective” dose or “therapeutically effective” dose of a drug refers to an amount that is non-toxic but sufficient to produce the desired effect. The “effective” dose varies among subjects, and even within subjects over time, depending on the individual’s age and general condition, mode of administration, etc. Therefore, it is not always possible to determine the exact “effective dose.” However, the appropriate “effective” dose in any individual case can be determined by those skilled in the art using routine experiments.

[0057] The term "parenteral" means that the drug is administered via a route other than the digestive tract, such as subcutaneous, intramuscular, intrathecal, or intravenous. As used herein, amino acid "substitution" refers to the replacement of one amino acid residue with another amino acid residue.

[0058] As used herein, the term “conservative amino acid substitution” is defined herein as an substitution within one of the following five groups: I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln; III. Polar, positively charged residues: His, Arg, Lys; Ornithine (Orn) IV. Large, aliphatic, nonpolar residues: Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine ​​(hCys) V. Large aromatic residues: Phe, Tyr, Trp, Acetylphenylalanine, Naphthylalanine (Nal) As used herein, the general terms "polyethylene glycol chain" or "PEG chain" refer to the general formula H(OCH2CH2) k This refers to a mixture of branched or linear ethylene oxide and water condensation polymers represented by OH (wherein k is at least 2). Where used herein, the terms "mini-PEG" or "OEG" refer to a structure:

[0059] [ka]

[0060] This defines functionalized polyethylene compounds that include the above. As used herein, the term "pegylated" and similar terms refer to compounds that have been modified from their native state by linking polyethylene glycol chains to the compound. A "pegylated polypeptide" is a polypeptide having a PEG chain covalently bonded to the polypeptide.

[0061] As used herein, “linker” or “spacer” refers to a bond, molecule, or group of molecules that connects two distinct entities to one another. A linker may provide an optimal distance between two entities or may supply an unstable bond that allows the two entities to separate from one another. Unstable bonds include photocleavable groups, acid-unstable moieties, base-unstable moieties, and enzymatically cleavable groups.

[0062] As used herein, “dimer” is a complex comprising two subunits covalently linked to one another via a linker. Where no specific terminology is used, the term “dimer” encompasses both homodimers and heterodimers. A homodimer comprises two identical subunits, while a heterodimer comprises two subunits that are different but substantially similar to one another.

[0063] In this specification, "C1~C n The term "alkyl" (wherein n can be 1 to 6) refers to a branched or linear alkyl group having 1 to a specified number of carbon atoms. Typical C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0064] As used herein, the term C16-C20 fatty acids refers to the structure: -CO(CH2) 15~18 The term CH3 is specified, and the term C16-C20 diacid refers to the structure: -CO(CH2) 14~18 The designation COOH, followed by the prefix "C16~C20", specifies the variable total number of carbon atoms in the compound encompassed by the designation. For example, C18 diacid has the structure: -CO(CH2) 16COOH is represented. As used herein, the general reference to acylated amino acids includes both amino acids having a fatty acid-acidified side chain and amino acids having a diacid-acidified side chain.

[0065] The physiological conditions disclosed herein are intended to include a temperature of approximately 35–40°C and a pH of approximately 7.0–7.4, and more typically, a pH of 7.2–7.4 and a temperature of 36–38°C. Since physiological pH and temperature are well-controlled within highly defined ranges in humans, the conversion rate from dipeptide / drug conjugates (prodrugs) to drugs exhibits high intra-patient and inter-patient reproducibility.

[0066] Where used herein, unless otherwise specified, the term “patient” is intended to encompass any warm-blooded vertebrate animal (including, but not limited to, livestock, horses, cats, dogs, and other pets) and humans, including individuals not receiving direct medical care. Abbreviation: Lowercase k = d-isomer of lysine γE = gamma, the l-isomer of glutamic acid. (mini PEG)2 = COCH2OCH2CH2OCH2CH2NH COC 16 H 32 CO2H = (C18 diacid) (N-Me)G = Sarcosine SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH(Sequence ID 7)=PTH Capital letter K = l-isomer of lysine Embodiment According to one embodiment, an improved analogue of parathyroid hormone (PTH) and a method for treating hypothyroidism and osteoporosis are provided. More specifically, the PTH analogues disclosed herein have an improved therapeutic index and extended duration of action compared to natural PTH and its known active fragments.

[0067] According to one embodiment, conjugate derivatives of parathyroid hormone and conjugate derivatives of PTH-related peptide (PTHrp) are provided, which, when administered to warm-blooded mammals including humans (Homo sapiens), have an extended in vivo duration of action and an improved therapeutic index compared to the unmodified parent peptide. In one embodiment, the conjugate derivative comprises an optionally acylated self-cleaved dipeptide linked to an N-terminal alpha-amine and acylation of the C-terminal amino acid. More specifically, in one embodiment, the modified parathyroid hormone (PTH) is a 33, 34, or 35 amino acid peptide comprising the sequence of SEQ ID NOs. 2, 3, or 30, respectively, having a fatty acid or diacid group (e.g., including C16-C20 fatty acids or C16-C20 diacids) covalently linked to the side chain or C-terminal amino acid of an amino acid selected from positions 13, 16, 19, 22, 26, and 33 (relative to SEQ ID NO: 7), or an amino acid sequence having at least 85%, 90%, 95%, or 97% sequence identity with SEQ ID NOs. 2, 3, 7, or 30 in aligned portions of the sequence being compared, and optionally further modified by covalent bonding of self-cleaving dipeptides. The self-cleaving dipeptide may be linked via an amide bond to any primary amine of the PTH peptide, which has a peptide side chain containing an amino acid at a position selected from, for example, positions 13, 16, 19, 22, 26, and 33 (for SEQ ID NO: 7). The amino acid side chain containing the self-cleaving dipeptide may optionally be linked to a fatty acid or diacitic acid group or other polymer to more effectively block the activity of the PTH peptide until the self-cleaving dipeptide is removed through a non-enzymatic self-cleavage mechanism. In one embodiment, the first amino acid of the dipeptide has a side chain acylated with a C16-C20 fatty acid or a C16-C20 diacitic acid.

[0068] In one embodiment, a self-cleaving dipeptide is covalently linked to the amino terminus of a PTH peptide via an amide bond, possibly with an N-terminal alpha-amine, and further comprises an acylated amino acid at one or more positions selected from the group consisting of positions 13, 16, 19, 22, 26, and 33 (relative to SEQ ID NO: 7), or as a C-terminal amino acid. In one embodiment, the modified PTH peptide of the disclosed PTH conjugate is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH (Sequence ID 7), SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHN (Sequence ID 31), SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (Sequence ID 32), SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFV (Sequence ID 33), SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVA (Sequence ID 34), SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVAL (Sequence ID 35), SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALG (Sequence ID 36), and SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGA (Sequence ID 37), The sequence comprises a sequence selected from the group consisting of the following, wherein the PTH peptide is modified, optionally by covalent bonding of a self-cleaved dipeptide at the N-terminal alphaamine of the PTH peptide, and by the attachment of an acyl group (e.g., a C14-C22 fatty acid or C14-C22 diacitor) of a size sufficient to bind to serum albumin to the amino acid side chain at positions 13, 16, 19, 22, 26, 33 or the C-terminal portion of any one of the peptides of SEQ ID NOs. 7, 31-37, optionally comprising the substitution of one or two acylated lysine residues at positions selected from the 13, 16, 19, 22, 26, 33 and C-terminal amino acids. In one embodiment, a PTH conjugate is provided in which any of the PTH peptides of SEQ ID NOs. 7, 31-37 is modified by acylated lysine substitution at the corresponding position of a native amino acid, or any analog thereof, at position 33 of the PTH peptide and / or the native amino acid of the C-terminal amino acid, or any analog thereof, and a self-cleaving dipeptide is covalently linked to the N-terminal amino acid of the PTH peptide via an amide bond, and optionally the first amino acid of the self-cleaving dipeptide is acylated, and optionally the acylated amino acid comprises a C14-C20 fatty acid, a C14-C20 diacid, a C16-C18 fatty acid, or a C16-C18 diacid.

[0069] According to this disclosure, a self-cleaving dipeptide comprises a combination of two amino acids linked to a primary amine of a PTH peptide such that, under physiological conditions, the dipeptide is spontaneously cleaved via a non-enzymatic degradation mechanism, releasing the dipeptide from the PTH peptide. In one embodiment, one of the amino acids of the self-cleaving dipeptide, optionally the first amino acid, is acylated with a C14-C20 fatty acid or C14-C20 diacid to further inhibit the activity of the PTH to which it is covalently linked. In one embodiment, the self-cleaving dipeptide is linked to a primary amine located in the side chain of the amino acid at position 13, 16, 19, 22, or 26 (for SEQ ID NO: 7) of the PTH peptide, or to an N-terminal primary amine. Thus, the presence of the self-cleaving dipeptide delays the ability of the conjugate form to interact with its target receptor until chemical cleavage releases the active form of PTH. According to this disclosure, one embodiment of the PTH conjugate of this disclosure is PTH(1-33), dK -1 (γE-(miniPEG)2-γE-COC 16 H 32 CO2H), N(Me)G 0 , K 33 (γE-2xOEG-γE-diacitic acid C18), its complete structure is: k(X)(N-Me)GSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHK(X)-OH (SEQ ID NO: 87) (In the formula, k is d-Lys; X is γE-(miniPEG)2-γE-COC 16 H 32 It is CO2H; γE is gamma, the l-isomer of glutamic acid; (miniPEG)2 is COCH2OCH2CH2OCH2CH2NH; COC 16 H 32 CO2H is a C18 diacid; (N-Me)G is sarcosine; SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHK is PTH(1-32; Sequence ID 7)+K33; K is l-Lys; -OH specifies that the C-terminal amino acid has a terminal carboxylic acid. That is the case.

[0070] In a particular embodiment, the molecular weight of the PTH conjugate of the present invention is 5873.9 daltons. In some embodiments, the present invention provides a PTH conjugate in which one or more miniPEGs in the conjugate are replaced. Non-limiting examples include:

[0071] [ka]

[0072] Includes. In some embodiments, the present invention relates to Tyr(OPO3H2):

[0073] [ka]

[0074] Cys(SO3H):

[0075] [ka]

[0076] , M(O):

[0077] [ka]

[0078] M(O)2:

[0079] [ka]

[0080] and γE:

[0081] [ka]

[0082] The present invention provides a PTH peptide containing one or more of the following: Advantageously, the cleavage rate of the self-cleaving dipeptide depends on the structure and stereochemistry of the dipeptide elements, as well as the strength of the nucleophile present on the dipeptide that induces cleavage to diketopiperazine or diketomorpholine-related entities. In one embodiment, based on a selected structure of the dipeptide, the non-enzymatic half-life (t1 / 2) of the dipeptide / drug conjugate may be selected to be between 1 and 720 hours under physiological conditions. The physiological conditions disclosed herein are intended to include a temperature of about 35 to 40°C and a pH of about 7.0 to 7.4, more typically including a pH of 7.2 to 7.4 and a temperature of 36 to 38°C. Since the physiological pH and temperature are tightly controlled within highly defined ranges, the conversion rate from the dipeptide / drug conjugate to the drug exhibits high intra-patient and inter-patient reproducibility. The chemical conversion rate determines the onset time and duration of the in vivo biological action. Therefore, the activation of the administered PTH analogs of this disclosure relies on an intramolecular chemical reaction that does not depend on additional chemical additives or enzymes, and the conversion rate is controlled by the intrinsic chemical properties of the dipeptide substituents.

[0083] In one embodiment, a self-cleaving dipeptide element is covalently bonded to a PTH peptide via an amide bond, and the dipeptide further comprises a depot polymer linked to the amino acid side chain of the self-cleaving dipeptide. In one embodiment, two or more depot polymers are linked to a single self-cleaving dipeptide element. In one embodiment, the depot polymer is selected to be biocompatible and of sufficient size so that the PTH peptide with the covalently attached dipeptide remains trapped at the injection site and / or remains unable to interact with its corresponding receptor when administered to a patient. Subsequent cleavage of the dipeptide releases it so that the PTH peptide interacts with its intended target. The selection of various combinations of substituents on the dipeptide element allows for the preparation of injectable compositions containing a dipeptide / PTH peptide mixture that releases the drug over a desired time frame. Suitable depot polymers include, but are not limited to, dextran, polylactide, polyglycolide, caprolactone-based polymers, poly(caprolactone), polyacid anhydride, polyamine, polyesteramide, polyorthoester, polydioxanone, polyacetal, polyketal, polycarbonate, polyphosphoester, polyester, polybutylene terephthalate, polyorthocarbonate, polyphosphazene, succinate, poly(malic acid), poly(amino acid), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, polysaccharides, chitin, chitosan, hyaluronic acid, and copolymers, terpolymers and mixtures thereof, as well as biodegradable polymers and copolymers thereof containing caprolactone-based polymers, polycaprolactone, and copolymers containing polybutylene terephthalate. In one embodiment, the depot polymer is selected from the group consisting of polyethylene glycol, dextran, polylactic acid, polyglycolic acid, and copolymers of lactic acid and glycolic acid, and in one specific embodiment, the depot polymer is polyethylene glycol. In one embodiment, the depot polymer comprises one or more polyethylene glycol chains linked to self-cleaving dipeptide elements, and the combined molecular weight of the depot polymer is 40,000 to 80,000 daltons.

[0084] In one embodiment, a self-cleaving dipeptide element is covalently bonded to a PTH peptide via an amide bond at the active site of PTH to form a prodrug derivative of the drug. In one embodiment, the first amino acid of the self-cleaving dipeptide has a D-stereochemical configuration. In one embodiment, the side chain of the first amino acid of the self-cleaving dipeptide is covalently linked to a portion that enhances the retention of the PTH analog in the patient's bloodstream. In one embodiment, the retention-enhancing portion linked to the first amino acid of the self-cleaving dipeptide contains an alkyl chain of sufficient size to bind to serum albumin when administered to a patient. In one embodiment, the side chain of the first amino acid of the self-cleaving dipeptide is covalently linked to an alkyl chain containing 14-30, 14-22, 16-20, 16, 18, 20, or 22 carbon atoms. In one embodiment, the first amino acid of the self-cleaving dipeptide is an alkylated or acylated amino acid with a D-stereochemical configuration. In one embodiment, the first amino acid of the self-cleaving dipeptide is covalently linked to a fatty acid or fatty diacid having a length of 14-30, 14-22, 16-20, 16, 18, 20, or 22 carbon atoms.

[0085] In one embodiment, a conjugate of a PTH peptide is provided, wherein a PTH peptide is covalently linked to a self-cleaving dipeptide. In one embodiment, the conjugate has a general structure of ABQ (wherein the formula, A is an amino acid or hydroxyl acid, and in some cases, the side chain of the amino acid or hydroxyl acid is covalently linked to a depot polymer or an alkyl or acyl chain; B is an N-alkylated amino acid; Q is an acylated or alkylated PTH peptide according to this disclosure, provided that if A is a non-acylated amino acid, then A is an amino acid with a D-stereochemical configuration. This includes. In further embodiments, for any of the PTH conjugates disclosed herein, either A or B of the AB dipeptide may be a non-coding amino acid, for example, an amino acid with a D-stereochemical configuration.

[0086] According to one embodiment, the self-cleaving dipeptide element (AB) has the structure:

[0087] [ka]

[0088] (In the formula, R1 is H, C1~C 18 Alkyl, C2~C 18 Alkenyl, (C1-C8 alkyl)OH, (C1-C8 alkyl)SH, (C2-C3 alkyl)SCH3, (C1-C4 alkyl)CONH2, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH2, (C1-C4 alkyl)NHC(NH2) + )NH2, (C0-C4 alkyl)(C5-C6 cycloalkyl),

[0089] [ka]

[0090] It comprises a side chain selected from the group consisting of the following, and optionally further comprises a C16-C30 carbon chain covalently linked to the side chain; R2 and R8 are independently H or C1-C6 alkyl groups; R4 is H, C1~C 18 Alkyl, C2~C 18 Alkenyl, (C1-C8 alkyl)OH, (C1-C8 alkyl)SH, (C2-C3 alkyl)SCH3, (C1-C4 alkyl)CONH2, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH2, (C1-C4 alkyl)NHC(NH2) + )NH2, (C0-C4 alkyl)(C5-C6 cycloalkyl),

[0091] [ka]

[0092] Selected from the group consisting of; R3 is selected from the group consisting of C1-C8 alkyl groups, or R4 and R3, together with the atom to which they are bonded, form a pyrrolidine or piperidine ring; R5 is either NH2 or OH; R 10 (where R1 and R2 are not H if R4 and R3, together with the atoms to which they are bonded, form a pyrrolidine ring.) Includes.

[0093] According to one embodiment, the self-cleaving dipeptide element (AB) has the structure:

[0094] [ka]

[0095] (In the formula, R1 is C1~C 18 The method comprises a side chain selected from the group consisting of alkyl, (C1-C8 alkyl)OH, (C1-C8 alkyl)SH, and (C1-C4 alkyl)COOH, and (C1-C4 alkyl)NH2, and optionally further comprises a C16-C30 fatty acid or C16-C30 diacid covalently linked to the side chain; R2 and R8 are independently H or C1-C6 alkyl groups; R4 is H, C1~C 18 Alkyl, C2~C 18 Alkenyl, (C1-C8 alkyl)OH, (C1-C8 alkyl)SH, (C2-C3 alkyl)SCH3, (C1-C4 alkyl)CONH2, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH2, (C1-C4 alkyl)NHC(NH2) + )NH2, (C0-C4 alkyl)(C5-C6 cycloalkyl),

[0096] [ka]

[0097] Selected from the group consisting of; R3 is selected from the group consisting of C1-C8 alkyl groups; R5 is NH2; R 10 (H, OH, or NH2) Includes.

[0098] According to one embodiment, the self-cleaving dipeptide element (AB) has the structure:

[0099] [ka]

[0100] (In the formula, R1 is C1~C 18 It comprises a side chain selected from the group consisting of alkyl, (C1-C8 alkyl)OH, (C1-C8 alkyl)SH, (C1-C4 alkyl)COOH, and (C1-C4 alkyl)NH2, and a C16-C30 fatty acid or C16-C30 diacid covalently linked to the side chain, possibly via a spacer; R2 and R8 are H, respectively; R4 is selected from the group consisting of H and C1-C8 alkyl groups; R3 is a C1-C6 alkyl group; R5 is NH2, and the spacer is gamma glutamate, gamma glutamate-gamma glutamate dipeptide, and gamma glutamate-[COCH2(OCH2OCH2) k -NH] q - Selected from the group consisting of gamma glutamate (wherein k is an integer selected from the range of 1 to 8 or 2 to 4, and q is an integer selected from the range of 1 to 4) This includes. In one embodiment, k is 2 or 4 and q is 1 or 2.

[0101] According to one embodiment, the self-cleaving dipeptide element (AB) has the structure:

[0102] [ka]

[0103] (In the formula, R1 comprises a (C1-C4 alkyl)NH2 side chain, and optionally a C16-C30 fatty acid or C16-C30 diacid is covalently linked to the side chain, optionally via a spacer; R2, R4, and R8 are independently H or C1-C4 alkyl groups; R3 is a C1-C6 alkyl group; R5 is NH2, and the spacer is gamma glutamate, gamma glutamate-gamma glutamate dipeptide, and gamma glutamate-[COCH2(OCH2OCH2) k -NH] q - Selected from the group consisting of gamma glutamate (wherein k is an integer selected from the range of 1 to 8 or 2 to 4, and q is an integer selected from the range of 1 to 4), and optionally, if R1 consists of (C1-C4 alkyl)NH2, the first amino acid of the self-cleaving dipeptide has a D-stereochemical configuration. This includes. In one embodiment, k is 2 or 4 and q is 1 or 2.

[0104] According to one embodiment, the self-cleaving dipeptide element (AB) has the structure:

[0105] [ka]

[0106] (In the formula, R1 is (C1~C8 alkyl)-CO(CH2) 14~20 CH3, (C1~C8 alkyl)S-CO(CH2) 14~20 CH3, (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3, (C1~C8 alkyl)-CO(CH2) 14~20 COOH, (C1~C8 alkyl)S-CO(CH2) 14~20 COOH and (C1~C4 alkyl)NH-CO(CH2) 14~20Selected from the group consisting of COOH; R2 and R8 are H, respectively; R4 is H, C1-C8 alkyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)SH, (C2-C3 alkyl)SCH3, (C1-C4 alkyl)CONH2, (C1-C4 alkyl)COOH, (C1-C4 alkyl)NH2, (C1-C4 alkyl)NHC(NH2) + )NH2, (C1-C4 alkyl)(C5-C6 cycloalkyl),

[0107] [ka]

[0108] Selected from the group consisting of; R3 is selected from the group consisting of C1-C6 alkyl groups, or R4 and R3, together with the atom to which they are bonded, form a pyrrolidine or piperidine; R5 is NH2; R 10 R4 is H, OH, or NH2, provided that R4 and R3, together with the atom to which they are bonded, form a pyrrolidine ring, in which case R2 is not H, and possibly the chemical cleavage half-life (t) of AB from the PTH peptide. 1 / 2 (Under physiological conditions, this is at least about 20-240 hours in a standard PBS solution.) Includes.

[0109] According to one embodiment, the self-cleaving dipeptide element (AB) has the structure:

[0110] [ka]

[0111] (In the formula, R1 is (C1~C4 alkyl)S-CO(CH2) 14~20 CH3, (C1~C4 alkyl)NH-CO(CH2) 14~20CH3, (C1~C4 alkyl)S-CO(CH2) 14~20 COOH and (C1~C4 alkyl)NH-CO(CH2) 14~20 Selected from the group consisting of COOH; R2 and R8 are H, respectively; R4 is selected from the group consisting of H and C1-C8 alkyl groups; R3 is selected from the group consisting of C1-C6 alkyl groups; R5 is NH2, and in some cases, the chemical cleavage half-life (t) of AB from the PTH peptide. 1 / 2 (Under physiological conditions, this is at least approximately 48–168 hours in standard PBS solution.) Includes. In one further embodiment, R1 is (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3, or (C1-C4 alkyl)NH-CO(CH2) 14~20 It is COOH; R2 and R8 are H, respectively; R4 is either H or CH3; R3 is a C1-C6 alkyl group; and R5 is NH2.

[0112] According to one embodiment, the self-cleaving dipeptide element (AB) has the structure:

[0113] [ka]

[0114] (In the formula, R1 is (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3, (C1~C4 alkyl)NH-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 CH3 and (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 Selected from the group consisting of COOH; R2 and R8 are H or C1-C4 alkyl, respectively; R4 and R3, together with the atoms to which they are bonded, form a piperidine ring; R5 is NH2, and in some cases, the chemical cleavage half-life (t) of AB from the PTH peptide. 1 / 2 ) is administered under physiological conditions in a standard PBS solution for at least about 72 to 144 hours, and the spacer contains gamma glutamate and -[COCH2(OCH2CH2) k -NH] q The spacer comprises one or more portions selected from, and optionally, gamma glutamate, gamma glutamate-gamma glutamate dipeptide, and gamma glutamate-[COCH2(OCH2OCH2) k -NH] q - Selected from the group consisting of gamma glutamate (wherein k is an integer selected from the range of 1 to 8 or 2 to 4, and q is an integer selected from the range of 1 to 4, and in some cases k is 2 or 4 and q is 1 or 2) Includes. In one further embodiment, R1 is (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3, (C1~C4 alkyl)NH-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 CH3 or (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 It is COOH; R2 and R8 are H, respectively; R4 is either H or CH3; R3 is a C1-C6 alkyl group; and R5 is NH2.

[0115] According to one embodiment, the self-cleaving dipeptide element (AB) has the structure:

[0116] [ka]

[0117] (In the formula, R1 is (C1~C4 alkyl)NH-CO(CH2) 16 COOH or (C1~C4 alkyl)NH-CO(CH2) 18 It is COOH; R2 and R8 are H, respectively; R4 is either H or CH3; R3 is CH3, R5 is NH2, and in some cases R1 is (C3~C4 alkyl)NH-CO(CH2) 16 (COOH is COOH, and R4 is CH3) Includes.

[0118] According to one embodiment, the self-cleaving dipeptide element (AB) has the structure:

[0119] [ka]

[0120] (In the formula, R1 is (C1~C4 alkyl)NH-[spacer]-CO(CH2) 16 COOH or (C1~C4 alkyl)NH-[spacer]-CO(CH2) 18 It is COOH; R2 and R8 are H, respectively; R4 is either H or CH3; R3 is CH3, R5 is NH2, and in some cases R1 is (C3~C4 alkyl)NH-[spacer]-CO(CH2) 16 COOH, R4 is CH3, and the spacer is gamma glutamate-[COCH2(OCH2OCH2)] k -NH] q - Gamma glutamate (wherein k is an integer selected from 1 to 8 or 2 to 4, q is an integer selected from 1 to 4, and in some cases k is 2 or 4, q is 1 or 2, and in some cases both k and q are 2)) Includes.

[0121] In one embodiment, the PTH peptide of the PTH conjugate disclosed herein is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLA PRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQX 35 (Sequence 20); AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRAX 35 (Sequence code 96); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALX 35 (Sequence 21); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 (Sequence 12); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHQFX 35 (Sequence 14); SVSEIQLMHNLX 12 X 13 HLX 16 X 17 MERVEWLRX 26 X 27 LQDX 31 HZ(sequence number 4); SVSEIQLMHNLGX 13 HLNSMERVEWLRX 26 X 27 LQDX 31 HZ(sequence number 5); SVSEIQLMHNLX 12 KHLX 56 X 17 MERVEWLRKKLQDVH-Z(sequence number 6); SVSEIQLMHX 10 LGX 13 HLX 16 SX 18 ERVEWLRX 26 X 27 LQDX 31 Hz, (sequence number 133); SVSEIQLMHX 10 LX 12 KHLX 16 X 17 X 18ERVEWLRKKLQDVH-Z;(Sequence ID 134); SVSEIQLMHX 10 LGKHLX 16 SX 18 ERVEWLRKKLQDVH-Z (Sequence ID 135); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDGH-Z (Sequence ID 22); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (Sequence ID 7); SVSEIQLMHNLGEHLNSMERVEWLRKKLQDVH-Z (Sequence ID 23); SVSEIQLMHNLGKHLNSMERVEWLREKLQDVH-Z (sequence number 24); or SVSEIQLMHNLGKHLNSMERVEWLRKELQDVH-Z (Sequence ID 25); (In the formula, Z is X 33 , X 33 F, battery 33 FX 35 , X 33 FVX 35 , X 33 FVAX 35 , X 33 FVALX 35 , X 33 FVALGX 35 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 And in some cases, Z is X 33 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35, or X 53 FVALGX 35 And in some cases, Z is X 33 F, battery 53 FX 35 , or X 33 and; X 10 and X 16 These are independently Asp, Gln, or Asn; X 12 It is aminoisobutyric acid (Aib) or Gly; X 56 It is aminoisobutyric acid (Aib) or Asn; X 17 It is aminoisobutyric acid (Aib) or Ser; X 18 is Met, Met(O), or Nleu; X 13 , X 26 and X 27 It was independently selected from the group consisting of Arg, Glu, Asp, and Lys; X 31 is Gly or Val; X 33 and X 35 Each of these includes an acylated or alkylated amino acid; X 53 It is Gln, Asp, Glu, or Asn, and in some cases X 53 is Gln or Asn, or X 53 is Asn, and in some cases, however X 12 , X 16 and X 17 Only one of them is Aib, and in some cases, the C-terminal amino acid is modified to replace the carboxyl terminus with an amide. It contains an amino acid sequence selected from the group consisting of the following.

[0122] In one embodiment, the PTH peptide of the PTH conjugate disclosed herein is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 (Sequence 12); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHQFX 35 (Sequence 14); SVSEIQLMHNLGX 13 HLNSMERVEWLRX 26 X 27 LQDX 31 HZ(sequence number 5); SVSEIQLMHX 10 LGKHLX 16 SX 18 ERVEWLRKKLQDVH-Z (Sequence ID 135); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDGH-Z (sequence number 22); or SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (Sequence ID 7); (In the formula, Z is X 33 F, battery 53 FX 35 , or X 33 and; X 10 and X 16 These are independently Asp, Gln, or Asn; X 18 is Met, Met(O), or Nleu; X 13 , X 26 , and X 27 It was independently selected from the group consisting of Arg, Glu, Asp, and Lys; X 31 is Gly or Val; X 33 and X 35 Each contains an acylated or alkylated amino acid, and may include X 33 is acylated Lys, and X 35 It is acylated Cys; X 53 (This can be Gln, Asp, Glu, or Asn) It contains an amino acid sequence selected from the group consisting of the following.

[0123] In one embodiment, the PTH peptide of the PTH conjugate disclosed herein is SVSEIQLMHNLGX 13 HLNSMERVEWLRX 26 X 27 LQDX 31 HZ (sequence number 5); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDGH-Z (sequence number 22); or SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (Sequence ID 7) (In the formula, Z is X 33 F, battery 53 FX 35 , or X 33 and; X 13 , X 26 , and X 27 It was independently selected from the group consisting of Arg, Glu, Asp, and Lys; X 31 is Gly or Val; X 33 and X 35 Each of these independently forms (C1~C4 alkyl)NH-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3, (C1~C4 alkyl)S-CO(CH2) 14~20 COOH, (C1~C4 alkyl)S-CO(CH2) 14~20 CH3, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 CH3, (C1~C4 alkyl)S-[spacer]-CO(CH2) 14~20 COOH, or (C1~C4 alkyl)S-[spacer]-CO(CH2) 14~20 It is an amino acid having a CH3 side chain; X 53 It is Gln, Asp, Glu, or Asn, and in some cases X 33 and X 35 (C1~C4 alkyl)NH-CO(CH2)14~20 COOH, (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH and (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 The spacer is an amino acid having a side chain selected from the group consisting of CH3, and the spacers are gamma glutamic acid, gamma glutamic acid-gamma glutamic acid dipeptide, and gamma glutamic acid-[COCH2(OCH2CH2)] k -NH] q - Gamma-glutamic acid (wherein k is an integer selected from the range of 1 to 8 or 2 to 4, and q is an integer selected from the range of 1 to 4) It comprises an amino acid sequence selected from the group consisting of the following. In one embodiment, k is 2 or 4 and q is 1 or 2.

[0124] In one embodiment, a PTH peptide conjugate exhibiting improved therapeutic index and improved in vivo retention time, SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAG SQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQX 35 (Sequence 20); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALX 35 (Sequence 21); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 (Sequence 12); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHQFX 35 (Sequence 14); SVSEIQLMHNLX 12 X 13 HLX 16 X 17 MERVEWLRX 26 X 27 LQDX 31 HZ(sequence number 4); SVSEIQLMHNLGX 13 HLNSMERVEWLRX 26 X 27 LQDX 31 HZ(sequence number 5); SVSEIQLMHNLX 12 KHLX 56 X 17 MERVEWLRKKLQDVH-Z(sequence number 6); SVSEIQLMHX 10 LGKHLX 16 SX 18 ERVEWLRKKLQDVH-Z (Sequence ID 135); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDGH-Z (Sequence ID 22); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (Sequence ID 7); SVSEIQLMHNLGEHLNSMERVEWLRKKLQDVH-Z (Sequence ID 23); SVSEIQLMHNLGKHLNSMERVEWLREKLQDVH-Z (sequence number 24); or SVSEIQLMHNLGKHLNSMERVEWLRKELQDVH-Z (Sequence ID 25); (In the formula, Z is X 33 , X 33 F, battery 33 FX 35 , X 33 FVX 35 , X 33 FVAX 35 , X 33 FVALX 35 , X 33 FVALGX 35 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 And in some cases, Z is X33 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 And in some cases, Z is X 33 F, battery 53 FX 35 , or X 33 and; X 10 and X 16 These are independently Asp, Gln, or Asn; X 12 It is aminoisobutyric acid (Aib) or Gly; X 56 It is aminoisobutyric acid (Aib) or Asn; X 17 It is aminoisobutyric acid (Aib) or Ser; X 18 is Met, Met(O), or Nleu; X 13 , X 26 , and X 27 It was independently selected from the group consisting of Arg, Glu, Asp, and Lys; X 31 is Gly or Val; X 33 and X 35 Each of these includes an acylated or alkylated amino acid; X 53 is Gln, Asp, Glu, or Asn, and in some cases, however X 12 , X 16 and X 17 Only one of them is Aib. It includes an amino acid sequence selected from the group consisting of; The self-cleaving dipeptide has a general structure AB- (In the formula, A is an acylated or alkylated amino acid; (B is an N-alkylated amino acid.) Includes; X 33 , X 35 Each of the acylated or alkylated amino acids in A is an amino acid containing a C16-C30 carbon chain covalently linked to its amino acid side chain, possibly via a spacer, and the self-cleaving dipeptide is linked to the PTH peptide through the formation of an amide bond between B and the N-terminal alphaamine of the PTH peptide. A conjugate is provided. In one embodiment, A and B are selected to provide a chemical cleavage half-life (t1 / 2) of A and B from the PTH peptide in a standard PBS solution under physiological conditions of at least about 24 to about 96 hours, or about 48 to about 96 hours, or about 72 to about 120 hours.

[0125] In one embodiment, a conjugate comprising a PTH peptide / PTHrP and a self-cleaving dipeptide, wherein the dipeptide is covalently bonded to the PTH / PTHr peptide via an amide bond, possibly at the N-terminal alpha-amine of the PTH peptide, and the PTH / PTHr peptide is SRRLKRAVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRATSEVSPNS KPSPNTKNHPVRFGSDDEGRYLTQETNKVETYKEQPLKTPGKKKKGKPG KRKEQEKKKRRTRSAWLDSGVTGSGLEGDHLSDTSTTSLELDSRRH-X 33 (Sequence number 39); SVSEIQLMHNLGX 13 HLNSMERVEWLRX 26 X 27 LQDX 31 HZ(sequence number 5); SVSEIQLMHNLX 12 KHLX 56 X 17 MERVEWLRKKLQDVH-Z(sequence number 6); and SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (Sequence ID 7); (In the formula, Z is X 33 , X 33 F, battery 33 FX 35 , X 33 FVX 35 , X 33 FVAX 35 , X 33 FVALX 35 , X 33 FVALGX35, X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 And in some cases, Z is X 33 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 and; X 12 It is aminoisobutyric acid (Aib) or Gly; X 56 It is aminoisobutyric acid (Aib) or Asn; X 17 It is aminoisobutyric acid (Aib) or Ser; X 13 , X 26 , and X 27 It was independently selected from the group consisting of Arg, Glu, Asp, and Lys; X 31 is Gly or Val; X 33 and X 35Each contains an acylated amino acid, and in some cases, X 33 and X 35 teeth,

[0126] [ka]

[0127] (In the formula, n is an integer selected from 1 to 4, R 50 (These are NH2, COOH, or SH) Independently selected from amino acids having the general structure of X, and in some cases, X 33 and X 35 The acylated amino acid is independently selected from lysine, ornithine, cysteine, or homocysteine, and the side chain of the acylated amino acid is covalently linked to a C16-C22 fatty acid or C16-C22 diacid, sometimes via a spacer; X 53 is Gln or Asn, depending on the case, however X 12 , X 16 and X 17 Only one of them is Aib. It includes an amino acid sequence selected from the group consisting of; The self-cleaving dipeptide has a general structure AB- (In the formula, A is an amino acid or acylated amino acid, which may be selected from cysteine ​​or lysine, and the side chain of the cysteine ​​or lysine is optionally covalently linked to a C16-C22 fatty acid or C16-C22 diacid via a spacer that optionally links the C16-C22 fatty acid or C16-C22 diacid to the amino acid side chain; B is an N-alkylated amino acid, and in some cases, B is N-methylglycine or N-methylalanine. including, A conjugate is provided.

[0128] In one embodiment, X 33 , X 35Each acylated amino acid of A is an amino acid containing a C16-C30 fatty acid or C16-C30 diacid, which is covalently linked to its amino acid side chain, possibly via a spacer, and the self-cleaving dipeptide is linked to the PTH peptide through the formation of an amide bond between B and the N-terminal alphaamine of the PTH peptide. In some cases, A has a D-stereochemical configuration. In one embodiment, a conjugate comprising a PTH peptide and a self-cleaving dipeptide, wherein the dipeptide is covalently linked to the PTH peptide, possibly via an amide bond at the N-terminal alphaamine of the PTH peptide. PTH peptide SVSEIQLMHNLGX 13 HLNSMERVEWLRX 26 X 27 LQDX 31 HZ(sequence number 5); SVSEIQLMHNLX 12 KHLX 56 X 17 MERVEWLRKKLQDVH-Z(sequence number 6); SVSEIQLMHX 10 LGKHLX 16 SX 18 ERVEWLRKKLQDVH-Z (sequence number 135); and SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (Sequence ID 7); (In the formula, Z is X 33 , X 33 F, battery 53 FX 35 is, or; Z 10 and X 16 These are independently Asp, Gln, or Asn; X 12 It is aminoisobutyric acid (Aib) or Gly; X 56 It is aminoisobutyric acid (Aib) or Asn; X 17 It is aminoisobutyric acid (Aib) or Ser; X 18is Met, Met(O), or Nleu; X 13 , X 26 , and X 27 It was independently selected from the group consisting of Arg, Glu, Asp, and Lys; X 31 is Gly or Val; X 33 and X 35 Each contains an acylated amino acid, and in some cases, X 33 and X 35 The side chain of the cysteine, homocysteine, ornithine, or lysine is independently selected from cysteine, homocysteine, ornithine, or lysine, and the side chain of the cysteine, homocysteine, ornithine, or lysine is covalently linked to a C16-C22 fatty acid or C16-C22 diacid, possibly through a spacer; X 53 is Gln or Asn, depending on the case, however X 12 , X 16 and X 17 Only one of them is Aib. It includes an amino acid sequence selected from the group consisting of; The self-cleaving dipeptide has a general structure AB- (In the formula, A is an acylated amino acid, optionally selected from cysteine ​​or lysine, wherein the side chain of the cysteine ​​or lysine is covalently linked to a C16-C22 fatty acid or C16-C22 diacid, optionally via a spacer; B is an N-alkylated amino acid, and in some cases, B is N-methylglycine or N-methylalanine. Includes; X 33 , X 35A conjugate is provided in which each of the acylated amino acids of A is an amino acid containing a C16-C30 fatty acid or a C16-C30 diacid, which is covalently linked to its amino acid side chain, possibly via a spacer, and the self-cleaving dipeptide is linked to the PTH peptide through the formation of an amide bond between B and the N-terminal alphaamine of the PTH peptide. Optionally, A has a D-stereochemical configuration.

[0129] In one embodiment, a conjugate comprising a PTH peptide and a self-cleaving dipeptide, wherein the dipeptide is covalently bonded to the PTH peptide via an amide bond, possibly by the N-terminal alpha-amine of the PTH peptide. PTH peptide SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (SEQ ID NO: 7), or a peptide different from SEQ ID NO: 7 due to one or two amino acid substitutions. (In the formula, Z is X 33 F, NFX 35 , or X 33 and; X 33 and X 35 Each of these is an amino acid containing a C16-C30 fatty acid or C16-C30 diacid, which is covalently linked to the side chain of an amino acid, and in some cases, X 33 and X 35 The side chain of the cysteine ​​or lysine is independently selected, and the side chain of the cysteine ​​or lysine is covalently linked to a C16-C22 fatty acid or C16-C22 diacid, possibly through a spacer; X 53 (This is either Gln or Asn) The sequence contains a self-cleaving dipeptide, structure:

[0130] [ka]

[0131] (In the formula, R1 comprises a side chain selected from the group consisting of C1-C8 alkyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)SH, (C1-C4 alkyl)COOH, and (C1-C4 alkyl)NH2, and optionally a C16-C30 carbon chain, wherein the C16-C30 side chain is covalently linked to the side chain, if present, optionally via a spacer; R2, R4, and R8 are independently H or C1-C4 alkyl groups; R3 is a C1-C6 alkyl group, or R3 and R4, together with the atom to which they are bonded, form a piperidine ring; R5 is NH2. A conjugate is provided, including [the specified element].

[0132] In one embodiment, a conjugate comprising a PTH peptide and one of the self-cleaving dipeptides disclosed herein, wherein the dipeptide is covalently bonded to the PTH peptide via an amide bond, optionally via the N-terminal alpha-amine of the PTH peptide. PTH peptide SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (SEQ ID NO: 7), or a peptide different from SEQ ID NO: 7 due to one, two, or three amino acid substitutions. (In the formula, Z is X 33 , X 33 F, battery 33 FX 35 , X 33 FVX 35 , X 33 FVAX 35 , X 33 FVALX 35 , X 33 FVALGX 35 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X53 FVALGX 35 And in some cases, Z is X 33 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 And in some cases, Z is X 33 , X 53 X 35 , or X 53 FX 35 and; X 33 and X 35 Each of these is an amino acid containing a C16-C30 carbon chain covalently linked to the side chain of an amino acid, sometimes via a spacer, and sometimes X 33 and X 35 The side chain of the cysteine ​​or lysine is independently selected, and the side chain of the cysteine ​​or lysine is covalently linked to a C16-C22 fatty acid or C16-C22 diacid, possibly through a spacer; X 53 (This is either Gln or Asn) A conjugate containing the sequence is provided. In one embodiment, the self-cleaving dipeptide has the structure:

[0133] [ka]

[0134] (In the formula, R1 comprises a side chain selected from the group consisting of (C1-C4 alkyl)NH2 and optionally a C16-C30 carbon chain, the C16-C30 carbon chain being covalently linked to the side chain, if present, and optionally via a spacer; R2, R4, and R8 are independently H or C1-C4 alkyl groups; R3 is a C1-C6 alkyl group, or R3 and R4, together with the atom to which they are bonded, form a piperidine ring; R5 is NH2. Includes.

[0135] In one embodiment, a conjugate comprising a PTH peptide and a self-cleaving dipeptide, wherein the dipeptide is covalently bonded to the PTH peptide via an amide bond, possibly by the N-terminal alpha-amine of the PTH peptide. PTH peptide SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (SEQ ID NO: 7), or a peptide different from SEQ ID NO: 7 due to one, two, or three amino acid substitutions. (In the formula, Z is X 33 , X 33 F, battery 33 FX 35 , X 33 FVX 35 , X 33 FVAX 35 , X 33 FVALX 35 , X 33 FVALGX 35 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 And in some cases, Z is X 33 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 And in some cases, Z is X33 , X 53 X 35 , or X 53 FX 35 and; X 33 and X 35 Each of these independently forms (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 CH3, (C1~C4 alkyl)NH-CO(CH2) 14~20 COOH, or (C1~C4 alkyl)NH-CO(CH2) 14~20 It is an amino acid containing a side chain selected from the group consisting of CH3; X 53 (is Asn) The sequence contains a self-cleaving dipeptide, structure:

[0136] [ka]

[0137] (In the formula, R1 is (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 CH3, (C1~C4 alkyl)NH-CO(CH2) 14~20 COOH and (C1~C4 alkyl)NH-CO(CH2) 14~20 Selected from the group consisting of CH3; R2, R4, and R8 are independently H or C1-C4 alkyl groups; R3 is a C1-C6 alkyl group, or R3 and R4, together with the atom to which they are bonded, form a piperidine ring; R5 is NH2, and in some cases, the first amino acid has a D-stereochemical configuration. A conjugate is provided that includes the following. In a further embodiment, R2 and R8 are both H, and R3 and R4 are independently C1-C4 alkyl groups.

[0138] In one embodiment, a PTH conjugate comprising a PTH peptide and a self-cleaving dipeptide, wherein the dipeptide is covalently bonded to the PTH peptide via an amide bond at the N-terminal alphaamine of the PTH peptide, and the PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (Sequence No. 7) (In the formula, Z is X 33 , X 33 F, battery 33 FV, NX 35 , or NFX 35 and; X 33 and X 35 Each of these is an amino acid (sometimes lysine or ornithine) containing a C16-C20 carbon chain covalently linked to the side chain of an amino acid, sometimes via a spacer, and sometimes X 33 and X 35 This is independently lysine acid containing C16-C20 fatty acids or C16-C20 diacides covalently linked to the lysine side chain, and in some cases, X 33 and X 35 The structure (C1~C4 alkyl)NH-[spacer]CO(CH2) is independent. 14~20 It is an amino acid that contains a side chain with a COOH group; X 53 (This is either Gln or Asn) The sequence contains a self-cleaving dipeptide, structure:

[0139] [ka]

[0140] (In the formula, R1 comprises a (C1-C4 alkyl)NH2 side chain and optionally a C16-C20 carbon chain, the C16-C20 carbon chain being covalently linked to the side chain, if present, and optionally via a spacer, and optionally acylated with a C16-C20 fatty acid or C16-C20 diacid, optionally via a spacer; R2, R4, and R8 are independently H or C1-C4 alkyl groups; R3 is a C1-C6 alkyl group; R5 is NH2, and in some cases, however, if R1 lacks the aforementioned C16-C20 carbon chain, the first amino acid of the dipeptide of formula I has a D-stereochemical configuration. A PTH conjugate is provided, which includes [this].

[0141] In one embodiment, a PTH conjugate comprising a PTH peptide and a self-cleaving dipeptide, wherein the dipeptide is covalently bonded to the PTH peptide via an amide bond at the N-terminal alphaamine of the PTH peptide, and the PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (SEQ ID NO: 7) (wherein Z is X) 35 NX 35 , or NFX 35 X 35 The structure is (C1~C4 alkyl)NH-[spacer]CO(CH2) 14~20 COOH, and in some cases (C4 alkyl)NH-[spacer]CO(CH2) 14~20 The dipeptide comprises a sequence of amino acids (which include a side chain having a COOH group), wherein the dipeptide has a structure AB (wherein A is Lys, epsilon-acylated Lys, epsilon-acylated dLys, or dLys, and B is N-methylglycine (sarcosine), and optionally acylated Lys or acylated dLys of A has a structure (C4 alkyl)NH-[spacer]CO(CH2) 14~20 The spacer comprises a side chain having a COOH group, and the spacer is gamma glutamate, gamma glutamate dimer, or gamma glutamate-[COCH2(OCH2CH2)] k -NH] q - Selected from the group consisting of gamma glutamate (wherein k is an integer selected from the range of 1 to 8 or 2 to 4, and q is an integer selected from 1 to 4, and in some cases k is 2 or 4 and q is 2), and in some cases the spacer is,

[0142] [ka]

[0143] A PTH conjugate is provided which contains gamma glutamic acid having the structure [COCH2(OCH2CH2)2-NH-COCH2(OCH2CH2)2-NH-gamma glutamic acid].

[0144] In one embodiment, a PTH conjugate comprising a PTH peptide and a self-cleaving dipeptide, wherein the dipeptide is covalently bonded to the PTH peptide via an amide bond at the N-terminal alphaamine of the PTH peptide, and the PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (SEQ ID NO: 7) (wherein Z is X) 35 NX 35 , or NFX 35 X 35 The structure is (C1~C4 alkyl)NH-[spacer]CO(CH2) 14~20 COOH, and in some cases (C4 alkyl)NH-[spacer]CO(CH2) 14~20 The dipeptide comprises a sequence of amino acids (including a side chain having COOH), and the dipeptide has a structure AB (wherein A is an amino acid with L or D stereochemical configuration, and (C1~C4 alkyl)NH-[spacer]CO(CH2) 14~20 COOH, and in some cases (C4 alkyl)NH-[spacer]CO(CH2) 14~20 The COOH side chain structure is included, B is N-methylglycine (sarcosine), and the spacer is gamma glutamate, gamma glutamate dimer, or gamma glutamate-[COCH2(OCH2CH2)] k -NH] q A PTH conjugate is provided comprising gamma-glutamic acid (wherein k is an integer selected from the range of 1 to 8 or 2 to 4, and q is an integer selected from the range of 1 to 4, and in some cases k is 2 or 4 and q is 2), and in some cases the spacer is gamma-glutamic acid-[COCH2(OCH2CH2)2NH]2-gamma-glutamic acid).

[0145] According to any of the conjugate embodiments disclosed herein, the spacer of the conjugate, if present, comprises an amino acid or a dipeptide. In one embodiment, the amino acid of the spacer is gamma glutamic acid. In one embodiment, the spacer comprises two amino acids separated by a polyethylene glycol polymer. According to any of the conjugate embodiments disclosed herein, the spacer has the structure: gamma glutamic acid-[COCH2(OCH2CH2) k -NH] q - May contain gamma glutamate (wherein k is an integer selected from the range of 1 to 8 or 2 to 4, and q is an integer selected from the range of 1 to 4). In one embodiment, k is 2 or 4 and q is 1 or 2. In one embodiment, k is 2 and q is 2 or 4. In one embodiment, k is 2 and q is 2. In one embodiment, k is 2 and q is 4. In one embodiment, k is 2 and q is 8. In one embodiment, k is 2 or 4 and q is 1. In one embodiment, k is 2 and q is 1. In one embodiment, k is 4 and q is 1. In one embodiment, k is 8 and q is 1. In one embodiment, k is 1 and q is an integer selected from 2 to 8 or 2 to 4. In one embodiment, k is 2 and q is an integer selected from 2 to 8 or 2 to 4. In one embodiment, k is 2 and q is 1.

[0146] In one embodiment, the PTH peptide of the conjugate is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX 33 (Sequence ID 2) or SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 (Array of Sequence ID 26) (In the formula, X 33 and X 35 Each of these is an amino acid containing a C16-C30 carbon chain covalently linked to the side chain of an amino acid, sometimes via a spacer, and sometimes X 33 and X 35Each of these is an amino acid that, independently and sometimes via a spacer, contains a side chain acylated with a C16-C20 fatty acid or a C16-C20 diacid. It contains a self-cleaving dipeptide, structure:

[0147] [ka]

[0148] (In the formula, R1 is (C1~C4 alkyl)NH-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3, (C1~C4 alkyl)NH-[spacer]CO(CH2) 14~20 COOH or (C1~C4 alkyl)NH-[spacer]CO(CH2) 14~20 CH3 is; R2, R4, and R8 are independently H or C1-C4 alkyl groups; R3 is a C1-C6 alkyl group; R5 is NH2, and in some cases R2 and R8 are both H, and R4 is either H or CH3. Includes.

[0149] In one embodiment, the PTH conjugate comprises a self-cleavable dipeptide covalently linked to the N-terminal alpha-amine of the PTH peptide, and the PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX 33 (Sequence ID 2) or SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 (Array of Sequence ID 26) (In the formula, X 33 and X 35 These are (C1~C4 alkyl)NH-CO(CH2) respectively 14~20 COOH, (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20COOH or (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 The spacer is an amino acid containing a side chain having a structure selected from the group consisting of CH3, and the spacer is gamma glutamate, gamma glutamate dimer, or gamma glutamate-[COCH2(OCH2CH2)] k -NH] q - Selected from the group consisting of gamma glutamate (wherein k is an integer selected from the range of 1 to 8 or 2 to 4, and q is an integer selected from the range of 1 to 4, and in some cases k is 2 and q is 2) Includes; Self-cleaving dipeptide, structure:

[0150] [ka]

[0151] (In the formula, R1 is (C1~C4 alkyl)NH-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH or (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 The structure is selected from the group consisting of CH3, and the spacer is gamma glutamate, gamma glutamate dimer, or gamma glutamate-[COCH2(OCH2CH2)] k -NH] q - Selected from the group consisting of gamma glutamate (wherein k is an integer selected from the range of 1 to 8 or 2 to 4, and q is an integer selected from the range of 1 to 4); R2, R4, and R8 are independently H or C1-C4 alkyl groups; R3 is a C1-C6 alkyl group; R5 is NH2, and in some cases R2 and R8 are both H, and R4 is either H or CH3. Includes.

[0152] In one embodiment, the PTH conjugate comprises a self-cleavable dipeptide covalently linked to the N-terminal alpha-amine of the PTH peptide, and the PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX 33 (Sequence number 2) array (In the formula, X 33 (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH or (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 The amino acid includes a side chain having a structure selected from the group consisting of CH3, and the spacer is gamma glutamate-[COCH2(OCH2CH2)] k -NH] q - Gamma glutamate (wherein k is an integer selected from 2 to 4, and q is 1 or 2, and in some cases both k and q are 2)) Includes; Self-cleaving dipeptide, structure:

[0153] [ka]

[0154] (In the formula, R1 is (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH or (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 The structure is selected from the group consisting of CH3, and the spacer is gamma glutamate-[COCH2(OCH2CH2)] k -NH] q -Gamma glutamate (wherein k is an integer selected from 2 to 4, and q is 1 or 2, and in some cases both k and q are 2); R2, R4, and R8 are independently H or C1-C4 alkyl groups; R3 is a C1-C6 alkyl group; R5 is NH2, and in some cases R2 and R8 are both H, R3 is a C1-C3 alkyl group, and R4 is H or CH3. Includes.

[0155] In one embodiment, a conjugate comprising a PTH peptide and a self-cleaving dipeptide covalently bonded to the N-terminal alphaamine of the PTH peptide via an amide bond, wherein the PTH peptide is Amino acid sequence of SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (In the formula, Z is X 33 F, battery 53 FX 35 , or X 33 and; X 33 and X 35 Each of these independently comprises cysteine, homocysteine, ornithine, d-lysine, or lysine, and the side chains of the cysteine, homocysteine, ornithine, d-lysine, or lysine residues are acylated or alkylated with a C14-C30 carbon chain; X 53 (This is either Gln or Asn) Includes; The self-cleaving dipeptide has a general structure:

[0156] [ka]

[0157] (In the formula, R1 is a polymer chain comprising a side chain selected from the group consisting of (C1-C4 alkyl)SH or (C1-C4 alkyl)NH2, and a C16-C30 carbon chain covalently linked to the side chain; R2, R4, and R8 are independently H or C1-C4 alkyl groups; R3 is a C1-C6 alkyl group; R5 is NH2, and in some cases R1 is (C1~C4 alkyl)NH-CO(CH2) 14~20(It is COOH; R2 and R8 are H respectively; R4 is H; R3 is CH3 and R5 is NH2) A conjugate is provided, including [the specified element].

[0158] According to one embodiment, a conjugate comprising a PTH peptide and a self-cleaving dipeptide, wherein the dipeptide is covalently linked to the N-terminal alphaamine of the PTH peptide via an amide bond, and further PTH peptide SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX 33 Or SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 The amino acid sequence (in the formula, X 33 and X 35 These are (C1~C4 alkyl)NH-gamma-glutamic acid-[COCH2(OCH2CH2) k -NH] q -Gamma glutamate-CO(CH2) 14~20 (An amino acid containing a COOH side chain) Includes; The self-cleaving dipeptide has a general structure:

[0159] [ka]

[0160] (In the formula, R1 is (C1~C4 alkyl)NH-gamma-glutamic acid-[COCH2(OCH2CH2)] k -NH] q -Gamma glutamate-CO(CH2) 14~20 It is COOH; R2 and R8 are H, respectively; R4 is either H or CH3; R3 is a C1-C3 alkyl group; R5 is NH2; k is an integer selected from 1, 2, 3, 4, 5, 6, 7, or 8; q is an integer selected from 1, 2, 3, 4, 5, and 6, and in some cases R4 is H, k is 2 or 4, q is 1 or 2, and in some cases k is 2 and q is 2. A conjugate is provided, including [the specified element].

[0161] According to one embodiment, a conjugate comprising a PTH peptide and a self-cleaving dipeptide, wherein the dipeptide is covalently linked to the N-terminal alphaamine of the PTH peptide via an amide bond, and the PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 The amino acid sequence (in the formula, X 35 (C1~C4 alkyl)NH-gamma-glutamic acid-[COCH2(OCH2CH2) k -NH] q -Gamma glutamate-CO(CH2) 14~20 (An amino acid containing a COOH side chain) Includes; The self-cleaving dipeptide has a general structure:

[0162] [ka]

[0163] (In the formula, R1 is (C4 alkyl)NH-gamma-glutamic acid-[COCH2(OCH2CH2)] k -NH] q -Gamma glutamate-CO(CH2) 14~20 It is COOH; R2, R4, and R8 are each H; R3 is CH3; R5 is NH2; k is an integer selected from the range of 1 to 4; q is an integer selected from 1 to 4, and in some cases k is 1 or 2, and q is 1, 2 or 4, and in some cases both k and q are 2. A conjugate is provided, including [the specified element].

[0164] According to one embodiment, a conjugate comprising a PTH peptide and a self-cleaving dipeptide, wherein the dipeptide is covalently linked to the N-terminal alphaamine of the PTH peptide via an amide bond, and the PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX 33 The amino acid sequence (in the formula, X 33 (C1~C4 alkyl)NH-gamma-glutamic acid-[COCH2(OCH2CH2) k -NH] q -Gamma glutamate-CO(CH2) 14~20 (An amino acid containing a COOH side chain) Includes; The self-cleaving dipeptide has a general structure:

[0165] [ka]

[0166] (In the formula, R1 is (C4 alkyl)NH-gamma-glutamic acid-[COCH2(OCH2CH2)] k -NH] q -Gamma glutamate-CO(CH2) 14~20 It is COOH; R2, R4, and R8 are each H; R3 is CH3; R5 is NH2; k is an integer selected from the range of 1 to 4; q is an integer selected from 1 to 4, and in some cases k is 1 or 2, and q is 1, 2 or 4, and in some cases both k and q are 2. A conjugate is provided, including [the specified element].

[0167] According to one embodiment, the present invention provides a PTH peptide selected from the group comprising SEQ ID NOs: 77, 78, 79, 84, 87, 95, 102, 108, 110, 127, or 128. In some embodiments, the PTH peptide is selected from the group comprising SEQ ID NOs: 87, 95, 108, 127, or 128. In some embodiments, the PTH peptide of the present invention is selected from SEQ ID NOs: 77 or 87. In one embodiment, the PTH peptide is SEQ ID NO: 77. In another embodiment, the PTH peptide is SEQ ID NO: 87. In one embodiment, the present invention encompasses the PTH peptides listed in Table 2. In another embodiment, the present invention encompasses the PTH peptides of Table 2 comprising an ornithine substitution made for one lysine in the sequence. In another embodiment, PTH peptides of Table 2 comprising two or more ornithines substituted for two or more lysine are provided herein.

[0168] According to some embodiments, the present invention provides a PTH peptide prodrug that, when administered to a patient requiring administration, provides less than half the amount of active drug relative to the prodrug after one week. According to some embodiments, the present invention provides a PTH peptide prodrug that, when administered to a patient requiring administration, provides less than one-third the amount of active drug relative to the prodrug after one week. According to some embodiments, the present invention provides a PTH peptide prodrug that, when administered to a patient requiring administration, provides less than one-quarter the amount of active drug relative to the prodrug after one week.

[0169] This disclosure also encompasses other conjugates to which the PTH conjugate of this disclosure is linked, optionally via covalent bonds and optionally via linkers, to additional conjugate portions. Bonding may be achieved by covalent chemical bonds, physical forces, e.g., electrostatic, hydrogen, ions, van der Waals, or hydrophobic or hydrophilic interactions. Various non-covalent coupling systems may be used, including biotin-avidin, ligand / receptor, enzyme / substrate, nucleic acid / nucleic acid-binding protein, lipid / lipid-binding protein, cell adhesion molecule partners; or any binding partners or fragments thereof that have affinity for one another.

[0170] The disclosed PTH peptide conjugates are considered suitable for any of the uses previously described for their corresponding parent PTHs. Therefore, the PTH conjugates may be administered to a patient to treat any disease or condition associated with insufficient levels of PTH (hypoparathyroidism), or diseases responsive to PTH treatment, such as osteoporosis. According to one embodiment, a method is provided for treating hypoparathyroidism, wherein a patient requiring such treatment is administered a composition or conjugate of any of those described herein in an amount effective to treat or prevent hypoparathyroidism, or to alleviate a medical condition associated with hypoparathyroidism. In one embodiment, the route of administration is parenteral, including subcutaneous. In another embodiment, the route of administration is oral. In yet another embodiment, the route of administration is pulmonary. In certain embodiments, the PTH peptides of the present invention are inhaled as an aerosol, mist, or powder formulation. The inhaled pharmaceutical compositions described herein may be delivered in the form of an aerosol spray from a pressurized pack or nebulizer, using a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. Such methods include the method described in U.S. Patent No. 6,468,798, which is incorporated herein by reference in whole. In certain embodiments, the dosage unit of the PTH peptide is determined by providing a valve for delivering a measured amount.

[0171] In certain embodiments of the present disclosure, a method for treating osteoporosis is described. The method comprises the step of administering one of the PTH conjugates of the present disclosure to a patient in need, optionally the PTH conjugate being administered in combination with a short-acting PTH agonist (e.g., the PTH peptide of SEQ ID NO: 7, or any of SEQ ID NOs: 31-37). In one embodiment, the patient is administered a composition comprising the PTH conjugate of the present disclosure and a second component selected from the group consisting of Teriparatide (Forteo®), SEQ ID NOs: 7, 31, 32, 33, 34, 35, 36, 37, and calcitonin. In one embodiment, a method for treating osteoporosis or osteopenia comprises the step of administering a composition comprising the PTH conjugate of the present disclosure to a subject in need by daily subcutaneous injection or daily oral administration.

[0172] In some embodiments, the subject requiring treatment has osteoporosis. In some embodiments, the subject requiring treatment has osteopenia. In certain embodiments, the subject requiring treatment is a postmenopausal woman. In some embodiments, the subject requiring treatment has glucocorticoid-induced osteoporosis. In certain embodiments, the subject requiring treatment has glucocorticoid-induced osteopenia. In another embodiment, a method for treating osteoporosis is provided, comprising the step of treating a subject requiring it by daily subcutaneous injection of the PTH conjugate of the Disclosure. In another embodiment, a method for treating osteoporosis is provided, comprising the step of treating a subject requiring it by every-other-day subcutaneous injection of the PTH conjugate of the Disclosure. In another embodiment, the Disclosure provides a method for treating osteoporosis, comprising the step of treating a subject requiring it by weekly or monthly subcutaneous injection of the PTH conjugate of the Disclosure.

[0173] Pharmaceutical compositions comprising the conjugates disclosed herein may be formulated and administered to patients using standard pharmaceutically acceptable carriers and routes of administration known to those skilled in the art. Accordingly, this disclosure also includes pharmaceutical compositions comprising one or more of the conjugates disclosed herein, or pharmaceutically acceptable salts thereof, in combination with a pharmaceutically acceptable carrier.

[0174] According to one embodiment, a pharmaceutical composition is provided comprising one of the novel dipeptide / PTH peptide conjugates disclosed herein, preferably sterile and preferably having a purity level of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and a pharmaceutically acceptable diluent, carrier, or excipient. Such a composition may contain the dipeptide / PTH peptide conjugate disclosed herein, wherein the resulting activator is present at a concentration of at least 0.1 to 10 mg / ml or higher. In one embodiment, the pharmaceutical composition comprises an aqueous solution that is sterile and optionally stored in various containers. According to one embodiment, the compounds disclosed herein may be used to prepare a pre-compounded solution that is ready for immediate injection. In other embodiments, the pharmaceutical composition comprises a lyophilized powder. The pharmaceutical composition may be further packaged as part of a kit comprising a disposable device for administering the composition to a patient. The container or kit may be labeled for storage at ambient room temperature or refrigerated temperature.

[0175] According to one embodiment, a pharmaceutical composition is provided in which a disclosed prodrug form of a PTH conjugate is selected to provide an optimized level of active PTH in a patient's blood / serum / plasma. In some embodiments, after an initial administration of one prodrug form of the PTH conjugate peptides of the Disclosure to a patient in need, the plasma concentration of the prodrug remains higher than the concentration of the released drug for at least 48–96 hours. In certain embodiments, after an initial administration with one prodrug form of the PTH conjugate peptides of the Disclosure, the plasma concentration of the prodrug remains higher than the plasma concentration of the active drug for at least 120 hours. In certain embodiments, the peptides of the Disclosure are administered to a patient in need, and the plasma concentration of the active peptide peaks 48–120 hours after the initial administration. In some embodiments, one prodrug form of the PTH conjugate peptides of the Disclosure is administered to a patient in need, and the plasma concentration of the active peptide remains above 75% of its Cmax one week after administration. In some embodiments, a prodrug form of one of the PTH conjugate peptides of the Disclosure is administered to a patient in need, and the plasma concentration of the resulting active peptide remains above 50% of its Cmax 10 days after administration. In some embodiments, a prodrug form of one of the PTH conjugate peptides of the Disclosure is administered to a patient in need, and the plasma concentration of the resulting active peptide remains above 25% of its Cmax 2 weeks after administration.

[0176] The PTH conjugates of this disclosure may be administered to a patient alone or in combination with other suitable components using any of the known standard routes. Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the recipient to whom the formulation is intended, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The term "parenteral" means by any route other than through the gastrointestinal tract, such as subcutaneous, intramuscular, intrathecal, or intravenous. Analogues of the present disclosure may be administered with or without the addition of pharmaceutically acceptable surfactants (such as soap or detergent), suspending agents (such as pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose), or emulsifiers and other pharmaceutical adjuvants, together with a physiologically acceptable diluent in a sterile liquid or mixture of liquids containing water, physiological saline, aqueous dextrose and related sugar solutions, alcohols (such as ethanol or hexadecyl alcohol), glycols (such as propylene glycol or polyethylene glycol), dimethyl sulfoxide, glycerol, ketals (such as 2,2-dimethyl-1,3-dioxolane-4-methanol), ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty acid glycerides.

[0177] The compounds of this disclosure may be administered in combination with short-acting PTH or PTH analogues. The peptides of this disclosure may be administered sequentially or co-administered with PTH compounds with relatively short biological half-lives to patients in need.

[0178] In other embodiments, the compounds of the Disclosure may be administered alone or in combination with other agents, such as calcitonin, bisphosphonates, SERMs (e.g., raloxifene), hormone replacement therapy (HRT), and bone resorption inhibitors including calcium, vitamin D1, vitamin D2, vitamin D3, vitamin D4, and estrogen. The compounds of the Disclosure may be co-administered with other agents. Alternatively, the compounds of the Disclosure may be administered sequentially with other agents; for example, the compounds of the Disclosure may be administered alone over a period of one week to one year, followed by the administration of another agent, either together with or in the absence of the compounds.

[0179] According to one embodiment, a pharmaceutical composition is provided comprising the PTH conjugate of the present disclosure and one or more bone resorption inhibitors, including calcitonin, bisphosphonates, SERMs (e.g., raloxifene), hormone replacement therapy (HRT), calcium, vitamin D1, vitamin D2, vitamin D3, vitamin D4, and estrogen. In one embodiment, such a composition is administered to a patient to treat osteopenia or osteoporosis, and optionally the pharmaceutical composition is formulated for oral administration.

[0180] Formulations suitable for oral administration may consist of (a) a liquid solution such as an effective amount of an analog of the present disclosure dissolved in a diluent such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient as a solid or granule; (c) a powder; (d) a suspension in a suitable liquid; and a suitable emulsion. According to one embodiment, a formulation suitable for oral administration comprises the PTH conjugate of the present disclosure and an absorption enhancer such as sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC). Sodium N-[8-(2-hydroxybenzoyl)amino]caprylate (SNAC) is a delivery agent that has been reported to enhance the permeability of molecules across a diverse spectrum, including proteins such as insulin and calcitonin, and other macromolecules such as heparin. According to one embodiment, a pharmaceutical composition comprising the PTH conjugate of the present disclosure and SNAC, optionally formulated as a tablet, is provided for oral delivery.

[0181] All therapeutic methods, pharmaceutical compositions, kits, and other similar embodiments described herein are intended to contain a dipeptide / PTH peptide complex comprising all pharmaceutically acceptable salts thereof.

[0182] In one embodiment, the kit comprises a device for administering a dipeptide / PTH peptide complex composition to a patient. The kit may further include various containers, such as vials, tubes, bottles, etc. Preferably, the kit also includes instructions for use. According to one embodiment, the device of the kit is an aerosol dispenser, and the composition is pre-packaged within the aerosol dispenser. In another embodiment, the kit comprises a syringe and needle, and in one embodiment, the prodrug composition is pre-packaged within the syringe or injection pen using a low-gauge needle having, for example, sizes 29 to 31.

[0183] According to one embodiment, a modified PTH peptide comprising a sequence different from SEQ ID NOs. 31, 32, or 33 by a lysine substitution at one or two positions selected from positions 13, 16, 19, 22, 26, and 33, comprising an acylated amino acid at one or two positions selected from positions 13, 16, 19, 22, 26, and 33, wherein the acylated amino acid is covalently bonded to its amino acid side chain, possibly via a spacer. A modified PTH peptide is provided, having linked C16-C20 fatty acids or C16-C20 diacids, wherein the PTH peptide further comprises a dipeptide AB (wherein A is an amino acid (e.g., Lys, ornithine, cysteine, or homocysteine), optionally an amino acid with a D-stereochemical configuration, optionally an acylated amino acid; B is an N-alkylated amino acid, optionally N-methylglycine, or N-methylalanine).

[0184] According to Embodiment 1, a conjugate comprising a PTH peptide and a self-cleaving dipeptide covalently bonded to the PTH peptide via an amide bond, wherein the self-cleaving dipeptide is optionally linked to the N-terminal alphaamine of the PTH peptide via an amide bond. The PTH peptide mentioned above SVSEIQLMHX 10 LGX 13 HLX 16 SX 18 ERVEWLRX 26 X 27 LQDX 31 Hz, (sequence number 133); SVSEIQLMHX 10 LX 12 KHLX 56 X 17 X 18 ERVEWLRKKLQDVH-Z;(Sequence ID 134); SVSEIQLMHX 10 LGKHLX 16 SX 18ERVEWLRKKLQDVH-Z (Sequence ID 135) and SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (Sequence ID 7); (In the formula, Z is X 33 , X 33 F, battery 33 FX 35 , X 33 FVX 35 , X 33 FVAX 35 , X 33 FVALX 35 , X 33 FVALGX 35 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 And in some cases, Z is X 33 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 and; X 10 and X 16 These are independently Asp, Gln, or Asn; X 12 It is Gly or Aib; X 56 It is aminoisobutyric acid (Aib) or Asn; X 17 It is aminoisobutyric acid (Aib) or Ser; X 18 is Met, Met(O), Leu, or Nleu; X13 , X 26 , and X 27 It was independently selected from the group consisting of Arg, Glu, Asp, and Lys; X 31 is Gly or Val; X 33 and X 35 Each comprises an acylated amino acid containing a C16-C30 fatty acid or C16-C30 diacid, which is covalently linked to the side chain of an amino acid, sometimes via a spacer, and the acylated amino acid is sometimes selected from the group consisting of Lys, dLys, ornithine, Cys, and homocysteine; X 53 is Gln or Asn, depending on the case, however X 12 , X 16 and X 17 (One or fewer of these are Aib, and in some cases the C-terminal amino acid is modified to replace the carboxyl terminus with an amide.) It includes an amino acid sequence selected from the group consisting of; The self-cleaving dipeptide has structure AB (wherein, A is an amino acid containing a C16-C30 fatty acid or C16-C30 diacid, possibly covalently linked to the side chain of an amino acid via a spacer, and possibly an acylated amino acid; (B is an N-alkylated amino acid.) A conjugate is provided, including [the specified element].

[0185] According to Embodiment 2, a conjugate of Embodiment 1 is provided in which A is selected from the group consisting of Lys, dLys, epsilon-acylated Lys, epsilon-acylated dLys, ornithine, epsilon-acylated ornithine, cysteine, S-acylated cysteine, homocysteine, and S-acylated homocysteine, and optionally A is selected from the group consisting of Lys, dLys, acylated Lys, and acylated dLys. In another embodiment, a conjugate of Embodiment 1 is provided in which A is dLys. In yet another embodiment, a conjugate of Embodiment 1 is provided in which A is epsilon-acylated dLys. In yet another embodiment, a conjugate of Embodiment 1 is provided in which A is selected from l-Lys or epsilon-acylated l-Lys.

[0186] According to Embodiment 3, Z is Lys; X 10 and X 16 is Asn;X 13 , X 26 , and X 27 Lys is X 17 is Ser; X 18 is Met; X 31 is Val; X 33 Lys is; A has a side chain via a gamma-Glu-COCH2(OCH2CH2)2NH-gamma-Glu spacer, COC 16 H 32 A conjugate of Embodiment 1 or 2 is provided, which is dLys acylated with CO2H; B is n-methylglycine.

[0187] According to Embodiment 4, a conjugate comprising a PTH peptide and a self-cleaving dipeptide covalently bonded to the PTH peptide via an amide bond at the N-terminal alpha-amine of a PTH protein, wherein the PTH peptide is I) SVSEIQLMHNLGX 13 HLNSMERVEWLRX 26 X 27 LQDX 31 HZ, (Sequence ID 5); SVSEIQLMHNLX 12 KHLX 56 X 17 MERVEWLRKKLQDVH-Z;(sequence number 6); and SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (Sequence ID 7); (In the formula, Z is X 33 , X 33 F, battery 33 FX 35 , X 33 FVX 35 , X 33 FVAX 35 , X 33 FVALX 35 , X 33 FVALGX 35 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 And; depending on the case, Z is X 33 X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 and; X 12 It is aminoisobutyric acid (Aib) or Gly; X 56 It is aminoisobutyric acid (AIb) or Asn; X 17 It is aminoisobutyric acid (AIb) or Ser; X 13 , X 26 , and X 27is independently selected from the group consisting of Arg, Glu, Asp and Lys, and optionally, X 13 , X 26 , and X 27 is independently selected from Glu and Lys; X 31 is Gly or Val; X 33 and X 35 each independently comprises an acylated amino acid comprising a C16-C30 fatty acid or a C16-C30 diacid covalently linked to the side chain of an amino acid, optionally via a spacer; X 53 is Gln or Asn, optionally, provided that only one of X 12 , X 16 and X 17 is Aib, and optionally, the C-terminal amino acid is modified to replace the carboxy terminus with an amide) or II) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX 35 (SEQ ID NO: 2); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 (SEQ ID NO: 12); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHQFX 35 (SEQ ID NO: 14); [[ID=四十二]] SVSEIQLMHNLX 12 X 13 HLX 16 X 17 MERVEWLRX 26 X 27 LQDX 31 HN(SEQ ID NO: 103); SVSEIQLMHNLGX 13 HLNSMERVEWLRX 26 [[ID=6三]]X 27 LQDX 31 HN(SEQ ID NO: 104); SVSEIQLMHNLX 12 KHLX 16 X 17MERVEWLRKKLQDVHN(sequence ID 105); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHN(Sequence ID 106); (In the formula, X 12 It is aminoisobutyric acid (AIb) or Gly; X 16 It is aminoisobutyric acid (AIb) or Asn; X 17 It is aminoisobutyric acid (AIb) or Ser; X 13 , X 26 , and X 27 It was independently selected from the group consisting of Arg, Glu, Asp, and Lys; X 31 is Gly or Val; X 35 (These are acylated amino acids containing C16-C30 fatty acids or C16-C30 diacids, which are covalently linked to the side chain of an amino acid, sometimes via a spacer.) It includes an amino acid sequence selected from the group consisting of, The PTH peptide of SEQ ID NOs. 2, 12, 14, 103, 104, 105, 106, or 107 is modified, possibly via a spacer, by substitution at position 13, 16, 19, 22, 26, 33, or at the C-terminal amino acid of the peptide of SEQ ID NOs. 103, 104, 105, 106, or 107 with lysine acylated with a C14-C20 fatty acid or a C14-C20 diacid; The self-cleaving dipeptide has structure AB (wherein, A is an amino acid, sometimes an amino acid with a D-stereochemical configuration, and sometimes the side chain of the "A" amino acid is acylated with a C16-C30 fatty acid or C16-C30 diacid, sometimes via a spacer; B is an N-alkylated amino acid, which may be N-(C1-C4)alkylated glycine, N-methylglycine, or N-methylalanine. Including; in some cases, however, X 33 and X 35The acylated amino acid and the "A" amino acid of the dipeptide of formula I are the same or different, and depending on the case, X 33 and X 35 The amino acid of the dipeptide of formula I is lysine, but the spacer, stereochemistry, or acylation group attached to the lysine side chain is different, and in some cases, "A" is epsilon-acylated dLys, and X 33 , and X 35 Each of these is an epsilon-acylated Lys, and in some cases the acylated group is linked via a spacer to a C16-C30 fatty acid or C16-C30 diacid. A conjugate is provided.

[0188] According to Embodiment 5, each of the acylated amino acids in the conjugate contains a C16-C30 fatty acid or C16-C30 diacid linked via a spacer, wherein the spacer is gamma glutamic acid and COCH2(OCH2CH2) k A conjugate is provided which comprises one or more linker moieties independently selected from the group consisting of NH (wherein k is an integer selected from the range of 1 to 8), wherein, optionally, if A is a non-acylated amino acid, A is an amino acid with a D-stereochemical configuration.

[0189] According to Embodiment 6, a conjugate of Embodiment 1 is provided in which A is selected from the group consisting of Lys, dLys, acylated Lys, acylated dLys, ornithine, acylated ornithine, cysteine, acylated cysteine, homocysteine, and acylated homocysteine, and optionally A is selected from the group consisting of Lys, dLys, acylated Lys, and acylated dLys.

[0190] According to Embodiment 7, Z is X 33 , X 53 X 35 , or X 53 FX 35 A conjugate is provided, which is one of the embodiments 1 to 6. According to Embodiment 8, a conjugate comprising a PTH peptide and a self-cleaving dipeptide covalently bonded to the PTH peptide via an amide bond at the N-terminal alpha-amine of a PTH protein, The PTH peptide mentioned above SVSEIQLMHNLGX 13 HLNSMERVEWLRX 26 X 27 LQDX 31 HZ(sequence number 5); SVSEIQLMHNLX 12 KHLX 56 X 17 MERVEWLRKKLQDVH-Z(sequence number 6); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (SEQ ID NO: 7), and peptides different from the peptide of SEQ ID NO: 7 by one, two, or three amino acid substitutions. (In the formula, Z is X 33 F, battery 53 X 35 , X 53 FX 35 , or X 33 and; X 12 It is aminoisobutyric acid (Aib) or Gly; X 56 It is aminoisobutyric acid (Aib) or Asn; X 17 It is aminoisobutyric acid (AIb) or Ser; X 13 , X 26 , and X 27 It was independently selected from the group consisting of Arg, Glu, Asp, and Lys; X 31 is Gly or Val; X 33 and X 35 Each contains an acylated amino acid; X 53 (This is either Gln or Asn) It includes an amino acid sequence selected from the group consisting of; The self-cleaving dipeptide has a general structure AB- (In the formula, A is an amino acid or acylated amino acid, and in some cases, the amino acid or acylated amino acid has a D-stereochemical configuration; (B is an N-alkylated amino acid.) Includes; X 33 , X 35 Each of the acylated amino acids of A is independently selected from amino acids containing C16-C30 fatty acids or C16-C30 diacids, which are covalently linked to the amino acid side chain, possibly via a spacer, and the self-cleaving dipeptide is linked to the PTH peptide either through the formation of an amide bond between B and the N-terminal alphaamine of the PTH peptide, or at any one of the positions 13, 16, 19, 22, 26, and 33, X 33 , X 35 and any of the acylated amino acids of A are gamma glutamic acid, gamma glutamic acid-gamma glutamic acid dipeptide, and gamma glutamic acid-[COCH2(OCH2CH2) k -NH] q - Independently selected from the group consisting of gamma glutamate (wherein k is an integer selected from 1 to 8, and q is an integer selected from 1 to 4); However, if A is a non-acylated amino acid, then A is an amino acid with a D-stereochemical configuration. A conjugate is provided.

[0191] According to Embodiment 9, the PTH peptide is sequence SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (Sequence ID 7) (In the formula, Z is X 33 F, battery 53 X 35 , X 53 FX 35 , or X 33 and; X 33 and X 35Each is independently an amino acid containing a C16 - C30 fatty acid or a C16 - C30 diacid covalently linked to the acidic side chain of the amino acid, optionally via a spacer; X 53 is Asn) including, provided that optionally, X 33 X 35 the acylated amino acids of X 33 X 35 and the "A" amino acid of the dipeptide of formula I are the same or different, provided that optionally, X 33 X 35 the acylated amino acids of X Conjugates of embodiment 8 are provided.

[0192] According to embodiment 10, the PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX 33 (SEQ ID NO: 16) or SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 (SEQ ID NO: 12) sequence (wherein X 33 and X 35 are each independently an amino acid containing a C16 - C30 fatty acid or a C16 - C30 diacid covalently linked to the acidic side chain of the amino acid, optionally via a spacer) Conjugates of any one of embodiments 4 - 9 are provided, including

[0193] According to embodiment 11, A, X 33 and X 35A conjugate is provided, one of embodiments 1 to 10, in which an acylated amino acid is independently selected from cysteine, homocysteine, ornithine, and lysine, and the side chain of the cysteine, homocysteine, ornithine, or lysine is covalently linked to a C16-C22 fatty acid or C16-C22 diacitate via a spacer, which may optionally include a gamma-glutamate bond.

[0194] According to Embodiment 12, A, X 33 and X 35 A conjugate is provided, which is one of embodiments 1 to 11, wherein the acylated amino acids are lysine or d-lysine, and the side chain of the lysine or d-lysine is covalently linked to a C16-C22 fatty acid or a C16-C22 diacitate via a spacer that may include a gamma-glutamate bond.

[0195] Embodiment 13 provides a conjugate from any one of Embodiments 1 to 12, wherein A is selected from the group consisting of Lys, dLys, epsilon-acylated Lys, and epsilon-acylated dLys, and the self-cleaving dipeptide is covalently linked to the N-terminal alphaamine of the PTH peptide via the carboxyl terminus of the B amino acid.

[0196] According to Embodiment 14, X 33 , X 35 Each of the acylated amino acids in A contains a C16-C30 fatty acid or a C16-C30 diacid covalently linked to the amino acid side chain via a spacer, and X 33 , X 35 And each spacer of A is gamma glutamate-gamma glutamate dipeptide, (Xaa)-[COCH2(OCH2CH2) k NH] q -Gamma-glutamic acid, gamma-glutamic acid-[COCH2(OCH2CH2) k NH] q -Gamma-glutamic acid (in the formula, Xaa is selected from Arg, Tyr(OPO3H2), and hCys(SO3H); k is an integer selected from the range of 1 to 8; q is an integer selected from the range 1 to 8, and in some cases k is 2 and q is selected from the range 1 to 4. A conjugate is provided which is independently selected from and optionally has a spacer of -{gamma-glutamic acid-[COCH2(OCH2CH2)2NH-COCH2(OCH2CH2)2]NH-gamma-glutamic acid}-.

[0197] According to Embodiment 15, the spacer has the structure: gamma glutamate-[COCH2(OCH2CH2) k -NH] q -Gamma-glutamic acid (in the formula, k is an integer selected from the range of 1 to 8; q is an integer selected from 1 to 8, and depending on the case, k is 2, 4, 6, or 8, and q is 1, and depending on the case, k is 2 or 4, and q is 2 or 4, and depending on the case, k is 2 or 4, and q is 1, and depending on the case, k is 2, and q is selected from 1 to 8, and depending on the case, k is 2, and q is 2. A conjugate is provided that includes any one of embodiments 1 to 14.

[0198] According to Embodiment 16, AB has the structure:

[0199] [ka]

[0200] (In the formula, R1 comprises a side chain selected from the group consisting of C1-C8 alkyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)SH, (C1-C4 alkyl)COOH, and (C1-C4 alkyl)NH2, and optionally a C16-C30 fatty acid or a C16-C30 diacid is covalently linked to the side chain, optionally via the spacer; R2, R4, and R8 are independently H or C1-C4 alkyl groups; R3 is a C1-C6 alkyl group; R5 is NH2, and the spacer contains an amino acid or dipeptide. A conjugate is provided that includes any one of embodiments 1 to 15.

[0201] According to Embodiment 17, the spacer is gamma glutamic acid, gamma glutamic acid-gamma glutamic acid dipeptide, and gamma glutamic acid-[COCH2(OCH2CH2) k -NH] q A conjugate is provided, which is selected from the group consisting of -gamma glutamate (wherein k is an integer selected from the range of 2 to 4, and q is 1 or 2), and optionally the spacer is -{gamma glutamate-[COCH2(OCH2CH2)2NH-COCH2(OCH2CH2)2]NH-gamma glutamate}-, and any one of embodiments 1 to 16 is provided.

[0202] According to Embodiment 18, the chemical cleavage half-life (t) of AB from the PTH peptide is 1 / 2 A conjugate is provided, which has a duration of at least about 48 to 168 hours in a standard PBS solution under physiological conditions, according to any one of Embodiments 1 to 17.

[0203] According to Embodiment 19, the chemical cleavage half-life (t) of AB from the PTH peptide is 1 / 2 A conjugate is provided, which has a incubation period of at least about 70–140 hours in a standard PBS solution under physiological conditions, according to any one of Embodiments 1 to 17.

[0204] According to Embodiment 20, the chemical cleavage half-life (t) of AB from the PTH peptide is 1 / 2 A conjugate is provided, which has a incubation period of at least about 90–120 hours in a standard PBS solution under physiological conditions, according to any one of Embodiments 1 to 17.

[0205] According to Embodiment 21, R1 is (C1~C4 alkyl)NH; R2 and R8 are H, respectively; R4 is either H or CH3; R3 is CH3, R5 is NH2. A conjugate of Embodiment 16 is provided.

[0206] According to Embodiment 22, R1 is (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 CH3, (C1~C4 alkyl)NH-CO(CH2) 14~20 COOH, or (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3 is; R2 and R8 are H, respectively; R4 is either H or CH3; R3 is CH3, R5 is NH2, and the [spacer] is gamma glutamate, gamma glutamate-gamma glutamate dipeptide, and gamma glutamate-[COCH2(OCH2CH2)] k NH] q - A connected part selected from the group consisting of gamma glutamate (wherein k is an integer selected from the range of 2 to 4, and q is an integer selected from the range of 1 to 8). A conjugate of Embodiment 16 is provided.

[0207] According to Embodiment 23, R1 is (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 A conjugate of Embodiment 22 is provided, which is COOH (wherein k is 2 or 4, q is 1, 2 or 4, and optionally both k and q are 2).

[0208] According to Embodiment 24, R1 is (C4 alkyl)NH-[gamma glutamate-[COCH2(OCH2CH2) k -NH] q -gamma glutamate]-CO(CH2) 14~20 COOH (where k is 2 to 4 and q is 1 or 2), A conjugate of Embodiment 22 is provided.

[0209] Embodiment 25 provides a conjugate of any one of Embodiments 1 to 24, wherein the first amino acid of the cleavable dipeptide is an amino acid with a D-stereochemical configuration. According to Embodiment 26, a conjugate comprising a PTH peptide and a self-cleaving dipeptide covalently linked to the N-terminal alpha-amine of the PTH peptide via an amide bond, The PTH peptide mentioned above SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX 33 (Sequence ID 16), SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 (SEQ ID NO: 12), or an amino acid sequence of a peptide different from both the peptide of SEQ ID NO: 16 and the peptide of SEQ ID NO: 12 by one or two amino acid substitutions (in the formula, X 33 and X 35 These are (C1~C4 alkyl)NH-CO(CH2) respectively 14~20 COOH, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3 or (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 (It is an amino acid that contains the CH3 side chain.) Includes; The self-cleaving dipeptide has a general structure:

[0210] [ka]

[0211] (In the formula, R1 is (C1~C4 alkyl)NH, (C1~C4 alkyl)NH-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3 or (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 CH3 is; R2 and R8 are H, respectively; R4 is either H or CH3; R3 is a C1-C3 alkyl group. R5 is NH2, and the spacer is gamma glutamate, gamma glutamate-gamma glutamate dipeptide, and gamma glutamate-[COCH2(OCH2CH2) k -NH] q - Selected from the group consisting of gamma glutamate (wherein k is an integer selected from the range of 2 to 4, and q is 1 or 2), and in some cases, the C-terminal amino acid is modified by replacing the carboxyl terminus with an amide. including, A conjugate is provided.

[0212] According to Embodiment 27, the PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 (SEQ ID NO: 12) Amino acid sequence ( During the ceremony, X 35 (C1~C4 alkyl)NH-[gamma-glutamic acid-[COCH2(OCH2CH2) k -NH] q -gamma glutamate]-CO(CH2) 14~20 It is an amino acid containing a COOH side chain, and in some cases, X 35 (C4 alkyl)NH-{gamma glutamate-[COCH2(OCH2CH2)2NH-COCH2(OCH2CH2)2]NH-gamma glutamate}-CO(CH2) 14~20(An amino acid containing a COOH side chain) Includes; The self-cleaving dipeptide has a general structure:

[0213] [ka]

[0214] (In the formula, R1 is (C1~C4 alkyl)NH-[gamma-glutamic acid-[COCH2(OCH2CH2) k -NH] q -gamma glutamate]-CO(CH2) 14~20 It is COOH; R2, R4, and R8 are each H; R3 is CH3; R5 is NH2; q is either 2 or 4, k is 2, and in some cases q is 2, and in some cases R1 is (C4 alkyl)NH-{gamma glutamate-[COCH2(OCH2CH2)2NH-COCH2(OCH2CH2)2]NH-gamma glutamate}-CO(CH2) 14~20 (COOH) This includes, however, X 35 The acylated amino acid and the "A" amino acid of the dipeptide of formula I may, in some cases, X 35 The amino acid of formula I and the "A" amino acid of the dipeptide are both lysine, but they differ in spacer, stereochemistry, or the acetylating group attached to the lysine side chain, and in some cases A is epsilon-acylated dLys, and X 35 (This is an epsilon-acylated Lys, and in some cases, the C-terminal amino acid is modified to replace the carboxyl terminus with an amide.) A conjugate of embodiment (claim) 26 is provided.

[0215] According to Embodiment 28, the PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX33 (SEQ ID NO: 16) Amino acid sequence (in the formula, X 33 (C1~C4 alkyl)NH-[gamma-glutamic acid-[COCH2(OCH2CH2) k -NH] q -gamma glutamate]-CO(CH2) 14~20 It is an amino acid containing a COOH side chain, and in some cases, (C4 alkyl)NH-{gamma-glutamic acid-[COCH2(OCH2CH2)2NH-COCH2(OCH2CH2)2]NH-gamma-glutamic acid}-CO(CH2) 16~18 (An amino acid containing a COOH side chain) Includes; The self-cleaving dipeptide has a general structure:

[0216] [ka]

[0217] (In the formula, R1 is (C1~C4 alkyl)NH-[gamma-glutamic acid-[COCH2(OCH2CH2) k -NH] q -gamma glutamate]-CO(CH2) 14~20 COOH, and possibly (C4 alkyl)NH-{gamma-glutamic acid-[COCH2(OCH2CH2)2NH-COCH2(OCH2CH2)2]NH-gamma-glutamic acid}-CO(CH2) 16~18 It is COOH; R2, R4, and R8 are each H; R3 is CH3; R5 is NH2; q is 2, k is either 2 or 4. including, A conjugate of Embodiment 26 is provided.

[0218] Embodiment 29 provides a conjugate of any one of Embodiments 1 to 28, wherein the first amino acid of the self-cleaving dipeptide has a D-stereochemical configuration, and optionally the spacer, if present, is -{gamma-glutamic acid-[COCH2(OCH2CH2)2NH-COCH2(OCH2CH2)2]NH-gamma-glutamic acid}-.

[0219] Embodiment 30 provides a pharmaceutical composition comprising one of the conjugates from Embodiments 1 to 29, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0220] According to Embodiment 31, a pharmaceutical composition of Embodiment 30 formulated for oral delivery is provided, further comprising N[8-(2-hydroxybenzoylaminocaprylate)]sodium and optionally formulated in tablet form.

[0221] Embodiment 32 provides a pharmaceutical composition of Embodiment 30 or 31, further comprising the peptide of SEQ ID NO: 7, SEQ ID NO: 31, or SEQ ID NO: 32, and optionally calcitonin.

[0222] Embodiment 33 provides a method for treating hypoparathyroidism, comprising the step of administering an effective amount of any one of the pharmaceutical compositions of Embodiments 30, 31, or 32 to a patient in need of treatment.

[0223] Embodiment 34 provides a liquid pharmaceutical composition of Embodiment 30 formulated for intravenous, subcutaneous, or intramuscular delivery. Embodiment 35 provides a method for treating osteoporosis or osteopenia, comprising the step of administering an effective amount of any one of the pharmaceutical compositions of Embodiments 30, 31, or 32 to a patient in need of treatment.

[0224] Embodiment 36 provides a method according to any one of Embodiments 33 to 35, in which the composition is administered daily, every other day, once a week, or once every two weeks. Embodiment 37 provides one of the methods described in Embodiments 33, 35 to 36, in which the composition is administered orally.

[0225] Embodiment 38 provides one of the methods described in Embodiments 33, 35 to 36, in which the composition is administered via pulmonary delivery. Embodiment 37 provides the use of any one of the conjugates of Embodiments 1 to 29 for treating hypoparathyroidism or alleviating symptoms associated with hypoparathyroidism. [Examples]

[0226] Example 1: Determination of model dipeptide cleavage rate (in PBS) Using a specific hexapeptide (HSRGTF-NH2; SEQ ID NO: 28) as a model peptide, the half-lives of various dipeptides linked to the hexapeptide via an amide bond were determined. The hexapeptide was assembled using a peptide synthesizer, and Boc-protected sarcosine and lysine were sequentially added to the model peptide bonding resin to produce peptide A (Lys-Sar-HSRGTF-NH2; SEQ ID NO: 29). Peptide A was cleaved by HF and purified by preparative HPLC. Preparative purification using HPLC: Purification was performed using HPLC analysis on a silica-based 1 × 25 cm Vydac C18 column (particle size 5 μm, pore size 300 Å). The equipment used was a Waters Associates Model 600 pump, Model 717 injector, and Model 486 UV detector. A wavelength of 230 nm was used for all samples. Solvent A contained 10% CH3CN / 0.1% TFA in distilled water, and solvent B contained 0.1% TFA in CH3CN. A linear gradient was used (0-100% over 2 hours). The flow rate was 10 ml / min, and the fraction size was 4 ml. Approximately 30 mg of pure peptide was obtained from about 150 mg of crude peptide.

[0227] Peptide A was dissolved in PBS buffer at a concentration of 1 mg / ml. The solution was incubated at 37°C. Samples were collected for analysis at 5, 8, 24, 31, and 47 hours. Dipeptide cleavage was quenched by lowering the pH with an equal volume of 0.1% TFA. The cleavage rate was qualitatively monitored by LC-MS and quantitatively tested by HPLC. Retention times and relative peak areas for the prodrug and parent model peptide were quantified using Peak Simple Chromatography software. Analysis using mass spectrometry Mass spectra were acquired using a Sciex API-III electrospray quadrupole mass spectrometer equipped with a standard ESI ion source. The ionization conditions used were as follows: positive ion mode ESI; ion spray voltage, 3.9 kV; orifice potential, 60 V. The spray curtain gas (trademark) used was nitrogen at a flow rate of 0.9 L / min. Mass spectra were recorded at 600–1800 Thompsons with a 0.5 Th / step and residence time of 2 milliseconds. The sample (approximately 1 mg / mL) was dissolved in a 50% acetonitrile aqueous solution containing 1% acetic acid and introduced by an external syringe pump at a flow rate of 5 μL / min. Prior to analysis, the peptide solubilized in PBS was desalted using a ZipTip solid-phase extraction tip containing 0.6 μL C4 resin, according to instructions provided by the manufacturer (Millipore Corporation, Billerica, Massachusetts). Analysis using HPLC HPLC analysis was performed using a Beckman System Gold Chromatography system equipped with a 214 nm UV detector and a 150 mm × 4.6 mm C8 Vydac column. The flow rate was 1 ml / min. Solvent A contained 0.1% TFA in distilled water, and solvent B contained 0.1% TFA in 90% CH3CN. A linear gradient was used (0% to 30% B over 10 minutes). Data were collected and analyzed using Peak Simple Chromatography software.

[0228] The rate constant for the dissociation of dipeptides from each prodrug was measured using the initial cleavage rate. The concentrations of the prodrug and model parent peptide were determined by peak immunization, "a" and "b", for each of the different recovery times. The zero-order dissociation rate constant of the prodrug was determined by plotting the logarithm of the prodrug concentration at various time intervals. The slope of this plot provides the rate constant "k". Equation t 1 / 2 The half-lives for cleavage of various prodrugs were calculated using =0.693 / k. The results generated from these experiments are provided in Table 1.

[0229] [Table 3]

[0230] Example 2: Synthesis of PTH conjugates PTH peptide analogs were assembled on 0.1 mmol of Fmoc-Lys(Mtt)-Wang resin using the ABI-433A peptide synthesizer and the Fmoc / Oxyma / DIC coupling protocol. Fmoc-Sar-OH and Boc-D-Lys(Boc)-OH were combined with the first amino acid (Ser) from the natural molecule. 1 It was sequentially coupled to the N-terminus of ). Lys 33 The Mtt side chain was deprotected, and the resulting free amine was used for further extension. This amine was sequentially coupled with two repeated additions of Fmoc-Glu-OtBu and Fmoc-NH-PEG2-CH2COOH, followed by Fmoc-Glu-OtBu, and finally mono-tert-butyl octadecanedioic acid. The peptide was chemically removed from the synthetic resin by treatment with a TFA solution containing 2.5% TIS, 2.5% 2-mercaptoethanol, 2.5% anisole, and 2.5% H2O with gentle stirring at room temperature for 2 hours. The resin was removed by filtration, and the peptide was precipitated by adding cold ether (50 ml). The peptide precipitate was recovered by centrifugation and washed with cold ether (3 × 50 ml).

[0231] The impure peptides were purified using a preparative reverse-phase HPLC column (Kinetex® 5 μm C8 100 Å LC column, 250 × 21.2 mm) at a flow rate of 15 mL / min, with 10-50% ACN aqueous solution (0.1% TFA). The pure peptides were evaluated by analytical LC-MS, and the pooled fractions were freeze-dried to obtain a final product as a white, fluffy solid.

[0232] In an example using fatty acid acylation with two different structures, the dipeptide-prodrug side chain was coupled at the final lysine as Boc-D-Lys(Fmoc)-OH. The lysine Fmoc side chain was deprotected by standard base treatment. The fatty acid side chain was assembled as Lys 33 This was done so that it would be described as follows: Lys 33 To attach a second, but different, fatty acid, a deprotected peptide resin is further used as described above for monolipid acylated peptide analogs, and Lys 33 The Mtt side chain was then deprotected, and the lysine side chain was used for further chemical extension.

[0233] For peptide analogues using the same structure with bilipid acylation at the N and C-terminal lysine residues, the last amino acid was added as Boc-D-Lys(Fmoc)-OH. After complete assembly of the resin, the Fmoc and Mtt protecting groups were removed so that the side chains could be simultaneously extended by the general coupling procedure described above. PEGylation Using the ABI-433A peptide synthesizer and the Fmoc / Oxyma / DIC coupling protocol, peptides [Cys] were synthesized on 0.1 mmol Rink Amide resin. 35 ]-PTH(1-35) was assembled. In the preparation of the peptide used for lipid acylation, the previously described TFA cleavage and ether precipitation resulted in impurities in Cys 35-Peptides were provided and purified by preparative HPLC. Thiol conjugation was carried out in 100 mM sodium citrate buffer, pH 4.0, containing 1 mM EDTA and 10 mM TCEP. Excess 40 k Maleimide and methoxy PEG were added, and the reaction mixture was stirred overnight at room temperature. Unreacted PEG reagent was removed by cation exchange chromatography using a linear salt gradient with SP-Sepharose resin and the same buffers containing 20 mM sodium acetate pH 4.0 as buffer A and 1 M NaCl as buffer B. The pooled fraction was desalted in a C2 SPE cartridge and lyophilized to obtain the desired PEGylated PTH. A list of compounds prepared according to this disclosure is provided in Table 2.

[0234] [Table 4-1]

[0235] [Table 4-2]

[0236] [Table 4-3]

[0237] [Table 4-4]

[0238] Example 3: Evaluation of cleavage half-life The PTH prodrug was dissolved in PBS buffer and adjusted to pH 7.4. The resulting solution was incubated at 37°C. Aliquots were taken at the designed time points and analyzed by LC-MS. Analysis was performed using an Agilent 1260 Infinity instrument with a Phenomenex Kinetex C8 2.6μ 100A (75 × 4.6 mm) column. A gradient of 10% to 80% acetonitrile in water containing 0.1% trifluoroacetic acid was applied over 10 minutes at a flow rate of 1 mL / min. Data were collected using absorption at 214 nm. Positive-mode MS data was acquired using an Agilent 6120 quadrupole LC / MS. The concentrations of the prodrug and drug were determined by their relative peak areas. The zero-order dissociation rate constant of the prodrug was determined by plotting the logarithm of the prodrug concentration at various time points. The gradient of this plot provides the rate constant "k". Then (them), equation t 1 / 2 The cleavage half-life was calculated based on =0.693 / k. Table 3 provides the cleavage half-lives for various embodiments of the PTH conjugate of this disclosure.

[0239] [Table 5]

[0240] Example 4: Bioassay design: Luciferase-based reporter gene assay for cAMP detection The ability of each PTH analog or prodrug to induce cAMP was measured using a firefly luciferase-based reporter assay. The induced cAMP production was directly proportional to the binding of PTH to its receptor. HEK293 cells cotransfected with luciferase genes linked to the PTH1 receptor and cAMP response element were used in the bioassay. The results are shown in Figures 3A, 3B, 4, and Table 4.

[0241] Cells were serum-depleted by culturing for 16 hours in Dulbecco's Minimum Essential Medium (Gibco, Life Technologies, Grand Island, New York) supplemented with 0.3% FetalClone III (HyClone, Logan, Utah), and then incubated with serial dilutions of PTH analogs or prodrugs for 5 hours at 37°C and 5% CO2 in a 96-well "Costar 3610" assay plate (Corning, Kennebank, Maine). At the end of incubation, 50 μL of Steady-Lite Plus (PerkinElmer, Waltham, Massachusetts) was added to each well. The plates were shaken briefly and incubated for 4 minutes, and the light output was measured using an EnSpire Alpha Multi-mode Plate Reader (PerkinElmer, Waltham, Massachusetts). The effective 50% concentration (EC50) was calculated using Origin 2019b software (OriginLab, Northampton, Massachusetts).

[0242] [Table 6]

[0243] Example 5: Pharmacological evaluation in normal rats Vehicle and compound SEQ ID NO: 102 (dose of 20, 40, and 80 nmol / kg) were subcutaneously injected into four groups of six rats each (sex: female; strain: Sprague Dawley; mean body weight: 267.3 g; age: 20-22 weeks; diet: standard solid feed). Blood was collected from each group of rats immediately before injection and at 6, 24, 48, and 72 hours after injection. The blood was rapidly rotated, serum was collected, and stored at -20°C. Calcium and phosphorus concentrations were determined using commercially available assays according to the manufacturer's instructions. The results are shown in Figures 5A and 5B.

[0244] Example 6: Pharmacological evaluation in normal mice The vehicle and compounds SEQ ID NO: 102, SEQ ID NO: 74, and SEQ ID NO: 77 (at doses of 20 or 40 nmol / kg, respectively) were subcutaneously injected into seven groups of eight mice each (sex: male; strain: C57Bl6 / J; mean body weight: 24.8 g; age: 8-10 weeks; diet: standard solid feed). Blood was collected from each group of mice immediately before injection and at 24 and 48 hours later. The blood was centrifuged, the serum was collected and stored at -20°C. Calcium concentrations were determined using commercially available assays according to the manufacturer's instructions. The results are shown in Figure 5C and demonstrate the in vivo efficacy in mice of PTH analogues containing an acylated amino acid at the C-terminus of the PTH peptide, which reduces serum calcium levels.

[0245] Example 7: Evaluation of pharmacokinetics in normal mice - Measurement of plasma peptide concentration by LC-MS Compounds SEQ ID NOs: 78, 79, and 84 were subcutaneously injected at a dose of 100 nmol / kg into mice (sex: male; strain: C57Bl6 / J; mean body weight: 34.8 g; age: 42-44 weeks; diet: standard solid feed). Each compound was injected into 20 mice, and blood was collected in EDTA-coated tubes from 4 mice in each treatment at 1, 4, 8, 24, and 48 hours. Plasma was collected after centrifugation and stored at -20°C.

[0246] Standard curve samples were prepared on the day of analysis by serial dilution using cd-1 mouse plasma. Aliquots (40 μl) of the standard curve and test samples were transferred to a 96-well plate and mixed with 160 μl of methanol:acetonitrile (ACN) (1:1, v / v) internal standard solution. After centrifugation for 10 minutes, the supernatant was diluted 2-fold with acidified (0.1% formic acid) ACN:water (3:1, v / v) and analyzed by LC-MS / MS.

[0247] A Shimadzu CBM-20A Nexera UPLC system and a CTC PAL autosampler constituted the front end of the LC-MS / MS system. Chromatography was performed using an Accurcore C8 column, 2.6 μm, 2.1 mm × 30 mm (Thermo 17226-032130), and a binary gradient program of 0.1% formic acid (aqueous solution) and 0.1% formic acid in ACN. Mobile phase solvent A consisted of microfiltrated water:formic acid (1000:1 v / v), and solvent B consisted of ACN:formic acid (1000:1). The flow rate was 0.8 ml / min, the column temperature was ambient temperature, and the injection volume was 5 μl. Two-needle rinses were performed with ACN:water (25:75, v / v) and ACN:isopropanol:acetone in 0.1% formic acid (5:4:1, v / v / v). The gradient cycle began at 15%B (concentration) and linearly increased to 65%B in 0.75 minutes. The column was washed with 98%B for 0.25 minutes and returned to the initial %B during a capture time of 1.20 minutes. The total cycle time for each injection, including re-equilibriumization to the initial %B, was approximately 3 minutes. The first 0.3 minutes of each run were converted to waste.

[0248] Mass spectrometry data were generated using Analyst software to control a Sciex API 6500+ triple quadrupole mass spectrometer (model 5060743-J) in positive ionization mode. Multiple reaction monitoring (MRM), collision energy, declustering potential, and collision exit potential settings for each test substance and internal standard (IS) are provided in Table 5. The concentrations of each analogue over time are shown in Figures 6A and 6B.

[0249] [Table 7]

[0250] Example 8: PTH analogue: Comparative pharmacokinetics in cynomolgus monkeys after single subcutaneous administration The pharmacokinetic profile of a PTH analog and the evaluation of serum calcium and phosphorus pharmacodynamic responses after a single subcutaneous administration were investigated in cynomolgus monkeys. Six monkeys in total—two males and four females—were 2–4 years old at the start of the study and weighed at least 2.5 kg. Experimental design: Route of administration, frequency, and duration The animals were assigned to the groups mentioned in Table 6 below. The animals were administered by subcutaneous injection. The doses were staggered.

[0251] [Table 8]

[0252] One dose was administered to the males in group 1, and approximately 4 days later, the remaining two females in group 1 and all animals in group 2 received a single dose. The first day of administration was designated as study day (SD) 1 for each animal. The dose was 0.3 mL / kg. Individual doses were calculated based on the animal's most recently recorded body weight. The animals were observed and data were recorded as shown in Table 7.

[0253] [Table 9]

[0254] At least 0.5 mL of blood samples were collected from all animals before administration and at 1, 2, 4, 8, 24, 48, 72, 96, 120, 144, 168, and 192 hours after administration. Animals were not fasted before collection. Blood was collected via the femoral vein (or another suitable site). Blood samples were maintained at 5±3°C (wet ice or equivalent) and centrifuged at 5±3°C within 1 hour of collection of each blood sample. The resulting plasma was transferred to a tube and then stored under conditions set to maintain -75±15°C until analysis. Pharmacokinetic evaluation Pharmacokinetic mean concentration-time data were analyzed using a non-compartmental method based on the route of administration (Phoenix® WinNonlin® version 7.0 or later). Where possible, the following parameters were calculated where data allowed: Cmax, Tmax, and AUC. Descriptive statistics were generated using Phoenix WinNonlin. result To minimize mortality in monkey studies while evaluating the pharmacokinetics of PTH analogs, the low-potency alanine-substituted PTH agonist SEQ ID NO: 93 PTH(1-33), A8, K33(γE-2xOEG-γE-diacitic C18) was tested in its two prodrug forms: K33(γE-2xOEG-γE-diacitic C18), N(Me)G0 containing SEQ ID NO: 94 PTH(1-33), A8, dK-1, and K33(γE-2xOEG-γE-diacitic C18), N(Me)G0 containing SEQ ID NO: 95 PTH(1-33), A8, dK-1(γE-2xOEG-γE-diacitic C18), N(Me)G0. Substitution at position 8 with alanine reduced in vitro potency by nearly 100-fold.

[0255] The results of these pharmacokinetic (PK) studies are provided in Figures 7A and 7B, which measure the concentrations of the administered prodrug form and its activated drug form (produced after in vivo non-enzymatic cleavage of the dipeptide prodrug element) over time. Monkeys were administered a single subcutaneous dose of 25 nmol / kg of a prodrug PTH analog (SEQ ID NO: 94 in Figure 7A and peptide 19 in Figure 7B), and the concentrations of prodrugs SEQ ID NO: 94 and SEQ ID NO: 95, along with their corresponding cleavage products, SEQ ID NO: 93 ("Drug"), were measured over the following 192 hours after administration.

[0256] Specifically, Figure 7A shows the time course after a single dose of the prodrug (SEQ ID NO: 94) and its PTH analogue, SEQ ID NO: 94 PTH(1-33), A8, K33(γE-2xOEG-γE-diacitic acid C18) dK -1 N(Me)G 0; and a graph demonstrating the detection levels of its activated form, SEQ ID NO: 93 PTH(1-33), A8, K33(γE-2xOEG-γE-dioxide C18). Figure 7B shows the time course of the prodrug PTH analog SEQ ID NO: 95 PTH(1-33), A8, K33(γE-2xOEG-γE-dioxide C18)dK after a single dose of the prodrug (SEQ ID NO: 95). -1 (γE-2xOEG-γE-Dioxide C18), N(Me)G 0 The graph demonstrates the detection levels of its activated form: SEQ ID NO: 93 PTH(1-33), A8, K33(γE-2xOEG-γE-diacitic acid C18). The data demonstrates the accumulation of the active form over time, corresponding to the decrease in the prodrug form, resulting in a relatively constant amount of the active form over the extended period.

[0257] Example 9: Serum calcium levels in C57BL / 6J mice measured 24 and 48 hours after subcutaneous administration of SEQ ID NO: 77 at 20 or 40 nM / kg. Female C57BL / 6J mice were randomly assigned to three groups (n=8 / group) and administered a single subcutaneous injection of either a vehicle (sterile PBS solution) or SEQ ID NO: 77 (20 or 40 nmol / kg) in PBS. SEQ ID NO: 77 was prepared at a concentration of 150 μM in PBS, pH 7.4. Blood samples were collected from each mouse via tail puncture immediately before administration (0 hours) and after administration (24 and 48 hours) for serum calcium determination.

[0258] Serum Ca concentration was determined using a commercially available colorimetric assay, following the manufacturer's recommendation (Calcium LiquiColor No. 10155, Stanbio Laboratory). The Ca assay contains a colorimetric reagent (catalog no. 0156) and a Ca standard (10 mg / dl; catalog no. 0157). Absorbance (Ab) was measured at a wavelength of 650 nm. The manufacturer's linearity is provided down to 15 mg / dl. The result was calculated as follows: Ab(unknown) / Ab(standard) × 10⁻⁶ Serum calcium levels were recorded at time 0, 24 hours, and 48 hours for both the control and treatment groups, and these are summarized in Tables 8-10. Mice that received a single subcutaneous dose of 40 nmol / kg of SEQ ID NO: 77 showed significantly elevated serum Ca levels at 24 and 48 hours post-treatment compared to controls or those that received 20 nmol / kg of SEQ ID NO: 77 (Figure 14A). This difference was statistically significant at 24 hours when considering initial baseline levels (Figure 14B). There was an increase in serum Ca 24 hours after administration of 40 nmol / kg. This increase of 0.631 nmol / μL (2.524 mg / dl) from a starting level of 2.4105 nmol / μL (9.6421 mg / dl) represents a 26.2% higher concentration.

[0259] The percentage change in serum Ca levels for each test group is summarized in Table 11. The increase in serum Ca levels in the 40 nmol / kg dose group was almost twice as high as the increase (13.8%) in the lower dose of 20 nmol / kg in SEQ ID NO: 77. At this dose, starting from a level of 2.2704 nmol / μl (9.0816 mg / dl), there was an increase of 0.3131 nmol / μl (1.252 mg / dl). The increase in serum Ca in the vehicle treatment was smaller than in any of the groups treated with SEQ ID NO: 77. In the 9.7% vehicle group, the increase in serum Ca represented a value of 0.2174 nmol / μL (0.898 mg / dl), starting from a level of 2.2512 nmol / μL (9.0049 mg / dl). In all treatments, serum Ca returned to approximately the starting level 48 hours after treatment.

[0260] [Table 10]

[0261] [Table 11]

[0262] [Table 12]

[0263] [Table 13]

[0264] Example 10: Serum calcium, inorganic phosphate, body weight, food intake, and drug levels in Sprague Dawley rats after single individual subcutaneous administration of SEQ ID NO: 77 or SEQ ID NO: 87 This experiment measured serum calcium and inorganic phosphorus profiles after subcutaneous injection of 30 or 60 nmol / kg of the parathyroid hormone analog prodrug SEQ ID NO: 87 in female Sprague-Drawley rats. Furthermore, the pharmacokinetics (PK) of the prodrug SEQ ID NO: 87 and its conversion to the active SEQ ID NO: 77 were measured in the group receiving 30 nmol / kg of SEQ ID NO: 87 and compared with the group receiving 30 nmol / kg of SEQ ID NO: 77. Female Sprague-Drawley rats were randomly assigned to four groups of 5 or 6 rats each. Formulations were prepared from lyophilized powders of SEQ ID NO: 87 and SEQ ID NO: 77, as well as diluents and PBS to obtain 50 μM solutions of SEQ ID NO: 77 in PBS and 100 μM and 50 μM solutions of SEQ ID NO: 87 in PBS, which were maintained on moist ice during administration. Dosage was based on body weight and recorded before administration. Each subject received a single subcutaneous injection of either a vehicle (sterile PBS solution; n=6), SEQ ID NO: 77 (30 nmol / kg; n=5), or SEQ ID NO: 87 (30 or 60 nmol / kg; n=5 or 6, respectively). Blood samples were collected from each rat via tail puncture immediately before administration (0 hours) and after administration (24, 48, 72, 96, 120, and 168 hours). These samples were used to determine serum calcium and inorganic phosphorus in the vehicle and all SEQ ID NO: 87-treated animals. Separate blood samples were collected for PK determination in the SEQ ID NO: 87 and SEQ ID NO: 77 30 nmol / kg groups, and additional blood samples were collected in these two PK groups at 2 and 7 hours. Data are summarized in Figures 15, 16A, 16B, 17A, and 17B, and Tables 12 and 13.

[0265] Serum levels of SEQ ID NO: 87 and SEQ ID NO: 77 were measured by LC-MS. Pharmacokinetic results are summarized in Figure 15. SEQ ID NO: 77 and SEQ ID NO: 87 were observed to increase plasma concentrations after a single dose of 30 nmol / kg. The prodrug form of the PTH agonist (SEQ ID NO: 87) demonstrated a Cmax at 24 hours, which decreased relative to 1 / 3 at 48 hours and 2 / 3 at 72 hours, returning to baseline levels at 1 week. SEQ ID NO: 77 was observed after administration of SEQ ID NO: 87 through in vivo conversion and release of the N-terminal dipeptide. This peaked at 24 hours at a concentration slightly lower than 20% of SEQ ID NO: 87. This was maintained at approximately the same level at 48 hours, reaching a concentration comparable to SEQ ID NO: 87 at 72 hours, and returning to baseline at 1 week. Similar PK analysis of serum concentrations of SEQ ID NO: 77 after administration at the same 30 nmol / kg dose as SEQ ID NO: 87 revealed a more rapid emergence of concentrations above 100 nM at 2 hours post-administration, which peaked at 7 hours and was maintained throughout 24 hours, but decreased to approximately 1 / 3 of Cmax at 48 hours and to baseline levels at 96 hours. The peak-to-trough ratio when comparing SEQ ID NO: 77 concentrations within day 1 compared to 96 hours demonstrated a difference depending on whether it was achieved by direct administration of the peptide or as a result of conversion from SEQ ID NO: 87.

[0266] The results showed that Sequence ID No. 87 was converted in vivo to Sequence ID No. 77 at a rate consistent with the in vitro determination, and the peptide was maintained in rats for nearly a week, demonstrating accelerated clearance of the lipid acylated peptide compared to primates. In administrations where the prodrug and drug could be measured by LC-MS, changes in serum calcium and phosphorus were within the physiological range and did not adversely affect the rats. Serum calcium and inorganic phosphorus measurement Following the manufacturer's recommendations (Stanbio laboratory: Calcium LiquiColor No. 10155; Phosphorous Liqui-UV, No. 0830), blood samples recovered via tail puncture were used to determine total calcium (Ca) and inorganic phosphorus (Pi) in serum using commercially available colorimetric assays. A 96-well plate spectrophotometer (SpectraMax M5, Molecular Devices) was used for detection. Serum Ca and Pi were measured on days 0, 1, 2, 4, 5, and 7. Absorbance (Ab) was measured at 650 nm for Ca and 340 nm for Pi. The manufacturer's linearity is provided down to 15 mg / dl for Ca and 10 mg / dl for Pi. The results were calculated as follows: Ab(unknown) / Ab(standard) × 10⁻⁶ Serum calcium levels were observed to be significantly elevated in the 48–96 hour time range compared to vehicle treatment, returning to levels comparable to control values ​​at 120 and 240 hours (Figure 16A, Figure 16B, and Table 12). Measurements of serum calcium after administration of SEQ ID NO: 87 demonstrated an elevation within the physiological range, peaking at 3 days. The lowest dose of the peptide provided a slightly enhanced elevation that was not statistically significant compared to higher doses.

[0267] [Table 14]

[0268] A small, statistically significant increase in serum phosphate levels was observed only when evaluated in comparison to vehicle-treated rats. This difference was attributed to lower starting concentrations in vehicle-treated rats (Figures 17A, 17B, and Table 13).

[0269] [Table 15]

[0270] Example 11: Assay of prodrugs and their conversion to drugs by LCMS & in vitro assay The physiological activity of the prodrug against its drug form was evaluated using a proprietary commercial assay in accordance with the agreement with Eurofins-Discover Rx Corporation win a cAMP Hunter® Teriparatide Bioassay (95-0118Y2). Eleven-point agonist-style dose curves were performed for each individual peptide or incubation condition. All samples were run in triplicate at each dose. The results in Table 14 are provided in Figures 8A-8C, and these results support the conclusion that SEQ ID NO: 87 is a high nM potency complete PTH agonist (Figure 8A), and that extension of SEQ ID NO: 87 by an uncleavable dipeptide composed of SEQ ID NO: 79 (Figure 8B) or a dipeptide composed of SEQ ID NO: 77 (Figure 8C) inactivates PTH agonism.

[0271] [Table 16]

[0272] The conversion of prodrugs to drugs was evaluated by LC-MS analysis, the methodology provided in Example 3. Peak area and percentage prodrug calculations are shown in Table 15. Individual chromatographic spectra are shown in Figures 9A to 9F. The reaction rate was calculated by fitting the experimental results to a zero-order equation that demonstrated high correlation fit at an R² value of 0.9988 (Figure 10).

[0273] [Table 17]

[0274] The conversion of prodrugs to drugs was further evaluated using a proprietary commercial assay in accordance with the agreement between Eurofins-Discover Rx Corporation and cAMP Hunter® Teriparatide Bioassay (95-0118Y2). Eleven-point agonist-style dose curves were created for each individual peptide or incubation condition. All samples were run in triplicate at each dose. Analysis of the same incubation samples, evaluated for relative prodrug / drug concentrations by LC-MS (Figures 9A-9F and 10), was performed using the cAMP Hunter® Teriparatide Bioassay. The results in Table 16 are provided in Figure 11, and these results support the conclusions determined above by LC-MS.

[0275] [Table 18]

[0276] Example 12: In vitro assay of Met(0) and deamidated analogs The physiological activity of PTH analogs site-specifically modified by oxidation to methionine sulfoxide (SEQ ID NOs. 109 and 110) or deamidated from natural Asn or Gln to their respective carboxylic acids Asp or Glu (SEQ ID NOs. 1118, 120, 122, and 124) was evaluated using a proprietary commercial assay in accordance with the Eurofins-Discover Rx Corporation win a cAMP Hunter® Teriparatide Bioassay (95-0118Y2). An agonist-style 11-point dose curve was constructed for each individual peptide at each concentration. All samples were run in triplicate at each dose. Results are shown in Tables 17, 18, and 19, and Figures 12, 13A, and 13B.

[0277] [Table 19]

[0278] [Table 20]

[0279] [Table 21]

[0280] Example 13: Serum calcium, phosphate, body weight, food intake, and drug levels in Sprague Dawley rats after repeated subcutaneous administration of SEQ ID NO: 77 at 20 nmol / kg, 20 nmol / kg, or SEQ ID NO: 87 at 40 nmol / kg. This experiment measured the effects of repeated daily administration of SEQ ID NOs: 77 and 87 on serum calcium and phosphate in Sprague-Dawley rats. Female Sprague-Dawley rats were housed individually or in pairs and randomly assigned to one of the four groups (n=5-6 / group) listed in Table 20, after which their body weight was measured.

[0281] [Table 22]

[0282] Rats were subcutaneously injected once daily for 7 days with either vehicle (sterile 0.9% NaCl solution; n=5), SEQ ID NO: 77 (20 nmol / kg; n=6), or SEQ ID NO: 87 (20 or 40 nmol / kg; n=5 and 6, respectively). After discontinuation of treatment, the animals were monitored at 9 and 11 days (washout period).

[0283] To determine total calcium (Ca) and inorganic phosphorus (Pi) in serum using commercially available colorimetric assays, blood samples were collected from all animals via tail puncture for serum calcium and phosphate analysis before (0 hours) and after (days 1, 2, 3, 4, and 7) administration of vehicle, SEQ ID NO: 77, or SEQ ID NO: 87. Body weight and food intake were measured on days 0, 1, 3, 5, and 7. Ca was measured on days 0, 1, 2, 3, 4, 7, 9, and 11. Pi was determined in the same samples except on days 9 and 11. Serum SEQ ID NO: 87 and SEQ ID NO: 77 measurements were performed by LC-MS, and the data are shown in Figures 18A and 18B.

[0284] Serum Ca levels increased in all three treatment groups throughout the treatment period and returned to initial levels at the end of the washout period (Figure 18A). Changes in serum Pi were not significantly different except on day 3, when the levels in the SEQ ID NO: 77 group were significantly different from those in the vehicle and SEQ ID NO: 87 rats treated with 40 nmol / kg (Figure 18B).

[0285] Plasma levels of SEQ ID NO: 77 and SEQ ID NO: 87 demonstrated dose-proportional increases after administration (Figures 19A and 19B). Each peptide reached steady-state levels after four daily doses. The relative concentration of SEQ ID NO: 77 was approximately one-quarter of the level of SEQ ID NO: 87. The steady-state relative concentration of SEQ ID NO: 77 induced from the administration of the prodrug SEQ ID NO: 87 was approximately 70% of the relative concentration achieved by direct administration of SEQ ID NO: 77.

[0286] Example 14: Measurement of serum calcium, body weight, and food intake in Sprague Dawley rats after subcutaneous administration of SEQ ID NO: 87 at 4, 8, or 12 nmol / kg. This experiment measured the effects of repeated daily administration of 4, 8, and 12 nmol / kg of SEQ ID NO: 87 over 28 days in female Sprague-Dawley rats. Changes in body weight and food intake were also measured.

[0287] Rats were randomly assigned to four groups (n=10) and administered either a vehicle or SEQ ID NO: 87 (4, 8, or 12 nmol / kg) subcutaneously once daily for 21 days. Body weight and food intake were measured on days 0 (immediately before peptide administration), 1, 3, 7, 10, 14, 17, 21, 24, 28, 31, and 35. Serum calcium was measured at the start of the experiment and on days 7, 14, 21, 28, 29, 30, 31, 32, and 34. Blood samples were collected via tail puncture to determine total calcium in serum using commercially available colorimetric assays. Serum samples were collected for pharmacokinetic measurements of SEQ ID NO: 87 and the resulting drug SEQ ID NO: 77.

[0288] All rats in the study, including vehicle-treated animals, experienced a slight increase in total body weight of less than 2% based on a starting body weight of 272.6 g. Cumulative food intake increased dose-proportionally, in proportion to the relative increase in body weight, compared to vehicle-treated rats.

[0289] In all treated rats, including the vehicle control, serum Ca levels increased throughout the study (Figure 20A). Absolute increases were 0.636, 1.356, 1.452, and 2.072 mg / dL for vehicle, 4, 8, and 12 nmol / kg treated rats, respectively. All serum Ca levels remained within the normal range, with a percentage increase of 6.6% in vehicle-treated rats. Increases of 14.7%, 16.7%, and 23% were recorded for increasing doses of SEQ ID NO: 87 (4, 8, and 12 nmol / kg, respectively). The relative increase in Ca within this dose range was less than proportional to the dose increase, as a three-fold increase in dose from 4 to 12 nmol / kg demonstrated a relative increase of less than twofold (a 56% increase) in serum Ca. On day 34, serum Ca levels in all SEQ ID NO: 87 rats were decreased (Figure 20B). Vehicle-treated animals remained unchanged, with Ca levels elevated by 6.9% compared to baseline. Low-dose treated rats showed a 6.9% increase in serum Ca compared to baseline. Medium and high-dose rats showed serum Ca increases of 11.7% and 12.2%, respectively. All animals appeared healthy.

[0290] After steady-state levels were achieved with daily administration, weekly determination of plasma concentrations of SEQ ID NO: 87, and subsequently SEQ ID NO: 77, was initiated on day 7. Both peptides showed dose-proportional increases, with each of the three doses of the prodrug (SEQ ID NO: 87) being four times higher than the resulting drug (SEQ ID NO: 77). Absolute and relative concentrations of the two peptides across the three doses were maintained for a period of 7–28 days with repeated daily administration. Clearing of these two peptides was monitored by LCMS over the following 7-day period. Plasma concentrations of each peptide were measured at 2, 7, and 24 hours post-administration during the first 24-hour period after the last administration. The results demonstrated that, once steady-state concentrations were achieved, the daily drug exposure changes with each subsequent administration were minimal. These results are shown in Figures 21 and 22.

[0291] Example 15: Measurement of serum calcium levels in parathyroidectomized Sprague-Dawley rats after repeated subcutaneous administration of SEQ ID NO: 87 at 15, 25, or 40 nmol / kg. This experiment measured serum calcium profiles in female Sprague-Dawley rats surgically treated with parathyroid hormone analog prodrugs, SEQ ID NO: 87, after repeated subcutaneous injections. Rats were randomly assigned to five groups and subcutaneously injected with either a vehicle (sterile 0.9% NaCl solution; n=6) or SEQ ID NO: 87 (10, 25, 40 nmol / kg; n=8) for four consecutive days. Two separate vehicle-treated groups of rats were tested. The first group represented control rats (sham controls) that were surgically managed like the rest of the rats but had not had their parathyroids removed. The second vehicle-controlled group represented rats that were surgically managed in the same manner as the SEQ ID NO: 87-treated rats. All rats were placed on a prescribed diet with specific calcium concentrations post-surgery and one week prior to the study.

[0292] To determine total calcium (Ca) in serum using a commercially available colorimetric assay according to the manufacturer's recommendations (Stanbio Laboratory: Calcium LiquiColor No. 0155), blood samples were collected via tail puncture before (0 hours) and after (24, 48, 72, 96, 144 hours) administration of vehicle or SEQ ID NO: 87. A 96-well plate spectrophotometer (SpectraMax M5, Molecular Devices) was used for detection. The calcium assay contained a colorimetric reagent (catalog no. 0156) and a calcium standard (10 mg / dL; catalog no. 0157). Absorbance (Ab) was measured at a wavelength of 650 nm. The manufacturer provided linearity down to 15 mg / dL. Serum calcium was measured immediately before injection (time 0), followed by 24, 48, 72, and 96 hours. The result (mg / dL) was calculated as follows: Ab(unknown) / Ab(standard) × 10⁻⁶.

[0293] Siamese control rats demonstrated significantly elevated starting serum calcium levels compared to vehicle-treated PTx rats. Starting calcium levels were 1.495 nmol / μl compared to 1.085 nmol / μl in the vehicle-controlled PTx rat group, representing a relatively high concentration of 37.8%. While the difference between the two control groups was largely maintained throughout the experiment, both groups showed an increase of approximately 0.3 nmol / μl. SEQ ID NO: 87 treated rats initiated treatment with intermediate serum calcium concentrations compared to the two vehicle-treated control groups: 1.185, 1.354, and 1.251 nmol / μl for the treatment groups of 10, 25, and 40 nmol / kg. These represented levels 9.2%, 24.8%, and 15.3% higher, respectively, than the equivalent PTx rats that comprised the vehicle-treated groups. A clear dose-response was observed in PTx-treated rats 48 hours later and immediately before the third dose, with the highest dose of 40 nmol / kg slightly exceeding that of sham control rats. This relationship was maintained at 72 hours, with the intermediate dose of 25 nmol / kg slightly different from that of sham control rats. At 144 hours, 72 hours after the last dose, PTx rats continued to show dose-proportional differences in serum calcium. The difference between the starting levels of PTx-vehicle-treated rats and the intermediate-dose group doubled throughout this treatment period, and for the highest-dose group, it more than tripled to the point where it exceeded the sham control by just over 0.2 nmol, the results of which are shown in Figure 23.

[0294] Example 16: Measurement of serum and urinary calcium levels in parathyroidectomized Sprague-Dawley rats after repeated subcutaneous administration of SEQ ID NO: 87 at 15, 25, or 40 nmol / kg. This experiment measured serum calcium profiles after repeated subcutaneous injections of the parathyroid hormone analog prodrug, SEQ ID NO: 87, in female Sprague-Dawley rats surgically treated with parathyroidectomy. Rats were randomly assigned to five groups and subcutaneously injected with either a vehicle or SEQ ID NO: 87 (10, 25, or 40 nmol / kg; n=8) for 10 consecutive days. Two separate vehicle-treated groups of rats were tested. The first group represented control rats (sham controls) that were surgically managed like the rest of the rats but had not had their parathyroids removed. The second vehicle-controlled group represented rats that were surgically managed in the same manner as the SEQ ID NO: 87-treated rats. All rats were placed on a prescribed diet with specific calcium concentrations post-surgery and one week prior to the test.

[0295] Blood samples were collected via tail puncture to determine total calcium (Ca) in serum using a commercially available colorimetric assay according to the manufacturer's recommendations (Stanbio Laboratory: Calcium LiquiColor No. 0155). A 96-well plate spectrophotometer (SpectraMax M5, Molecular Devices) was used for detection. The calcium assay contained a colorimetric reagent (catalog no. 0156) and a calcium standard (10 mg / dL; catalog no. 0157). Absorbance (Ab) was measured at a wavelength of 650 nm. The manufacturer provided linearity down to 15 mg / dL. Serum calcium was measured immediately before injection (time 0), and subsequently at 24, 48, 72, and 96 hours. The results (mg / dL) were calculated as follows: Ab(unknown) / Ab(standard) x 10.

[0296] Siamese control rats demonstrated significantly elevated starting serum calcium levels compared to vehicle-treated PTx rats. Starting calcium levels were 5.896 mg / dl in PTx rats compared to 4.66 mg / dl in the vehicle control group. This represented a relatively high concentration of 26.5%. This difference between the two control groups was largely maintained throughout the experiment. SEQ ID NO: 87 treated rats started treatment at intermediate serum calcium concentrations compared to the two vehicle control groups: 4.5, 5.028, and 5.108 mg / dl for treatment groups of 10, 25, and 40 nmol / kg (Figure 24). This represented a 3.4% decrease in the lowest dose group and 7.9% and 9.6% increases in the intermediate and high dose groups, respectively. Two days later and immediately before the third dose, all SEQ ID NO: 87 treated rats showed virtually identical serum calcium levels to the samese control rats. At days 4 and 7, medium and high-dose PTx rats showed serum calcium levels consistent with or exceeding those observed in sham control rats. A dose-proportional difference was observed in rats treated with SEQ ID NO: 87, with the highest-dose rats having the largest size and the lowest-dose rats having the lowest serum calcium. PTx-treated rats maintained elevated serum calcium levels comparable to sham-treated rats at days 10–11. From day 14 and beyond, PTx rats, including vehicle-treated rats, returned to consistent serum calcium levels. These calcium levels were similar to those of sham rats at day 21, with serum calcium levels decreasing equally compared to the difference present at day 0 (start of the study).

[0297] Phosphate levels were determined using sampling at approximately the same time as serum calcium, differing only in the last day of the washout period. While the response to SEQ ID NO: 87 was comparable to that previously described for serum calcium, phosphate levels were lower at each dose compared to vehicle-treated PTx rats (Figure 25). As treatment time increased, phosphate levels in SEQ ID NO: 87-treated rats were consistent with those in sham control rats. Siam control rats demonstrated significantly lower starting serum phosphate levels compared to vehicle-treated PTx rats. Starting phosphate levels were 7.003 mg / dl compared to 11.317 mg / dl in the vehicle control group of PTx rats. This represented a relatively low concentration of 38.1%. The difference between the two control groups was maintained throughout the experiment. SEQ ID NO: 87 treated rats were initiated with intermediate serum phosphate concentrations compared to two vehicle control groups: 11.412, 10.673, and 8.152 mg / dl for treatment groups of 10, 25, and 40 nmol / kg, respectively. These represented a 1.0% increase at the low dose and a 5.7% and 28% decrease at the medium and high doses, respectively. After 7 days, all SEQ ID NO: 87 PTx treated rats showed serum phosphate levels comparable to those of sham control rats. Efficacy was fully maintained throughout observations at day 10, one day after the last dose. After 2 weeks, the medium and high-dose rats showed relatively decreased phosphate levels, while the lowest-dose rats showed an increase compared to their peak at day 10. After 25 days from the start of the study, all PTx rats lined up as the group with elevated phosphate compared to sham control rats.

[0298] Example 17: Comparative pharmacokinetics in cynomolgus monkeys after single subcutaneous administration of SEQ ID NO: 87 The pharmacokinetic profile of PTH analog SEQ ID NO: 87 was tested after a single subcutaneous administration to cynomolgus monkeys using the methodology detailed in Example 8. A total of three monkeys were used for each test dose. The monkeys were between 2 and 4 years old and weighed at least 2.5 kg at the start of the study. The dose was 0.3 mL / kg. Individual doses were calculated based on the animal's most recently recorded body weight.

[0299] At least 0.5 mL of blood samples were collected from all animals before administration and at 6, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, and 336 hours after administration. Animals were not fasted before collection. Blood was collected via the femoral vein (or another suitable site). Blood samples were maintained at 5±3°C (wet ice or equivalent) and centrifuged at 5±3°C within one hour of collection of each blood sample. The resulting plasma was transferred to a tube and then stored under conditions set to maintain -75±15°C until analysis. Pharmacokinetic evaluation Pharmacokinetic mean concentration-time data were analyzed using a non-compartmental method (Phoenix® WinNonlin® version 7.0 or later) based on the route of administration. Where possible, the following parameters were calculated where data allowed: Cmax, Tmax, and AUC. Descriptive statistics were generated using Phoenix WinNonlin. result The results of these pharmacokinetic (PK) studies are provided in Figures 26A and 26B, where the concentrations of the administered prodrug form and its active drug form (produced after in vivo non-enzymatic cleavage of the dipeptide prodrug element) were measured over time. Monkeys were administered single subcutaneous doses of the prodrug PTH analogue 87 at concentrations of 2.5, 3.75, and 5 nmol / kg, and the concentration of the prodrug (SEQ ID NO: 87, Figure 26A) was measured along with the concentration of the corresponding cleavage product, SEQ ID NO: 77 ("the drug"), over the next 336 hours after administration. There was a dose-proportional increase in the concentration of SEQ ID NO: 87, peaking at 24 hours but not returning to the starting concentration until 1 week later (Figure 26A). The peptide SEQ ID NO: 77 resulting from the conversion from SEQ ID NO: 87 peaked at 48–72 hours, and its concentration remained approximately 2 / 3 of the Cmax concentration when assessed at 1 week. The concentration of SEQ ID NO: 77 remained elevated compared to the starting concentration even 2 weeks after administration (Figure 26B). In some embodiments, the present invention may be described as follows. [Aspect 1] A conjugate comprising a PTH peptide and a self-cleaving dipeptide covalently bonded to the PTH peptide via an amide bond, The PTH peptide is SVSEIQLMHX 10 LGX 13 HLX 16 SX 18 ERVEWLRX 26 X 27 LQDX 31 Hz, (sequence number 133); SVSEIQLMHX 10 LX 12 KHLX 56 X 17 X 18 ERVEWLRKKLQDVH-Z;(Sequence ID 134); SVSEIQLMHX 10 LGKHLX 16 SX 18 ERVEWLRKKLQDVH-Z (Sequence ID 135) and SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (Sequence ID 7); (In the formula, Z is X) 33 , X 33 F, battery 33 FX 35 , X 33 FVX 35 , X 33 FVAX 35 , X 33 FVALX 35 , X 33 FVALGX 35 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 And in some cases, Z is X 33 , X 53 X 35 , X 53 FX 35 , X53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 and; X 10 and X 16 These are independently Asp, Gln, or Asn; X 12 It is Gly or Aib; X 56 It is aminoisobutyric acid (Aib) or Asn; X 17 It is aminoisobutyric acid (Aib) or Ser; X 18 is Met, Met(O), Leu, or Nleu; X 13 , X 26 , and X 27 It was independently selected from the group consisting of Arg, Glu, Asp, and Lys; X 31 is Gly or Val; X 33 and X 35 Each comprises an acylated amino acid containing a C16-C30 fatty acid or C16-C30 diacid, which is covalently linked to the side chain of an amino acid, possibly via a spacer, and the acylated amino acid is possibly selected from the group consisting of Lys, dLys, ornithine, Cys, and homocysteine; X 53 is Gln or Asn, depending on the case, however X 12 , X 16 and X 17 (One or fewer of these are Aib, and in some cases the C-terminal amino acid is modified to replace the carboxyl terminus with an amide.) It includes an amino acid sequence selected from the group consisting of; The self-cleaving dipeptide has structure AB (In the formula, A is an amino acid; (B is an N-alkylated amino acid.) including, The above conjugate. [Aspect 2] Z is X 33 , X 53 X 35 , X 53 FX 35 , X 53 FVX 35 , X 53 FVAX 35 , X 53 FVALX 35 , or X 53 FVALGX 35 The conjugate described in Embodiment 1. [Aspect 3] A conjugate comprising a PTH peptide and a self-cleaving dipeptide covalently bonded to the PTH peptide via an amide bond, The PTH peptide is SVSEIQLMHNLGX 13 HLNSMERVEWLRX 26 X 27 LQDX 31 HZ(sequence number 5); SVSEIQLMHX 10 LGKHLX 16 SX 18 ERVEWLRKKLQDVH-Z (Sequence ID 135); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (SEQ ID NO: 7), and peptides different from the peptide of SEQ ID NO: 7 by one, two, or three amino acid substitutions. (In the formula, Z is X 33 F, battery 53 X 35 , X 53 FX 35 , or X 33 and; X 10 and X 16 These are independently Asp, Gln, or Asn; X 18 is Met, Met(O), Leu, or Nleu; X 13 , X 26 , and X 27It was independently selected from the group consisting of Arg, Glu, Asp, and Lys; X 31 is Gly or Val; X 33 and X 35 Each contains an acylated amino acid; X 53 (This is either Gln or Asn) It includes an amino acid sequence selected from the group consisting of; The self-cleaving dipeptide has a general structure AB- (In the formula, A is an amino acid or an acylated amino acid; (B is an N-alkylated amino acid.) Includes, X 33 , X 35 Each acylated amino acid of A is independently selected from amino acids containing C16-C30 fatty acids or C16-C30 diacides, which are covalently linked to the amino acid side chain, possibly via a spacer, and the self-cleaving dipeptide is linked to the PTH peptide through the formation of an amide bond between B and the N-terminal alphaamine of the PTH peptide, and further, X 33 , X 35 And each of the arbitrary spacers of A contains gamma glutamate and COCH2(OCH2CH2) k It includes one or more linker parts independently selected from the group consisting of NH (wherein k is an integer selected from 1 to 8); However, if A is a non-acylated amino acid, the above conjugate is one in which A is an amino acid with a D-stereochemical configuration. [Aspect 4] The PTH peptide has the sequence SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (Sequence ID 7) (In the formula, Z is X 33 F, battery 53 X 35 , X 53 FX 35 , or X 33 and; X 33 and X 35Each of these is an amino acid containing a C16-C30 fatty acid or C16-C30 diacid, which is covalently linked to the acid side chain of an amino acid, independently and sometimes via a spacer; X 53 (is Asn) A conjugate according to any one of embodiments 1 to 3, including the conjugate described above. [Aspect 5] PTH ​​peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX 33 (Sequence ID 16) or SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 (Sequence number 12) array (In the formula, X 33 and X 35 These are amino acids containing C16-C22 fatty acids or C16-C22 diacides that are covalently linked to the side chain of an amino acid, independently and sometimes via a spacer. A conjugate according to any one of embodiments 1 to 3, including the conjugate described above. [Aspect 6] The conjugate according to any one of aspects 1 to 5, wherein A is selected from the group consisting of Lys, dLys, acylated Lys, and acylated dLys, and the self-cleaving dipeptide is covalently linked to the N-terminal alphaamine of the PTH peptide. [Aspect 7]X 33 , X 35 Each of the acylated amino acids in A contains a C16-C30 fatty acid or C16-C30 diacid covalently linked to the amino acid side chain via a spacer, and X 33 , X 35 And each spacer of A is gamma glutamate-gamma glutamate dipeptide, (Xaa)-[COCH2(OCH2CH2) k NH] q -Gamma-glutamic acid and gamma-glutamic acid-[COCH2(OCH2CH2) k NH] q -Gamma-glutamic acid (in the formula, Xaa is selected from Arg, Tyr(OPO3H2), and hCys(SO3H); k is an integer selected from the range of 1 to 8; q is an integer selected from the range 1 to 8, and in some cases k is 2 and q is selected from the range 1 to 4. A conjugate according to any one of embodiments 1 to 6, which is independently selected from the above. [Aspect 8] A, X 33 and X 35 The acylated amino acids are independently selected from cysteine, homocysteine, ornithine, lysine, and d-lysine, and the side chains of cysteine, homocysteine, ornithine, lysine, or d-lysine are covalently linked to a C16-C22 fatty acid or C16-C22 diacid, sometimes via a spacer, and A, X 33 and X 35 A conjugate according to any one of embodiments 1 to 7, wherein each of the arbitrary spacers contains a gamma-glutamate bond. [Aspect 9] A, X 33 and X 35 The conjugate according to any one of embodiments 1 to 8, wherein the acylated amino acid is independently selected from lysine or d-lysine, and the side chain of lysine or d-lysine is covalently linked to a C16-C22 fatty acid or C16-C22 diacitate via a spacer, which may include a gamma-glutamate bond. [Aspect 10] The acylated amino acid of A is d-lysine, and X 33 and X 35 The conjugate according to embodiment 9, wherein each of the acylated amino acids is lysine. [Aspect 11] A, X 33 and X 35 The spacer has the structure: gamma glutamate-[COCH2(OCH2CH2) k NH] q A conjugate according to embodiment 9 or 10, each independently selected from compounds containing gamma glutamic acid (wherein k is an integer selected from 2, 4, or 8, and q is an integer selected from 1, 2, 4, or 8). [Aspect 12] The conjugate according to aspect 11, wherein k is 2 and q is 2 or 4. [Aspect 13] AB has structure: [ka] (In the formula, R1 comprises a side chain selected from the group consisting of C1-C8 alkyl, (C1-C4 alkyl)OH, (C1-C4 alkyl)SH, (C1-C4 alkyl)COOH, and (C1-C4 alkyl)NH2, and optionally C16-C30 fatty acids or C16-C30 diacids, and optionally gamma glutamic acid, gamma glutamic acid-gamma glutamic acid dipeptide, and gamma glutamic acid-[COCH2(OCH2CH2)] k NH] q -Gamma-glutamic acid (in the formula, k is an integer selected from the range of 1 to 8; q is an integer selected from the range 1 to 8, and in some cases k is 2 and q is selected from the range 1 to 8. It is covalently connected to the side chain via a spacer selected from the group consisting of; R2, R4, and R8 are independently H or C1-C4 alkyl groups; R3 is a C1-C6 alkyl group; R5 is NH2. A conjugate according to any one of embodiments 1 to 5, including the conjugate described therein. [Aspect 14] Chemical cleavage half-life of AB from PTH peptide (t 1 / 2 The conjugate according to embodiment 13, wherein the time is at least about 48 to 168 hours in a standard PBS solution under physiological conditions. [Aspect 15] R1 is (C1~C4 alkyl)NH; R2 and R8 are H, respectively; R4 is either H or CH3; R3 is CH3; R5 is NH2. The conjugate described in embodiment 13. [Aspect 16] R1 is (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 CH3, (C1~C4 alkyl)NH-CO(CH2)14~20 COOH, or (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3 is; R2 and R8 are H, respectively; R4 is either H or CH3; R3 is CH3, R5 is NH2, and the [spacer] is gamma glutamate, gamma glutamate-gamma glutamate dipeptide, and gamma glutamate-[COCH2(OCH2CH2)] k NH] q - A connected part selected from the group consisting of gamma glutamate (wherein k is an integer selected from the range of 2 to 4, and q is an integer selected from the range of 1 to 8). The conjugate described in embodiment 13. [Aspect 17] R1 is (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH, and the [spacer] is structured gamma glutamate-[COCH2(OCH2CH2)] k NH] q - The conjugate according to embodiment 16, which is a linking portion containing gamma glutamic acid (wherein k is 2 or 4 and q is 1, 2 or 4). [Aspect 18] R1 is (C4 alkyl)NH-{gamma glutamate-[COCH2(OCH2CH2)2NH-COCH2(OCH2CH2)2]NH-gamma glutamate}-CO(CH2) 14~20 COOH The conjugate described in embodiment 16. [Aspect 19] The conjugate according to any one of aspects 1 to 18, wherein the first amino acid of the cleavable dipeptide is an amino acid with a D-stereochemical configuration. [Aspect 20] A conjugate comprising a PTH peptide and a self-cleaving dipeptide covalently linked to the N-terminal alphaamine of the PTH peptide via an amide bond, The PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX 33(Sequence ID 16), SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 (SEQ ID NO: 12), or a peptide that is different from both the peptide of SEQ ID NO: 16 and the peptide of SEQ ID NO: 12 by one or two amino acid substitutions (in the formula, X 33 and X 35 These are (C1~C4 alkyl)NH-CO(CH2) respectively 14~20 COOH, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3 or (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 (It is an amino acid that contains the CH3 side chain.) It contains the amino acid sequence; The self-cleaving dipeptide has a general structure: [ka] (In the formula, R1 is (C1~C4 alkyl)NH, (C1~C4 alkyl)NH-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 COOH, (C1~C4 alkyl)NH-CO(CH2) 14~20 CH3 or (C1~C4 alkyl)NH-[spacer]-CO(CH2) 14~20 CH3 is; R2 and R8 are H, respectively; R4 is either H or CH3; R3 is a C1-C3 alkyl group. R5 is NH2. Includes R1, X 33 and X 35 The spacer contains gamma glutamate, gamma glutamate-gamma glutamate dipeptide, and gamma glutamate-[COCH2(OCH2CH2) k NH] q- Selected independently from the group consisting of gamma glutamate (wherein k is an integer selected from the range of 2 to 4, and q is an integer selected from the range of 1 to 4), The above conjugate. [Aspect 21] PTH ​​peptide, SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX 35 (Sequence No. 12) (In the formula, X 35 (C1~C4 alkyl)NH-{gamma-glutamic acid-[COCH2(OCH2CH2)2NH-COCH2(OCH2CH2)2]NH-gamma-glutamic acid}-CO(CH2) 14~20 (An amino acid containing a COOH side chain) It contains the amino acid sequence; Self-cleaving dipeptides, general structure: [ka] (In the formula, R1 is (C1~C4 alkyl)NH-{gamma glutamate-[COCH2(OCH2CH2)2NH-COCH2(OCH2CH2)2]NH-gamma glutamate}-CO(CH2) 14~20 It is COOH; R2, R4, and R8 are each H; R3 is CH3; R5 is NH2. A conjugate according to embodiment 20, including the conjugate described in embodiment 20. [Aspect 22] PTH ​​peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX 33 (Sequence ID 16) (In the formula, X 33 (C1~C4 alkyl)NH-{gamma-glutamic acid-[COCH2(OCH2CH2) k -NH] q -gamma glutamate}-CO(CH2) 14~20 (An amino acid containing a COOH side chain) It contains the amino acid sequence; Self-cleaving dipeptides, general structure: [ka] (In the formula, R1 is (C1~C4 alkyl)NH-{gamma glutamate-[COCH2(OCH2CH2) k NH] q -gamma glutamate}-CO(CH2) 16~18 It is COOH; R2, R4, and R8 are each H; R3 is CH3; R5 is NH2; q is either 2 or 4, k is 2) The conjugate described in embodiment 19, including the conjugate described in embodiment 19. [Aspect 23]X 33 (C4 alkyl)NH-{gamma glutamate-[COCH2(OCH2CH2)2NH-COCH2(OCH2CH2)2]NH-gamma glutamate}-CO(CH2) 16 It is an amino acid that contains a COOH side chain; R1 is (C4 alkyl)NH-{gamma glutamate-[COCH2(OCH2CH2)2NH-COCH2(OCH2CH2)2]NH-gamma glutamate}-CO(CH2) 16 COOH The conjugate described in embodiment 22. [Aspect 24] The conjugate according to any one of aspects 21, 22, or 23, wherein the first amino acid of the self-cleaving dipeptide has a D-stereochemical configuration. [Aspect 25] A pharmaceutical composition comprising a conjugate according to any one of aspects 1 to 24, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. [Aspect 26] The pharmaceutical composition according to aspect 25, further comprising N[8-(2-hydroxybenzoylaminocaprylate)]sodium, and optionally formulated in tablet form, for oral delivery. [Aspect 27] The pharmaceutical composition according to aspect 25 or 26, further comprising the peptide of SEQ ID NO: 7, SEQ ID NO: 31, or SEQ ID NO: 32, and optionally calcitonin. [Aspect 28] A method for treating hypoparathyroidism, comprising the step of administering an effective amount of the pharmaceutical composition according to aspect 23, 24, or 25 to a patient in need of treatment. [Aspect 29] A method for treating osteoporosis or osteopenia, comprising the step of administering an effective amount of the pharmaceutical composition according to aspect 22, 23, or 24 to a patient in need of treatment. [Aspect 30] The method according to aspect 28 or 29, wherein the composition is administered once a week. [Aspect 31] The method according to aspect 28 or 29, wherein the composition is administered daily. [Aspect 32] The method according to any one of aspects 28 to 31, wherein the composition is administered orally. [Aspect 33] Use of the composition according to any one of aspects 25 to 27 for treating hypoparathyroidism. [Aspect 34] Use of the composition according to any one of aspects 25 to 27 for treating osteoporosis or osteopenia.

Claims

1. A conjugate comprising a PTH peptide and a self-cleaving dipeptide covalently bonded to the PTH peptide via an amide bond to the N-terminal alpha-amine of the PTH peptide, The PTH peptide is SVSEIQLMHNLGX １３ HLNSMERVEWLRX ２６ X ２７ LQDX ３１ H-Z (Sequence ID 5); SVSEIQLMHX １０ LGKHLX １６ SX １８ ERVEWLRKKLQDVH-Z (Sequence ID 135); SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (SEQ ID NO: 7), and peptides different from the peptide of SEQ ID NO: 7 due to one, two, or three amino acid substitutions. (In the formula, Z is X ３３ F, X ５３ X ３５ , X ５３ FX ３５ , or X ３３ ; and X １０ and X １６ These are independently Asp, Glun, or Asn; X １８ is Met, Met(O), Leu, or Nleu; X １３ , X ２６ , and X ２７ It is independently selected from the group consisting of Arg, Glu, Asp, and Lys; X ３１ is Gly or Val; X ３３ and X ３５ Each contains an acylated amino acid; X ５３ (This is either GLN or Asn) It includes an amino acid sequence selected from the group consisting of; The self-cleaving dipeptide has a general structure A-B- (In the formula, A is an acylated amino acid; (B is an N-alkylated amino acid.) Includes, X ３３ , X ３５ Each acylated amino acid of A is independently selected from amino acids containing C16-C30 fatty acids or C16-C30 diacids covalently linked to the amino acid side chain via a spacer, and the self-cleaving dipeptide is linked to the PTH peptide through the formation of an amide bond between B and the N-terminal alpha amine of the PTH peptide, and further, X ３３ , X ３５ And each spacer of A contains gamma glutamate and COCH ２ (OCH ２ CH ２ ) ｋ It includes one or more linker parts independently selected from the group consisting of NH (wherein k is an integer selected from 1 to 8), The above conjugate.

2. The PTH peptide has the sequence SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH-Z (SEQ ID NO: 7) (In the formula, Z is X ３３ F, X ５３ X ３５ , X ５３ FX ３５ , or X ３３ And; X ３３ and X ３５ Each of these is an amino acid containing a C16-C30 fatty acid or a C16-C30 diacid, independently covalently linked to the acid side chain of an amino acid via a spacer; X ５３ (This is Asn) The conjugate according to claim 1, including the following:

3. PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX ３３ (Sequence ID 16) or SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX ３５ (Array of sequence number 12) (In the formula, X ３３ and X ３５ Each of these is an amino acid containing a C16-C22 fatty acid or C16-C22 diacid, which is independently covalently linked to the side chain of an amino acid via a spacer. The conjugate according to claim 1 or 2, including the conjugate described in claim 1 or 2.

4. A, X ３３ , and X ３５ The conjugate according to any one of claims 1 to 3, wherein the acylated amino acids are the same.

5. X ３３ , X ３５ And each acylated amino acid of A contains a C16-C30 fatty acid or C16-C30 diacid covalently linked to the amino acid side chain via a spacer, X ３３ , X ３５ And each spacer of A is gamma glutamate-gamma glutamate dipeptide, (Xaa)-[COCH ２ (OCH ２ CH ２ ) ｋ NH] ｑ - Gamma-glutamic acid and gamma-glutamic acid - [COCH] ２ (OCH ２ CH ２ ) ｋ NH] ｑ - Gamma glutamate (in the formula, Xaa is Arg, Tyr(OPO ３ H ２ ), and hCys (SO ３ Selected from H); k is an integer selected from the range of 1 to 8; (q is an integer selected from the range of 1 to 8.) A conjugate according to claim 1 or 2, independently selected from the above.

6. A, X ３３ and X ３５ The acylated amino acids are independently selected from cysteine, homocysteine, ornithine, lysine, and d-lysine, and the side chains of cysteine, homocysteine, ornithine, lysine, or d-lysine are covalently linked to a C16-C22 fatty acid or C16-C22 diacid via a spacer, A, X ３３ and X ３５ The conjugate according to claim 1 or 2, wherein each of the spacers contains a gamma-glutamate bond.

7. A, X ３３ and X ３５ The conjugate according to any one of claims 1 to 6, wherein the acylated amino acid is independently selected from lysine or d-lysine, and the side chain of lysine or d-lysine is covalently linked to a C16-C22 fatty acid or a C16-C22 diacitic acid through a spacer containing a gamma-glutamic acid bond.

8. The acylated amino acid of A is d-lysine, and X ３３ and X ３５ The conjugate according to claim 7, wherein each of the acylated amino acids is lysine.

9. A, X ３３ and X ３５ The spacer has the structure: gamma glutamate-[COCH] ２ (OCH ２ CH ２ ) ｋ NH] ｑ - A conjugate according to claim 7 or 8, each independently selected from compounds comprising gamma glutamic acid (wherein k is an integer selected from 2, 4, or 8, and q is an integer selected from 1, 2, 4, or 8).

10. The conjugate according to claim 5 or 9, wherein k is 2 and q is 2 or 4.

11. A-B is structure: 【Chemistry 1】 (In the formula, R １ is C １ to C ８ alkyl, (C １ to C ４ alkyl)OH, (C １ to C ４ alkyl)SH, (C １ to C ４ alkyl)COOH, and (C １ to C ４ alkyl)NH ２ and contains a side chain selected from the group consisting of, and C16-C30 fatty acids or C16-C30 diacids are gamma-glutamic acid, gamma-glutamic acid-gamma-glutamic acid dipeptide, and gamma-glutamic acid-[COCH ２ (OCH ２ CH ２ ) ｋ NH] ｑ -gamma-glutamic acid (wherein, k is an integer selected from the range of 1 to 8; (q is an integer selected from the range of 1 to 8.) It is covalently connected to the side chain via a spacer selected from the group consisting of; R ２ 、 R ４ and R ８ are independently H, or C １ ~C ４ alkyl; R ３ is C １ ~C ６ It is alkyl; R ５ NH ２ (is) A conjugate according to any one of claims 1 to 3, including the conjugate described in any one of claims 1 to 3.

12. Half-life of chemical cleavage of A-B from PTH peptide (t １／２ The conjugate according to claim 11, wherein the duration is at least 48 to 168 hours in a standard PBS solution under physiological conditions.

13. R １ However, (C) is covalently linked to C16-C30 fatty acids or C16-C30 diacids. １ ~C ４ Alkyl)NH; R ２ and R ８ Each of these is H; R ４ is H, or CH ３ And; R ３ ga CH ３ And; R ５ NH ２ That is, The conjugate according to claim 11.

14. R １ But (C １ ~C ４ Alkyl)NH-[Spacer]-CO(CH ２ ) １４～２０ COOH, (C １ ~C ４ Alkyl)NH-[Spacer]-CO(CH ２ ) １４～２０ CH ３ , (C １ ~C ４ Alkyl)NH-CO(CH ２ ) １４～２０ COOH, or (C １ ~C ４ Alkyl)NH-CO(CH ２ ) １４～２０ CH ３ And; R ２ and R ８ Each of these is H; R ４ is H, or CH ３ And; R ３ ga CH ３ And, R ５ NH ２ The [spacer] consists of gamma glutamate, gamma glutamate-gamma glutamate dipeptide, and gamma glutamate-[COCH] ２ (OCH ２ CH ２ ) ｋ NH] ｑ - A linked part selected from the group consisting of gamma glutamate (wherein k is an integer selected from the range of 2 to 4, and q is an integer selected from the range of 1 to 8). The conjugate according to claim 11.

15. R １ But (C １ ~C ４ Alkyl)NH-[Spacer]-CO(CH ２ ) １４～２０ COOH, and [spacer] is structured gamma glutamate-[COCH ２ (OCH ２ CH ２ ) ｋ NH] ｑ - The conjugate according to claim 14, comprising a linking portion containing gamma glutamic acid (wherein k is 2 or 4 and q is 1, 2 or 4).

16. R １ But (C ４ Alkyl)NH-{gamma-glutamic acid-[COCH ２ (OCH ２ CH ２ ) ２ NH-COCH ２ (OCH ２ CH ２ ) ２ ]NH-gamma-glutamic acid}-CO(CH ２ ) １４～２０ The conjugate according to claim 14, wherein it is COOH.

17. The conjugate according to any one of claims 1 to 16, wherein the first amino acid of the cleavable dipeptide is an amino acid with a D-stereochemical configuration.

18. A conjugate comprising a PTH peptide and a self-cleaving dipeptide covalently linked to the N-terminal alpha-amine of the PTH peptide via an amide bond, The PTH peptide is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX ３３ (Sequence No. 16), SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX ３５ (SEQ ID NO: 12), or a peptide that is different from both the peptide of SEQ ID NO: 16 and the peptide of SEQ ID NO: 12 by one or two amino acid substitutions (in the formula, X ３３ and X ３５ (C １ ~C ４ Alkyl)NH-CO(CH ２ ) １４～２０ COOH, (C １ ~C ４ Alkyl)NH-[Spacer]-CO(CH ２ ) １４～２０ COOH, (C １ ~C ４ Alkyl)NH-CO(CH ２ ) １４～２０ CH ３ or (C １ ~C ４ Alkyl)NH-[Spacer]-CO(CH ２ ) １４～２０ CH ３ (It is an amino acid that includes the side chain.) It contains the amino acid sequence; The self-cleaving dipeptide has the following general structure: 【Chemistry 2】 (In the formula, R １ is, (C １ ~C ４ Alkyl)NH-CO(CH ２ ) １４～２０ COOH, (C １ ~C ４ Alkyl)NH-[Spacer]-CO(CH ２ ) １４～２０ COOH, (C １ ~C ４ Alkyl)NH-CO(CH ２ ) １４～２０ CH ３ or (C １ ~C ４ Alkyl)NH-[Spacer]-CO(CH ２ ) １４～２０ CH ３ And; R ２ and R ８ Each of these is H; R ４ is H, or CH ３ And; R ３ is C １ ~C ３ It is alkyl, R ５ NH ２ (is) Includes R １ , X ３３ and X ３５ The spacer contains gamma glutamate, gamma glutamate-gamma glutamate dipeptide, and gamma glutamate-[COCH] ２ (OCH ２ CH ２ ) ｋ NH] ｑ - Gamma glutamate (wherein k is an integer selected from the range of 2 to 4, and q is an integer selected from the range of 1 to 4) is independently selected from the group, The above conjugate.

19. PTH peptide, SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFX ３５ (Sequence No. 12) (In the formula, X ３５ is (C １ ~C ４ Alkyl)NH-{gamma-glutamic acid-[COCH ２ (OCH ２ CH ２ ) ２ NH-COCH ２ (OCH ２ CH ２ ) ２ ]NH-gamma-glutamic acid}-CO(CH ２ ) １４～２０ (An amino acid containing a COOH side chain) It contains the amino acid sequence; Self-cleaving dipeptides have a general structure: 【Transformation 3】 (In the formula, R １ is (C １ ~C ４ Alkyl)NH-{gamma-glutamic acid-[COCH ２ (OCH ２ CH ２ ) ２ NH-COCH ２ (OCH ２ CH ２ ) ２ ]NH-gamma-glutamic acid}-CO(CH ２ ) １４～２０ It is COOH; R ２ , R ４ and R ８ Each of these is H; R ３ CH ３ And; R ５ NH ２ (is) The conjugate according to claim 18, including the following:

20. PTH peptide, SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHX ３３ (Sequence No. 16) (In the formula, X ３３ is (C １ ~C ４ Alkyl)NH-{gamma-glutamic acid-[COCH ２ (OCH ２ CH ２ ) ｋ -NH] ｑ -gamma glutamate}-CO(CH ２ ) １４～２０ (An amino acid containing a COOH side chain) It contains the amino acid sequence; Self-cleaving dipeptides have a general structure: 【Chemistry 4】 (In the formula, R １ is (C １ ~C ４ Alkyl)NH-{gamma-glutamic acid-[COCH ２ (OCH ２ CH ２ ) ｋ NH] ｑ -gamma glutamate}-CO(CH ２ ) １６～１８ It is COOH; R ２ , R ４ and R ８ Each of these is H; R ３ CH ３ And; R ５ NH ２ And; q is 2 or 4, k is 2. The conjugate according to claim 17, including the following:

21. X ３３ But (C ４ Alkyl)NH-{gamma-glutamic acid-[COCH ２ (OCH ２ CH ２ ) ２ NH-COCH ２ (OCH ２ CH ２ ) ２ ]NH-gamma-glutamic acid}-CO(CH ２ ) １６ It is an amino acid that contains a COOH side chain; R １ But (C ４ Alkyl)NH-{gamma-glutamic acid-[COCH ２ (OCH ２ CH ２ ) ２ NH-COCH ２ (OCH ２ CH ２ ) ２ ]NH-gamma-glutamic acid}-CO(CH ２ ) １６ COOH The conjugate according to claim 20.

22. The conjugate according to any one of claims 19, 20, or 21, wherein the first amino acid of the self-cleaving dipeptide has a D-stereochemical configuration.

23. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 22, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

24. The pharmaceutical composition according to claim 23, further comprising N[8-(2-hydroxybenzoylaminocaprylate)]sodium, formulated into a tablet form, and formulated for oral delivery.

25. The pharmaceutical composition according to claim 23 or 24, further comprising the peptide of SEQ ID NO: 7, SEQ ID NO: 31, or SEQ ID NO: 32, and calcitonin.

26. A pharmaceutical composition according to any one of claims 23 to 25 for treating hypoparathyroidism.

27. A pharmaceutical composition according to any one of claims 23 to 25 for treating osteoporosis or osteopenia.

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