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DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with GLENMARK PHARMACEUTICALS LTD, Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 30 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri, Dr T.V. Radhakrishnan and Dr B. K. Kulkarni, etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him Open superstar worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international, etc in organic chemistry are some most read blogs He has hands on experience in initiation and developing novel routes for drug molecules and implementation them on commercial scale over a 30 year tenure till date Dec 2017, Around 35 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 9 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 50 Lakh plus views on dozen plus blogs, He makes himself available to all, contact him on +91 9323115463, email, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 19 lakh plus views on New Drug Approvals Blog in 216 countries...... , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc

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Efmoroctocog alfa, エフモロクトコグアルファ;

(Heavy chain)
(Lignt chain)
(disulfide bridges: H153-H179, H248-H329, H528-H554, H630-H711, H938-H964, H1005-H1009, H1127-H1275, H1280-H1432, H1444-L6, H1447-L9, H1479-H1539, H1585-H1643, L41-L101, L147-L205)

Efmoroctocog alfa

Protein chemical formulaC9736H14863N2591O2855S78

Protein average weight220000.0 Da (Apparent, B-domain deleted)


CAS: 1270012-79-7


2015/11/19 ema APPROVED elocta

Image result for Efmoroctocog alfa

Image result for Efmoroctocog alfa

Efmoroctocog alfa is a fully recombinant factor VIII-Fc fusion protein (rFVIIIFc) with an extended half-life compared with conventional factor VIII (FVIII) preparations, including recombinant FVIII (rFVIII) products such as Moroctocog alfa[1]. It is an antihemorrhagic agent used in replacement therapy for patients with haemophilia A (congenital factor VIII deficiency). It is suitable for all age groups. Haemophilia A is a rare bleeding disorder associated with a slow clotting process caused by the deficiency of factor VIII. Patients with this disorder are more susceptible to recurrent bleeding episodes and excessive bleeding following minor traumatic injuries or surgical procedures [1]. Prophylactic treatment may dramatically improve the management of severe haemophilia A in the future by reducing joint bleeding and other hemorrhages that cause chronic pain and disability to patients [12]. Prophylaxis has also shown to reduce the formation of neutralizing anti-FVIII antibodies, or inhibitors [2].

Factor VIII is a blood coagulant factor involved in the intrinsic pathway to form fibrin, or a blood clot. Efmoroctocog alfa is a first commercially available rFVIII-Fc fusion protein (rFVIIIFc) where the conjugated molecule of rFVIII to polyethylene glycol is covalently fused to the dimeric Fc domain of human immunoglobulin G1, a long-lived plasma protein [FDA Label]. The B domain of factor VIII is deleted. In animal models of haemophilia, efmoroctocog alfa demonstrated an approximately two-fold longer t½ than commercially available rFVIII products [1].

Other drug products with similar structure and function to Efmoroctocog alfa include Moroctocog alfa, which is produced by recombinant DNA technology and is identical in sequence to endogenously produced Factor VIII, but does not contain the B-domain, which has no known biological function, and Antihemophilic factor human, which is purified endogenous Factor VIII from human pooled blood and contains both A- and B-subunits.

It is commonly marketed as Elocta or Eloctate for intravenous injection. To date, no confirmed inhibitory autoantibodies were seen in previously treated patients included in clinical studies and treatment-emergent adverse events were generally consistent with those expected in the patient populations being studied [1]. The extended half-life of efmoroctocog alfa provides several clinical benefits for patients, including reduced frequency of injections required and improved adherence to prophylaxis [1].

Haemophilia A is an inherited sex-linked disorder of blood coagulation in which affected males (very rarely females) do not produce functional coagulation FVIII in sufficient quantities to achieve satisfactory haemostasis. The incidence of congenital haemophilia A is approximately 1 in 10,000 births. Disease severity is classified according to the level of FVIII activity (% of normal) as mild (>5% to <40%), moderate (1% to 5%) or severe (<1%). This deficiency in FVIII predisposes patients with haemophilia A to recurrent bleeding episodes in joints, muscles or internal organs, either spontaneously or as a result of accidental or surgical trauma. Without adequate treatment these repeated haemarthroses and haematomas lead to long-term sequelae with severe disability. Other less frequent, but more severe bleeding sites, are the central nervous system, the urinary or gastrointestinal tract, eyes and the retro-peritoneum. Patients with haemophilia A are at high risk of developing major and life-threatening bleeds after surgical procedures, even after minor procedures such as tooth extraction. The development of cryoprecipitate and subsequently FVIII concentrates, obtained by fractionation of human plasma, provided replacement FVIII and greatly improved clinical management and life expectancy of patients with haemophilia A. Current treatment approaches focus on either prophylactic or on demand factor replacement therapy with plasma-derived FVIII or recombinant FVIII products. In the short term, prophylaxis can prevent spontaneous bleeding and in the long term, prophylaxis can prevent bleeding into joints that will eventually lead to debilitating arthropathy. Prophylaxis with FVIII concentrates is currently the preferred treatment regimen for patients with severe haemophilia A, especially in very young patients. The majority of patients receiving prophylaxis are treated 3-times weekly or every other day at a dose of 25–40 international units (IU)/kg (or 15–25 IU/kg in an intermediate dose regimen), although an escalating dose regimen is also used. However, on-demand treatment is still the predominant replacement approach in many countries. The most serious complication in the treatment of haemophilia A is the development of neutralising antibodies (inhibitors) against FVIII, rendering the patient resistant to replacement therapy and thereby increasing the risk of unmanageable bleeding, particularly arthropathy, and disability.

ELOCTA (efmoroctocog alfa) is a recombinant human coagulation factor VIII Fc fusion protein (rFVIIIFc) consisting of B-domain deleted FVIII covalently attached to the Fc domain of human immunoglobulin G1 (IgG1) thus aiming at prolongation of plasma half-life. It has been developed as a long-acting version of recombinant FVIII (rFVIII) for the control and prevention of bleeding episodes, routine prophylaxis, and perioperative management (surgical prophylaxis) in individuals with hemophilia A. ELOCTA is formulated as powder for intravenous administration in a single-use vial. Each single-use vial contains nominally 250, 500, 750, 1000, 1500, 2000, or 3000 International Units (IU) of rFVIIIFc for reconstitution with a solvent (Sterile Water for Injections), which is provided in a pre-filled syringe. In 2013, national scientific advice was sought from the United Kingdom Medicines and Healthcare Products Regulatory Agency (MHRA), Swedish Medicinal Products Agency, and German Paul-Ehrlich-Institute. No substantial deviations from the advices provided could be identified. On 2 April 2014, the Paediatric Committee (PDCO) of the European Medicines Agency adopted a favourable opinion on the modification of an agreed paediatric investigation plan (PIP) (P/0077/2014) and a partially completed compliance procedure was finalised on 16-18 July 2014 (EMEA-C1-001114-PIP01-10-MO2). Completed studies, Study 997HA301 and Study 8HA02PED, and the initiation of Study 8HA01EXT are considered compliant with EMA Decision P/0077/2014.

The active substance of ELOCTA, efmoroctocog alfa, is a recombinant human coagulation factor VIII, Fc fusion protein (rFVIIIFc) comprising B-domain deleted (BDD) human FVIII covalently linked to the Fc domain of human immunoglobulin G1(IgG1). It has been developed as a long-acting version of recombinant FVIII (rFVIII). ELOCTA is formulated as a sterile, non-pyrogenic, preservative-free, lyophilized, white to off-white powder to cake for intravenous administration in a single-use vial. Each single-use vial contains nominally 250, 500, 750, 1000, 1500, 2000, or 3000 International Units (IU) of rFVIIIFc for reconstitution with liquid diluent (Sterile Water for Injection), which is provided in a pre-filled syringe. The finished medicinal product consists of a package containing a rFVIIIFc drug product vial, a pre-filled diluent (SWFI) syringe and medical devices (a plunger rod, a vial adapter (used as a transfer device during reconstitution), an infusion set, alcohol swabs, plasters and gauze pad for intravenous administration).

Structure The active substance of Elocta, efmoroctocog alfa, is a recombinant human coagulation factor VIII, Fc fusion protein (rFVIIIFc) comprised of a single molecule of B-domain deleted human Factor VIII (BDD FVIII) fused to the dimeric Fc region of human IgG1 with no intervening linker sequence.

The rFVIIIFc protein has a molecular weight of approximately 220 kDa. rFVIIIFc is synthesized as 2 polypeptide chains, one chain consisting of BDD FVIII fused to the N-terminal of human IgG1 Fc domain the other chain consisting of the same Fc region alone. The two subunits of rFVIIIFc, FVIIIFc single chain and Fc single chain, are associated through disulfide bonds in the hinge region of Fc as well as through extensive noncovalent interactions between the Fc fragments.

Characterisation rFVIIIFc was extensively characterised by physicochemical methods in accordance with guideline ICH Q6B. The structural characterisation and the physicochemical properties confirmed the expected properties for a recombinant FVIIIFc product. In general, the characterization performed was considered appropriate for this complex fusion molecule. The panel of tests was comprehensive and covered most of its structural and functional attributes. The comparability between representative batches from development and commercial manufacture (including process validation batches) as well as with rFVIIIFc reference materials was demonstrated. The biological activity was analysed by the FVIII one stage clotting assay (activated partial thromboplastin time (aPTT)), the FVIII chromogenic assay and the FcRn binding assay. Additional in vitro functional tests were performed comprising the binding to von Willebrand factor and the generation of Factor Xa. Since it is anticipated that the potency of modified products measured by the one stage clotting assay (aPTT) may be dependent on the choice of the aPTT reagent, the ISTH recommends for all new FVIII products to perform a study including assay variations (different aPTT reagents) for FVIII testing when using the coagulation assay. Respective studies were provided by the Applicant in Module 5 (no significant dependence on the aPTT reagent was observed). REF 3


Submission details Type of submission: New biological entity Decision: Approved Date of decision: 18 June 2014 Active ingredient: Efmoroctocog alfa (rhu2)3

Product name: Eloctate Sponsor’s name and address: Biogen Idec Australia Pty Ltd Suite 1, Level 5 123 Epping Rd North Ryde, NSW 2113 Dose form: Powder for injection and diluent Strengths: 250 international units (IU), 500 IU, 750 IU, 1000 IU, 1500 IU, 2000 IU and 3000 IU Containers: Type I glass vial (powder) and pre-filled syringe (diluent) Pack size: Single Approved therapeutic use: Eloctate is a long-acting antihaemophilic factor (recombinant) indicated in adults and children ( ≥ 12 years) with haemophilia A (congenital factor VIII deficiency) for: · control and prevention of bleeding episodes · routine prophylaxis to prevent or reduce the frequency of bleeding episodes · perioperative management (surgical prophylaxis) Eloctate does not contain von Willebrand factor, and therefore is not indicated in patients with von Willebrand’s disease. Route of administration: Intravenous (IV) infusion Dosage: Refer to the Product Information (PI; Attachment 1) ARTG numbers: 210521 (250 IU), 210519 (500 IU), 210523 (750 IU), 210525 (1000 IU), 210522 (1500 IU), 210524 (2000 IU), 210520 (3000 IU). 2 recombinant human 3 The ingredient name at the time of submission and registration was Efraloctocog alfa, The name was subsequently changed on 20 February 2015 to harmonise to the International Non-proprietary Name (INN) Efmoroctocog alfa. The AusPAR document has been amended by replacing the previous name efraloctocog alfa with approved INN efmoroctocog alfa.

  1. Frampton JE: Efmoroctocog Alfa: A Review in Haemophilia A. Drugs. 2016 Sep;76(13):1281-1291. doi: 10.1007/s40265-016-0622-z. [PubMed:27487799]
  2. Tiede A: Half-life extended factor VIII for the treatment of hemophilia A. J Thromb Haemost. 2015 Jun;13 Suppl 1:S176-9. doi: 10.1111/jth.12929. [PubMed:26149020]

///////////Efmoroctocog alfa, Peptide, ema 2015


Abaloparatide, абалопаратид , أبالوباراتيد , 巴罗旁肽 ,

Chemical structure for Abaloparatide



BA058; BIM-44058; CAS  247062-33-5

MW 3960.5896, MF C174 H300 N56 O49

абалопаратид [Russian] [INN]
أبالوباراتيد [Arabic] [INN]
巴罗旁肽 [Chinese] [INN]

NAME………C2.29-methyl(22-L-glutamic acid(F>E),23-L-leucine(F>L),25-L-glutamic acid(H>E),26-L-lysine(H>K),28-L-leucine(I>L),30-L-lysine(E>K),31-L-leucine(I>L))human parathyroid hormone-related protein-(1-34)-proteinamide
L-Alaninamide, L-alanyl-L-valyl-L-seryl-L-alpha-glutamyl-L-histidyl-L-glutaminyl-L-leucyl-L-leucyl-L-histidyl-L-alpha-aspartyl-L-lysylglycyl-L-lysyl-L-seryl-L-isoleucyl-L-glutaminyl-L-alpha-aspartyl-L-leucyl-L-arginyl-L-arginyl-L-arginyl-L-alpha-glutamyl-L-leucyl-L-leucyl-L-alpha-glutamyl-L-lysyl-L-leucyl-L-leucyl-2-methylalanyl-L-lysyl-L-leucyl-L-histidyl-L-threonyl-

L-Alaninamide, L-alanyl-L-valyl-L-seryl-L-α-glutamyl-L-histidyl-L-glutaminyl-L-leucyl-L-leucyl-L-histidyl-L-α-aspartyl-L-lysylglycyl-L-lysyl-L-seryl-L-isoleucyl-L-glutaminyl-L-α-aspartyl-L-leucyl-L-arginyl-L-arginyl-L-arginyl-L-α-glutamyl-L-leucyl-L-leucyl-L-α-glutamyl-L-lysyl-L-leucyl-L-leucyl-2-methylalanyl-L-lysyl-L-leucyl-L-histidyl-L-threonyl-

  1. C2.29-methyl(22-L-glutamic acid(F>E),23-L-leucine(F>L),25-L-glutamic acid(H>E),26-L-lysine(H>K),28-L-leucine(I>L),30-L-lysine(E>K),31-L-leucine(I>L))human parathyroid hormone-related protein-(1-34)-proteinamide

Biologic Depiction

Abaloparatide biologic depiction
IUPAC Condensed









FDA 4/28/2017

To treat osteoporosis in postmenopausal women at high risk of fracture or those who have failed other therapies
Drug Trials Snapshot

2D chemical structure of 247062-33-5

Image result for AbaloparatideImage result for Abaloparatide


BIM-44058 is a 34 amino acid analog of native human PTHrP currently in phase III clinical trials at Radius Health for the treatment of postmenopausal osteoporosis. Radius is also developing a microneedle transdermal patch using a 3M drug delivery system in phase II clinical trials. The drug candidate was originally developed at Biomeasure (a subsidiary of Ipsen), and was subsequently licensed to Radius and Teijin Pharma.

Abaloparatide (brand name Tymlos; formerly BA058) is a parathyroid hormone-related protein (PTHrP) analog drug used to treat osteoporosis. Like the related drug teriparatide, and unlike bisphosphonates, it is an anabolic (i.e., bone growing) agent.[1] A subcutaneous injection formulation of the drug has completed a Phase III trial for osteoporosis.[2] This single study found a decrease in fractures.[3] In 28 April 2017, it was approved by Food and drug administration (FDA) to treat postmenopausal osteoporosis.

Image result for Abaloparatide


Medical use

Abaloparatide is indicated to treat postmenopausal women with osteoporosis who are more susceptible to bone fractures.[2]


The dose recommended is 80mcg subcutaneous injection once a day, administered in the periumbilical area using a prefilled pen device containing 30 doses.[4]

Warnings and Precautions

Preclinical studies revealed that abaloparatide systemic daily administration leads to a dose- and time-dependent increase in the incidence of osteosarcoma in rodents.[5] However, whether abaloparatide-SC will cause osteosarcoma in humans is unknown. Thus, the use of abaloparatide is not recommended for individuals at increased risk of osteosarcoma. Additionally, its use is not advised for more than 2 years during a patient’s lifetime.[4][6]

Image result for Abaloparatide

Side Effects

The most common side effects reported by more than 2% of clinical trials subjects are hypercalciuria, dizziness, nausea, headache, palpitations, fatigue, upper abdominal pain and vertigo.[4]


Abaloparatide is 34 amino acid synthetic analog of PTHrP. It has 41% homology to parathyroid hormone (PTH) (1-34) and 76% homology to parathyroid hormone-related protein (PTHrP) (1-34).[7] It works as an anabolic agent for the bone, through selective activation of the parathyroid hormone 1 receptor (PTH1R), a G protein-coupled receptor (GPCR) expressed in the osteoblasts and osteocytes. Abaloparatide preferentially binds the RG conformational state of the PTH1R, which in turn elicits a transient downstream cyclic AMP signaling response towards to a more anabolic signaling pathway.[8][9]


Preclinical studies

Abaloropatide was previously known as BA058 and BIM-44058 while under development. The anabolic effects of abaloparatide on bone were demonstrated in two preclinical studies conducted in ovarectomized rats. Both studies showed increased cortical and trabecular bone volume and density, and trabecular microarchitecture improvement in vertebral and nonvertebral bones after short-term[10] and long-term[11] daily subcutaneous injection of abaloparatide compared to controls. Recent studies indicated a dose-dependent increased in bone mass and strength in long-term abalorapatide treatment.[12] However, it was also indicated that prolonged abalorapatide-SC treatment leads to increased incidence of osteosarcoma.[5] To date, there is no yet evidence for increased risk of bone tumors due to prolonged abalorapatide systemic administration in humans. Based on this preclinical data, the FDA does not advised the use of abaloparatide-SC for more than 2 years, or in patients with history of Paget disease and/or other conditions that exacerbates the risk of developing osteosarcoma.[4]

Clinical Trials

Phase II trials were initiated in 2008. A 24-week randomized trial was conducted in postmenopausal women with osteoporosis (n=222) assessing bone mass density (BMD) changes as the primary endpoint.[13] Significant BMD increase at doses of 40 and 80 mcg were found in the lumbar spine, femur and hips of abaloparatide-treated participants compared to placebo. Additionally, abaloparatide showed superior anabolic effects on the hips compared to teriparatide.[14]

In the phase III (2011-2014) Abaloparatide Comparator Trial in Vertebral Endpoints (ACTIVE) trial, a 18-months randomized, multicenter, double-blinded, placebo-controlled study evaluated the long-term efficacy of abaloparatide compared to placebo and teriparatide in 2,463 postmenopausal women (± 69 years old).[2] Women who received daily injections of abaloparatide experienced substantial reduction in the incidence of fractures compared to placebo. Additionally, greater BMD increase at 6, 12 and 18 months in spinal, hips and femoral bones was observed in abaloparatide compared to placebo and teriparatide-treated subjects.[3]

Participants who completed 18 months of abaloparatide or placebo in the ACTIVE study were invited to participate in an extended open-labeled study – ACTIVExtend study (2012-2016).[15] Subjects (n=1139) received additional 2 years of 70 mg of alendronate, Vitamin D (400 to 800 IU), and calcium (500–1000 mg) supplementation daily. Combined abaloparatide and alendronate therapy reduced significantly the incidence of vertebral and nonvertebral fractures.[16]

A clinical trial assessing the effectiveness of abaloparatide in altering spinal bone mineral density (BMD) in male subjects is expected to start in the first quarter of 2018. If successful, Radius Health aims to submit a sNDA to expand the use of abaloparatide-SC to treat men with osteoporosis.[17]

In addition to the injectable form of abaloparatide, a transdermal patch is also in development.[1]


As previously noted, abaloparatide-SC is manufactured by Radius Health, Inc. (Nasdaq: RDUS), a biomedical company based in Waltham, Massachusetts. This company is focused on the development of new therapeutics for osteoporosis, cancer and endocrine diseases. Abaloparatide is the only drug currently marketed by Radius Health. RDUS reported that sales for abaloparatide were $3.5million for the third quarter of 2017.[17] The company announced a net loss of $57.8 million, or $1.31 per share for the third quarter of 2017, compared to $19.2 million for the same quarter of 2016.[18] The net loss most likely reflects the substantial expenses associated with the preparation and launching of abaloparatide into the US market in May 2017.

In July 2017, Radius Health licensed rights to Teijin Limited for abaloparatide-SC manufacture and commercialization in Japan. Teijin is developing abaloparatide-SC under agreement with Ipsen Pharma S.A.S., and is conducting a phase III clinical trial in Japanese patients with osteoporosis.[19]

Regulatory Information

Radius Health filed a Marketing Authorization Application (MAA) in November 2015,[20] which was validated in December, 2015, and still under regulatory assessment by the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA). As in July 2017, the CHMP issued a second Day-180 List of Outstanding Issues, which Radius is addressing with the CHMP.[17]

In February 2016 a NDA was filed to the FDA, Radius NDA for abaloparatide-SC was accepted in May, 2016.[21] A Prescription Drug User Fee Act (PDUFA) date was initially granted in March 30, 2016, but then extended to June 30, 2017.[22] As previously stated, abaloparatide injection was approved for use in postmenopausal osteoporosis on April 28, 2017.[6]

Intellectual Property

Radius Health currently holds three patents on abaloparatide-SC, with expiration dates from 2027-2028.[23] The patents relate to the drug composition (US 8148333), and the drug delivery methods (US 7803770 B2 and US 8748382-B2).

As previously mentioned, Teijin Limited was granted use of Radius Health intellectual property in July 2017, for the development, manufacture and commercialization of abaloparatide-sc in Japan.


  1. A peptide of the formula:

    [Glu22, 25, Leu23, 2831, Lys26, Aib29, Nle30]hPTHrP(1-34)NH2;
    [Glu22, 25, Leu23, 28, 3031, Lys26, Aib29]hPTHrP(1-34)NH2; [Glu22, 25,29, Leu23, 28, 30, 31, Lys26]hpTHrP(1-34)NH2; [Glu22, 25, 29, Leu23, 28, 31, Lys26, Nle30]hPTHrP(1-34)NH2; [Ser1, Ile5, Met8, Asn10, Leu11, 23, 28, 31, His14, Cha15, Glu22, 25, Lys26, 30, Aib29]hPTHrP (1-34)NH2; [Cha22, Leu23, 28, 31, Glu25, 29, Lys26, Nle30]hPTHrP(1-34)NH2; [Cha7, 1115]hPTHrP(1-34)NH2; [Cha7, 8, 15]hPTHrP(1-34)NH2; [Glu22, Leu23, 28, Aib25, 29, Lys26]hpTHrP(1-34)NH2; [Aib29]hPTHrP(1-34)NH2; [Glu22, 25, Leu23, 28, 31, Lys26, Aib29, 30]hPTHrP(1-34)NH2; [Glu22, 25, Leu23, 28, 31, Lys26, Aib29]hPTHrP(1-34)NH2; [Glu22, 25, Leu23, 28, 31, Aib26, 29, Lys30] hPTHrP(1-34)NH2; or [Leu27, Aib29]hPTH(1-34)NH2; or a pharmaceutically acceptable salt thereof.





EP5026436A Title not available
US3773919 Oct 8, 1970 Nov 20, 1973 Du Pont Polylactide-drug mixtures
US4767628 Jun 29, 1987 Aug 30, 1988 Imperial Chemical Industries Plc Polylactone and acid stable polypeptide
WO1994001460A1* Jul 13, 1993 Jan 20, 1994 Syntex Inc Analogs of pth and pthrp, their synthesis and use for the treatment of osteoporosis
WO1994015587A2 Jan 5, 1994 Jul 21, 1994 Steven A Jackson Ionic molecular conjugates of biodegradable polyesters and bioactive polypeptides
WO1997002834A1* Jul 3, 1996 Jan 30, 1997 Biomeasure Inc Analogs of parathyroid hormone
WO1997002834A1* 3 Jul 1996 30 Jan 1997 Biomeasure Inc Analogs of parathyroid hormone
WO2008063279A2* 3 Oct 2007 29 May 2008 Radius Health Inc A stable composition comprising a bone anabolic protein, namely a pthrp analogue, and uses thereof
US5695955 * 23 May 1995 9 Dec 1997 Syntex (U.S.A.) Inc. Gene expressing a nucleotide sequence encoding a polypeptide for treating bone disorder
US20030166836 * 6 Nov 2002 4 Sep 2003 Societe De Conseils De Recherches Et D’application Scientefiques, S.A.S., A France Corporation Analogs of parathyroid hormone
US20050282749 * 14 Jan 2005 22 Dec 2005 Henriksen Dennis B Glucagon-like peptide-1 (GLP-1); immunotherapy; for treatment of obesity
Tymlos abaloparatide 4/28/2017 To treat osteoporosis in postmenopausal women at high risk of fracture or those who have failed other therapies
Drug Trials Snapshot
Clinical data
Trade names Tymlos
Synonyms BA058, BIM-44058
Routes of
Subcutaneous injection
ATC code
  • none
Legal status
Legal status
  • Investigational
CAS Number
PubChem CID
Chemical and physical data
Formula C174H299N56O49
Molar mass 3,959.65 g·mol−1
3D model (JSmol)

/////////FDA 2017, Abaloparatide, TYMLOS, RADIUS HEALTH, PEPTIDE, BA058, BIM 44058; 247062-33-5, абалопаратид أبالوباراتيد 巴罗旁肽 





Elamipretide biologic depiction




CAS 736992-21-5

Chemical Formula: C32H49N9O5

Molecular Weight: 639.8

  • A free radical scavenger and antioxidant that localizes in the inner mitochondrial membrane.
  • Mitochondrial Protective Agent to Improve Cell Viability
  1. Elamipretide
  2. bendavia
  3. UNII-87GWG91S09
  4. 736992-21-5
  5. MTP 131
  6. RX 31
  7. SS 31
  8. 87GWG91S09
  9. L-Phenylalaninamide, D-arginyl-2,6-dimethyl-L-tyrosyl-L-lysyl-
  10. SS-31 peptide
  11. Arg-Dmt-Lys-Phe-NH2
  12. D-Arg-Dmt-Lys-Phe-NH2
  13. SS31 peptide
  14. Elamipretide [USAN:INN]
  15. MTP-131
  16. Elamipretide (USAN/INN)
  17. arginyl-2,’6′-dimethyltyrosyl-lysyl-phenylalaninamide
  18. CHEMBL3833370
  19. SCHEMBL15028020
  20. CTK2H1007

Elamipretide is a cardiolipin peroxidase inhibitor and mitochondria-targeting peptide, Improves Left Ventricular and Mitochondrial Function. In vitro: Elamipretide significantly increases enzymatic activities of both complexes to near normal levels.

Background Information

Elamipretide is a cardiolipin peroxidase inhibitor and mitochondria-targeting peptide, Improves Left Ventricular and Mitochondrial Function. In vitro: Elamipretide significantly increases enzymatic activities of both complexes to near normal levels. long-term therapy with elamipretide reduces ROS formation, attenuated mPTP openings, and significantly decreases the levels of cytosolic cytochrome c and active caspase-3, thus suppressing a major signaling pathway for apoptosis. Elamipretide represents a new class of compounds that can improve the availability of energy to failing heart and reduce the burden of tissue injury caused by excessive ROS production. [1] In vivo: Fourteen dogs with microembolization-induced HF are randomized to 3 months monotherapy with subcutaneous injections of elamipretide (0.5 mg/kg once daily. Elamipretide has been shown to enhance ATP synthesis in multiple organs, including heart, kidney, neurons, and skeletal muscle. [1] ……by MedChemexpress Co., Ltd.

Elamipretide (also known as SS-31 and Bendavia)[1][2] is a small mitochondrially-targeted tetrapeptide (D-Arg-dimethylTyr-Lys-Phe-NH2) that appears to reduce the production of toxic reactive oxygen species and stabilize cardiolipin.[3]

Stealth Peptides, a privately held company, was founded in 2006 to develop intellectual property licensed from several universities including elamipretide; it subsequently changed its name to Stealth BioTherapeutics.[4][5]

Acute coronary syndrome; Age related macular degeneration; Cardiac failure; Corneal dystrophy; Diabetic macular edema; Lebers hereditary optic atrophy

  • Originator Stealth Peptides
  • Developer Stealth BioTherapeutics
  • Class Eye disorder therapies; Ischaemic heart disorder therapies; Oligopeptides; Peptides; Small molecules
  • Mechanism of Action Free radical scavengers; Mitochondrial permeability transition pore inhibitors
  • Phase II/III Barth syndrome
    • Phase II Acute kidney injury; Corneal disorders; Heart failure; Leber’s hereditary optic atrophy; Mitochondrial disorders; Reperfusion injury
    • Phase I/II Diabetic macular oedema; Dry age-related macular degeneration; Mitochondrial myopathies
    • Phase I Age-related macular degeneration
    • No development reported Chronic heart failure; Diabetes mellitus; Eye disorders; Neurodegenerative disorders

    Most Recent Events

    • 29 Jun 2017 Initial efficacy and adverse events data from phase II MMPOWER-2 trial in Mitochondrial-myopathies released by Stealth
    • 02 Jun 2017 Stealth BioTherapeutics completes a phase II trial in Heart failure in Germany and Serbia (SC) (NCT02814097)
    • 01 May 2017 Phase-II/III clinical trials in Barth syndrome (In children, In adolescents, In adults, In the elderly) in USA (SC) (NCT03098797)

Novel crystalline salt (eg hydrochloride, mesylate and tosylate salts) forms of D-Arg-Dmt-Lys-Phe-NH2 (referred to as MTP-131 or elamipretide ) and composition comprising them are claimed. See WO2016190852 , claiming therapeutic compositions including chromanyl compounds, variants and analogues and uses thereof. Stealth BioTherapeutics (formerly known as Stealth Peptides) is developing elamipretide, which targets mitochondria, for the potential iv/sc treatment of cardiac reperfusion injury, acute coronary syndrome, acute kidney injury, mitochondrial myopathy, skeletal muscle disorders and congestive heart failure.

Also, the company is developing an oral formulation of elamipretide , which targets mitochondria and reduces the production of excess reactive oxygen species, for treating chronic heart failure. In January 2015, a phase II trial was ongoing . In July 2016, a phase II trial was initiated in Latvia, Spain and Hungary .

Further, the company is developing an ophthalmic formulation of elamipretide , a mitochondria targeting peptide, for treating ocular diseases including diabetic macular edema, age-related macular degeneration and fuchs’ corneal endothelial dystrophy and Leber’s hereditary optic neuropathy.

In April 2016, a phase II trial was initiated for LHON . Family members of the product case of elamipretide ( WO2007035640 ) hold protection in the EU until 2026 and expires in the US in 2027 with US154 extension.

Acute coronary syndrome; Age related macular degeneration; Cardiac failure; Corneal dystrophy; Diabetic macular edema; Lebers hereditary optic atrophy






; MTP-131; D-Arg-Dmt-Lys-Phe-Nth). Compound

1 has been shown to affect the mitochondrial disease process by helping to protect organs from oxidative damage caused by excess ROS production and to restore normal ATP production.


US 20110082084

WO 2011091357

WO 2012129427

WO 2013059071

WO 2013126775

US 20140378396

US 20140093897

WO 2015134096

WO 2015100376

WO 2015060462

US 20150010588


WO 2015197723




The invention relates to a process for solution-phase synthesis of D- Arginyl-2,6-dimethyl-L-tyrosyl-L-lysyl-L-phenylalaninamide (abbreviated H-D-Arg-(2,6-Dimethyl)Tyr-L-Lys-L-Phe-NH2, development code SS-31 , MTP-131 , X-31) of Formula (I), an active ingredient developed by Stealth BioTherapeutics under the investigational drug brand names Bendavia® and Ocuvia®, for both common and rare diseases including a mitochondrial targeted therapy for ischemia reperfusion injury.

Formula (I)


The product belongs to the class of so-called “Szeto-Schiller peptides”. Szeto-Sciller peptides or “SS peptides” are small, aromatic-cationic, water soluble, highly polar peptides, such as disclosed in US 6703483 and US 7576061 , which can readily penetrate cell membranes. The aromatic-cationic peptides include a minimum of two amino acids, and preferably include a minimum of four amino acids, covalently joined by peptide bonds. The maximum number of amino acids is about twenty amino acids covalently joined by peptide bonds. As described by EP 2012/2436390, optimally, the number of amino acids present in the SS peptides is four.

Bendavia® is being tested for the treatment of ischemia reperfusion injury in patients with acute myocardial infarction (AMI), for the treatment of acute kidney injury (AKI) and renal microvascular dysfunction in hypertension, for the treatment of skeletal muscle dysfunction, for the treatment of mitochondrial myopathy and for the treatment of chronic heart failure. Trials are ongoing to assess the Ocuvia’s potential to treat Leber’s Hereditary Optic Neuropathy (LHON) a devastating inherited disease that causes sudden blindness, often in young adults.

Mitochondria are the cell’s powerhouse, responsible for more than 90% of the energy our bodies need to sustain life and support growth. The energetics from mitochondria maintains healthy physiology and prevents disease. In many common and rare diseases, dysfunctional mitochondria are a key component of disease progression.

D-Arginyl-2,6-dimethyl-L-tyrosyl-L-lysyl-L-phenylalaninamide is a cell-permeable and mitochondria-targeted peptide that showed antioxidant activity and was concentrated in the inner mitochondrial membrane. Compound (< 1 nM) significantly reduced intracellular reactive oxygen species, increased mitochondrial potential and prevented tBHP-induced apoptosis in both N2A and SH-SY5Y neuronal cell lines. In rats, intraperitoneal treatment (1 and 3 mg/kg) 1 day prior to unilateral ureteral obstruction and every day thereafter for 14 days significantly decreased tubular damage, macrophage infiltration and interstitial fibrosis. Compound (3 mg/kg i.p. qd for 2 weeks) also prevented apoptosis and insulin reduction in mouse pancreatic islets caused by streptozotocin.

Further studies performed in a G93A mouse model of amyotrophic lateral sclerosis (ALS) demonstrated that the compound (5 mg/kg/day i.p. starting at 30 days of age) led to a significant delay in disease onset.

Potentially useful for the treatment of ALS and may be beneficial in the treatment of aging and diseases associated with oxidative stress.

In the last few years the peptide H-D-Arg-(2,6-Dimethyl)Tyr-L-Lys-L-Phe-NH2, shown in Fig 1 , and its therapeutic activity have been disclosed and

claimed by in several patent applications.

EP 2436390, US 201 10245182 and US 201 10245183 claim topical anesthetic compositions for application to the skin for pain management or anti-skin aging agents, respectively, comprising Szeto-Schiller peptides; SS-31 is specifically claimed as active ingredient. Sequence of solid-phase synthesis is indicated as the preferred preparation process.

US 7718620 claims a process of treating or preventing ischemia-reperfusion injury of the kidney in a mammal by administrating an effective amount of an aromatic-cationic peptide. SS-31 is specifically claimed as active ingredient.

WO2005/001023 discloses a generical process and carrier complexes for delivering molecules to cells comprising a molecule and an aromatic cationic peptide of type D-Arg-Dmt-Lys-Phe-NH2. The tetrapeptide SS-31 is

specifically claimed as product useful for the process at claim 18.

WO2012/1741 17 and WO2014/210056 claim therapeutic compositions based on SS peptides and the aromatic-cationic peptide D-Arg-Dmt-Lys-Phe-NH2 as active agent.

WO 2013/086020, WO 2004/070054 and WO 2005/072295 provide processes for preventing mithochondrial permeability transition and reducing oxidative damage in a mammal, a removed organ, or a cell in need thereof and specifically claims the process wherein the peptide does not have mu-opioid receptor agonist activity, i.e., D-Arg-Dmt-Lys-Phe-NH2.

WO 2009/108695 discloses a process for protecting a kidney from renal injury which may be associated with decreased or blocked blood flow in the subject’s kidney or exposure to a nephrotoxic agent, such as a radiocontrast dye. The processes include administering to the subject an effective amount of an aromatic-cationic peptide to a subject in need thereof and one of the selected peptide is D-Arg-Dmt-Lys-Phe-NH2.

US 6703483 discloses a detailed procedure for the preparation of novel analogs of DALDA [H-Tyr-D-Arg-Phe-Lys-NH2], namely H-Dmt-D-Arg-Phe-Lys-NH2 using the solid-phase techniques and /?-methylbenzhydrylamine

resin and protocols that have been extensively used by inventor’s laboratory.

Most prior art processes for preparing the compound typically comprise conventionally performed peptide solid-phase synthesis with further purification by chromatography in order to obtain the requested purity for therapeutic use.

It is well known that solid-phase synthesis followed by chromatographic purification is time consuming, very expensive and very difficult to be scaled up on industrial scale, so the need of developing a process for large scale production is obvious. The compound is isolated as organic acid salt, as acetate or trifluoro acetate.

eddy et al., Adv. Exp. Med. Biol, 2009, 61 1 , 473 generally describes the liquid-phase synthesis of antioxidant peptides of Figure 1 and similar others (SS-02, SS-20), involving routinely used side chain protecting groups for amino acid building blocks. The guanidine group was protected with NO2 and the ε-ΝΗ2 of Lys was protected by Cbz or 2-Cl-Cbz. These peptides were

synthesized using Boc/Cbz chemistry and BOP reagent coupling. Starting with the C-terminal Lys residue protected as H-Lys(2-Cl-Cbz)-NH2, (prepared

from the commercially available Boc-Lys(2-Cl-Cbz)-OH in two steps by amidation with NH4HCO3 in the presence of DCC/HOBt following a literature procedure [Ueyama et all, Biopolymers, 1992, 32, 1535, PubMed: 1457730], followed by exposure to TFA). Selective removal of the 2-Cl-Cbz in the

presence of the NO2 group was accomplished using catalytic transfer hydrogenolysis (CTH) [Gowda et al., Lett. Pept. Sci., 2002, 9, 153].

A stepwise procedure by standard solution peptide synthesis for preparation of potent μ agonist [DmtJDALDA and its conversion into a potent δ antagonist H-Dmt-Tic-Phe-Lys(Z)-OH by substitution of D-Arg with Tic to enhance the δ opioid agonist activity is described by Balboni et al., J. Med.

Chem., 2005, 48, 5608. A general synthetic procedure for a similar tetrapeptide ([Dmt-D-Arg-Phe-Lys-NH2 is described by Ballet et al., J. Med.

Chem. 2011, 54, 2467.

Similar DALDA analog tetrapeptides were prepared by the manual solid-phase technique using Boc protection for the a-amino group and DIC/HOBt or HBTU/DIEA as coupling agent [Berezowska et al., J. Med. Chem., 2009, 52, 6941 ; Olma et al., Acta Biochim. Polonica, 2001, 48, 4, 1 121 ; Schiller at al., Eur. J. Med. Chem., 2000, 35, 895].

Despite the high overall yield in the solid-phase approach, it has several drawbacks for the scale-up process such as:

a. the application of the highly toxic and corrosive hydrogen fluoride for cleavage of the peptide from the resin,

b. low loading (0.3-0.35 mmol/g of resin) proved necessary for successful end-step, and

c. use of excess amounts of reagents (3-fold of DIC, 2.4-fold of HOBt, etc.) on each step [ yakhovsky et al., Beilstein J. Org. Chem., 2008, 4(39), 1 , doi: 10.376/bjoc.4.39]


The invention relates to a more efficient process avoiding either solid-phase synthesis or chromatographic purification, more suitable for large scale production. The process of the invention is described in Scheme A.

The following abbreviations are used:

Dmt = 2,6-dimethyl tyrosine; Z= benzyloxycarbonyl; MeSO3H = methane sulphonic acid; Boc = Tert-butyloxycarbonyl; NMM = N-methyl morpholine; TBTU= N,N,N’,N’-Tetramethyl-O-(benzotriazol- l-yl)uronium tetrafluoroborate; DMF = dimethyl formamide; TFA = trifluoroacetic acid

Scheme A shows the process for the solution phase synthesis of peptide

1 for assembly of the tetrapeptide backbone using O-Benzyl (Bzl) group and benzyloxycarbonyl (Z) group respectively, as the temporary protection for amino acids’ N-termini (Scheme Figure 2), followed by a final catalytic hydrogenolysis. The final product is isolated as organic acid salt, for example, acetic acid salt.

H-Phe-NH 2 + Boc-Lys(Z)-OH

Boc-Lys(Z)-Phe-NH 2


(V) I MeS03H/CH2CI2

Boc-DMTyr(Bzl)-OH + MeS03H.H-Lys(Z)-Phe-NH 2


Boc-DMTyr(Bzl)-Lys(Z)-Phe-NH 2



Z-D-Arg-ONa + H-DMTyr(Bzl)-Lys(Z)-Phe-NH 2.MeS03H

(X) (IX)




I H2, Pd/C




Scheme A

This process is a notable improvement with respect to the prior art and its advantages can be summarized as follows:

• The synthesis is performed in liquid phase allowing the scale up on industrial scale without need of special equipment; · The selection of the protecting group in the building blocks allows a straightforward synthesis with very simple deprotection at each step and minimize the formation of undesired by-product;

• Each intermediate can be crystallized allowing removal of impurities which are not transferred to the following step;

· The purity of each intermediate is very high and usually close to



Example 1: Preparation of Boc-Lys(Z)-Phe-NH2

Charge 200 mL of DMF, 44 g of Boc-Lys(Z)-OH and 15.6 g of H-Phe-NH2 in a flask. Stir the mixture at room temperature for 10 min. Add 19.2 g of

N-methylmorpholine and 32.1 g of TBTU successively at room temperature. Stir the mixture at room temperature for 1 h. Add 500 mL of water into the reaction mixture to precipitate the product at room temperature. Filter the mixture to isolate the solid product and wash the filter cake with water.

Transfer the filter cake into a flask containing 360 mL of ethyl acetate and heat the mixture at 50°C till all the solid is dissolved. Separate the organic phase of product and discard the small aqueous phase. Concentrate the organic phase at 40~45°C and under vacuum to remove the solvent till lots of solid is formed. Filter the residue to isolate the solid product. Transfer the filter cake into a flask containing 2000 mL of MTBE and heat the mixture at refluxing for 20 min. Then, cool down the mixture to room temperature. Filter the mixture to isolate the solid product. Dry the filter cake at 30 °C and under vacuum to give 35 g of solid product.

Example 2: Preparation of H-Lys(Z)-Phe-NH2.MeSC>3H

Charge 26.3 g of Boc-Lys(Z)-Phe-NH2, 200 mL of methylene chloride

and 9.6 g of methanesulfonic acid. Stir the mixture at 15-20 °C for 18 h. Add 100 mL of MTBE into the mixture and stir at 15-20 °C for 1 h. Filter the mixture to isolate the solid product. Dry the wet cake in air at room temperature to give 26.4 g of white solid product.

Example 3: Preparation of Boc-DMeTyr(Bzl)-Lys(Z)-Phe-NH2

Charge 8.4 g of Boc-DMeTyr(Bzl)-OH, 1 1 g of H-Lys(Z)-Phe-NH2.MeSO3H, 7.4 g of TBTU and 80 mL of THF in a flask. Stir the mixture

at room temperature for 15 min, and then cool down to 10°C. Add 6.36 g of N-methylmorpholine and stir the mixture at 20-25°C for 3 h. Add the reaction mixture into a flask containing 240 mL of water. Add 32 mL of methylene chloride into the mixture obtained in the previous operation of. Stir the resultant mixture at room temperature for 20 min. Filter the mixture to isolate the solid product and wash the filter cake with acetone (300 mL X 2). Dry the filter cake in air at room temperature to give 14.3 g of white solid product.

Example 4: Preparation of H-DMeTyr(Bzl)-Lys(Z)-Phe-NH2.MeS03H

Charge 14 g of Boc-BMeTyr(Bzl)-Lys(Z)-Phe-NH2, 280 mL of methylene chloride and 3.3 g of methanesulfonic acid in a flask. Stir the mixture at 18 ~ 22 °C for 10 h. Add 560 mL of heptanes into the mixture and stir the mixture at room temperature for 30 min. Filter the mixture to isolate the solid product. Dry the wet cake in air at room temperature to give 14 g of white solid product.

Example 5: Preparation of Z-D-Arg-DMeTyr(Bzl)-Lys(Z)-Phe-NH2

Charge 6.34 g of Z-D-Arg-ONa, 100 mL of DMF and 2.0 g of methanesulfonic acid in a flask. Stir the mixture at room temperature till a clear solution was formed. Add 14 g of H-DMeTyr(Bzl)-Lys(Z)-Phe-NH2.MeSO3H and cool down the mixture to 10°C. Add 6.15 g of TBTU and

9.67 g of N-methylmorpholine successively. Stir the mixture at room temperature for 4 h. Add aqueous solution of LiOH prepared by dissolving 2.9 g of LiOH.L O in 8 mL of water. Stir the mixture for 30 min. Add the resultant mixture slowly into a flask containing 420 mL of water under stirring. Add 56 mL of methylene chloride into the mixture. Filter the mixture to isolate the solid product. Transfer the filter cake into a flask containing 150 mL of acetic acid, and heat the mixture at 35-40 °C till most of the solid was dissolved. Add 450 mL of MTBE into the mixture and cool down the mixture under stirring to room temperature. Filter the mixture to isolate the solid product. Dry the filter cake in air at room temperature to give 17.3 g of the white solid product.

Example 6 Preparation of H-D-Arg-DMeTyr-Lys-Phe-NH2.3AcOH

Charge 2.0 g of Z-D-Arg-DMeTyr(Bzl)-Lys(Z)-Phe-NH2, 20 mL of acetic acid and 5% Pd/C catalyst (which is obtained by washing 5.0 g of 5% Pd/C containing 60% of water with 30 mL of acetic acid) in a flask. Change the atmosphere of the flask with hydrogen. Stir the mixture at room temperature and pressure of 1 atm of hydrogen for 2 h. Filter the mixture to remove the Pd/C catalyst and wash the filter cake with 10 mL of acetic acid. Combine the filtrate and washing solution and concentrate the solution at 20°C and under vacuum to remove most the solvent. Add 100 mL of acetonitrile into the residue and stir the mixture at room temperature for 20 min. Filter the mixture to isolate the solid product. Dry the filter cake at room temperature and under vacuum to give 0.7 g of the white product.


WO 2016001042


  1. Jump up^ “Recommended INN List 75” (PDF). WHO Drug Information30 (1): 111. 2016.
  2. Jump up^ “Elamipretide”. AdisInsight. Retrieved 24 April 2017.
  3. Jump up^ Kloner, RA; Shi, J; Dai, W (February 2015). “New therapies for reducing post-myocardial left ventricular remodeling.”Annals of translational medicine3 (2): 20. PMC 4322169Freely accessiblePMID 25738140.
  4. Jump up^ Valigra, Lori (April 9, 2012). “Stealth Peptides sees positive results from Bendavia”Boston Business Journal.
  5. Jump up^ Dolgin, Elie (11 February 2016). “New drugs offer hope for mitochondrial disease”STAT.
Patent ID

Patent Title

Submitted Date

Granted Date

Patent ID

Patent Title

Submitted Date

Granted Date

US2014294796 AROMATIC-CATIONIC PEPTIDES AND USES OF SAME 2012-12-05 2014-10-02
US2017129920 Process for Preparing D-Arginyl-2, 6-Dimethyl-L-Tyrosyl-L-Lysyl-L-Phenylalaninamide 2015-06-24


1: Alam NM, Mills WC 4th, Wong AA, Douglas RM, Szeto HH, Prusky GT. A mitochondrial therapeutic reverses visual decline in mouse models of diabetes. Dis Model Mech. 2015 Jul 1;8(7):701-10. doi: 10.1242/dmm.020248. Epub 2015 Apr 23. PubMed PMID: 26035391; PubMed Central PMCID: PMC4486862.

2: Szeto HH, Birk AV. Serendipity and the discovery of novel compounds that restore mitochondrial plasticity. Clin Pharmacol Ther. 2014 Dec;96(6):672-83. doi: 10.1038/clpt.2014.174. Epub 2014 Sep 4. Review. PubMed PMID: 25188726; PubMed Central PMCID: PMC4267688.

3: Dai W, Shi J, Gupta RC, Sabbah HN, Hale SL, Kloner RA. Bendavia, a mitochondria-targeting peptide, improves postinfarction cardiac function, prevents adverse left ventricular remodeling, and restores mitochondria-related gene expression in rats. J Cardiovasc Pharmacol. 2014 Dec;64(6):543-53. PubMed PMID: 25165999.

4: Eirin A, Ebrahimi B, Zhang X, Zhu XY, Woollard JR, He Q, Textor SC, Lerman A, Lerman LO. Mitochondrial protection restores renal function in swine atherosclerotic renovascular disease. Cardiovasc Res. 2014 Sep 1;103(4):461-72. doi: 10.1093/cvr/cvu157. Epub 2014 Jun 19. PubMed PMID: 24947415; PubMed Central PMCID: PMC4155472.

5: Liu S, Soong Y, Seshan SV, Szeto HH. Novel cardiolipin therapeutic protects endothelial mitochondria during renal ischemia and mitigates microvascular rarefaction, inflammation, and fibrosis. Am J Physiol Renal Physiol. 2014 May 1;306(9):F970-80. doi: 10.1152/ajprenal.00697.2013. Epub 2014 Feb 19. PubMed PMID: 24553434.

6: Brown DA, Hale SL, Baines CP, del Rio CL, Hamlin RL, Yueyama Y, Kijtawornrat A, Yeh ST, Frasier CR, Stewart LM, Moukdar F, Shaikh SR, Fisher-Wellman KH, Neufer PD, Kloner RA. Reduction of early reperfusion injury with the mitochondria-targeting peptide bendavia. J Cardiovasc Pharmacol Ther. 2014 Jan;19(1):121-32. doi: 10.1177/1074248413508003. Epub 2013 Nov 28. PubMed PMID: 24288396; PubMed Central PMCID: PMC4103197.

7: Birk AV, Chao WM, Bracken C, Warren JD, Szeto HH. Targeting mitochondrial cardiolipin and the cytochrome c/cardiolipin complex to promote electron transport and optimize mitochondrial ATP synthesis. Br J Pharmacol. 2014 Apr;171(8):2017-28. doi: 10.1111/bph.12468. PubMed PMID: 24134698; PubMed Central PMCID: PMC3976619.

8: Szeto HH. First-in-class cardiolipin-protective compound as a therapeutic agent to restore mitochondrial bioenergetics. Br J Pharmacol. 2014 Apr;171(8):2029-50. doi: 10.1111/bph.12461. Review. PubMed PMID: 24117165; PubMed Central PMCID: PMC3976620.

9: Zhao WY, Han S, Zhang L, Zhu YH, Wang LM, Zeng L. Mitochondria-targeted antioxidant peptide SS31 prevents hypoxia/reoxygenation-induced apoptosis by down-regulating p66Shc in renal tubular epithelial cells. Cell Physiol Biochem. 2013;32(3):591-600. doi: 10.1159/000354463. Epub 2013 Sep 6. PubMed PMID: 24021885.

10: Dai DF, Hsieh EJ, Chen T, Menendez LG, Basisty NB, Tsai L, Beyer RP, Crispin DA, Shulman NJ, Szeto HH, Tian R, MacCoss MJ, Rabinovitch PS. Global proteomics and pathway analysis of pressure-overload-induced heart failure and its attenuation by mitochondrial-targeted peptides. Circ Heart Fail. 2013 Sep 1;6(5):1067-76. doi: 10.1161/CIRCHEARTFAILURE.113.000406. Epub 2013 Aug 9. PubMed PMID: 23935006; PubMed Central PMCID: PMC3856238.

/////////////////////Elamipretide,  SS-31,  Bendavia, PEPTIDE


The greening of peptide synthesis


The greening of peptide synthesis


The synthesis of peptides by amide bond formation between suitably protected amino acids is a fundamental part of the drug discovery process. However, the required coupling and deprotection reactions are routinely carried out in dichloromethane and DMF, both of which have serious toxicity concerns and generate waste solvent which constitutes the vast majority of the waste generated during peptide synthesis. In this work, propylene carbonate has been shown to be a green polar aprotic solvent which can be used to replace dichloromethane and DMF in both solution- and solid-phase peptide synthesis. Solution-phase chemistry was carried out with Boc/benzyl protecting groups to the tetrapeptide stage, no epimerisation occurred during these syntheses and chemical yields for both coupling and deprotection reactions in propylene carbonate were at least comparable to those obtained in conventional solvents. Solid-phase peptide synthesis was carried out using Fmoc protected amino acids on a ChemMatrix resin and was used to prepare the biologically relevant nonapeptide bradykinin with comparable purity to a sample prepared in DMF.

Graphical abstract: The greening of peptide synthesis
Boc-Ala-Phe-OBn 5a    ref S1
Boc-Ala-OH (324 mg, 1.71 mmol) and HCl.H-Phe-OBn (500 mg, 1.71 mmol) were coupled according to the general coupling procedure. The residue was purified using flash column chromatography (35:65, EtOAc:PE) to give Boc-Ala-Phe-OBn 5a as a white crystalline solid (682 mg, 93%). RF = 0.34 (40:60, EtOAc:PE);
mp 95.6-96.3 °C;
[α]D 23 -27.7 (c 1.0 in MeOH);
IR (Neat) νmax 3347 (m), 3063 (w), 3029 (w), 2928 (m), 2852 (w), 1735 (w), 1684 (w) 1666 (w) and 1521 (s) cm-1;
1H NMR (400 MHz, CDCl3): δ = 7.36-7.31 (m, 3H, ArH), 7.29-7.24 (m, 2H, ArH), 7.26-7.21 (m, 3H, ArH), 7.04-6.97 (m, 2H, ArH), 6.72 (d J 7.7 Hz, 1H, Phe-NH), 5.16-5.10 (m, 1H, Ala-NH), 5.13 (d J 12.1 Hz, 1H, OCH2Ph), 5.07 (d J 12.1 Hz, 1H, OCH2Ph), 4.88 (dt, J 7.7, 5.9 1H, PheNCH), 4.11 (br, 1H, Ala-NCH), 3.13 (dd J 13.9, 6.1 Hz, 1H, CH2Ph), 3.08 (dd J 13.9, 6.1 Hz, 1H, CH2Ph), 1.41 (s, 9H, C(CH3)3), 1.29 (d J 6.6 Hz, 3H, CH3);
13C NMR (100 MHz, CDCl3): δ = 172.3 (C=O), 171.2 (C=O), 155.6 (NC=O), 135.7 (ArC), 135.1 (ArC), 129.5 (ArCH), 128.7 (ArCH), 128.6 (ArCH), 127.2 (ArCH), 80.2 (CMe3), 67.4 (OCH2Ph), 53.3 (Phe-NCH), 50.3 (Ala-NCH), 38.0 (CH2Ph), 28.4 (C(CH3)3), 18.5 (CH3);
MS (ESI) m/z 449 [(M+Na)+ , 100]; HRMS (ESI) m/z calculated for C24H30N2O5Na 449.2048 (M+Na)+ , found 449.2047 (0.6 ppm error).
S1 J. Nam, D. Shin, Y. Rew and D. L. Boger, J. Am. Chem. Soc., 2007, 129, 8747–8755; Q. Wang, Y. Wang and M. Kurosu, Org. Lett., 2012, 14, 3372–3375.
General procedure for peptide coupling reactions in PC To a suspension of an N-Boc-amino acid (1.0 eq.) and an amino acid or peptide benzyl ester (1.0 eq.) in PC (5 mL mmol-1), at 0 °C, was added a solution of HOBt (1.1 eq.) and i Pr2EtN (3.0 eq.) in a minimal quantity of PC. EDC (1.1 eq.) was added dropwise and the reaction mixture was allowed to stir at room temperature for 16h. The reaction mixture was then diluted using EtOAc (50 mL) and washed with 1M HClaq (3 × 25 mL), saturated Na2CO3 (3 × 25 mL) and H2O (3 × 25 mL). The organic layer was dried (MgSO4 ), filtered and concentrated in vacuo. Any residual PC was removed via short path distillation. Purification details for each peptide and characterising data are given in the supplementary information. General procedure for Boc deprotections in PC An N-Boc-peptide benzyl ester (1.0 eq.) was dissolved in a minimum amount of PC and trifluoroacetic acid (60 eq.) was added. The reaction mixture was allowed to stir for 3h. at room temperature before being concentrated in vacuo. Any residual PC was removed via short path distillation. Characterising data for each deprotected peptide are given in the supplementary information.
Procedure for Boc deprotection of dipeptide 5a using HCl in PC Boc-Ala-Phe-OBn 5a (50 mg, 0.117 mmol) was dissolved in PC (2.34 mL). MeOH (0.40 mL, 9.8 mmol) was added and the solution cooled to 0 o C. Acetyl chloride (0.67 mL, 9.36 mmol) was added dropwise and the solution allowed to stir at room temperature for 2h. Then, PC was removed by short path distillation. The residue was suspended in Et2O and stirred for 5 minutes before being filtered to give HCl.Ala-Ph-OBn as a white solid (32.4 mg, 76%).
Propylene carbonate 1 has been shown to be a green replacement for reprotoxic amide based solvents which are widely used in peptide synthesis. Both solution- and solidphase peptide synthesis can be carried out in propylene carbonate using acid and base labile amine protecting groups respectively. No significant racemisation of the activated amino acids occurs in propylene carbonate and the viability of solid-phase peptide synthesis in propylene carbonate was demonstrated by the synthesis of the nonapeptide bradykinin.

FDA approves Adlyxin (lixisenatide) 利西拉 to treat type 2 diabetes



07/28/2016 07:53 AM EDT
The U.S. Food and Drug Administration approved Adlyxin (lixisenatide), a once-daily injection to improve glycemic control (blood sugar levels), along with diet and exercise, in adults with type 2 diabetes.

July 28, 2016


The U.S. Food and Drug Administration approved Adlyxin (lixisenatide), a once-daily injection to improve glycemic control (blood sugar levels), along with diet and exercise, in adults with type 2 diabetes.

“The FDA continues to support the development of new drug therapies for diabetes management,” said Mary Thanh Hai Parks, M.D., deputy director, Office of Drug Evaluation II in the FDA’s Center for Drug Evaluation and Research. “Adlyxin will add to the available treatment options to control blood sugar levels for those with type 2.”

Type 2 diabetes affects more than 29 million people and accounts for more than 90 percent of diabetes cases diagnosed in the United States. Over time, high blood sugar levels can increase the risk for serious complications, including heart disease, blindness and nerve and kidney damage.

Adlyxin is a glucagon-like peptide-1 (GLP-1) receptor agonist, a hormone that helps normalize blood sugar levels. The drug’s safety and effectiveness were evaluated in 10 clinical trials that enrolled 5,400 patients with type 2 diabetes. In these trials, Adlyxin was evaluated both as a standalone therapy and in combination with other FDA-approved diabetic medications, including metformin, sulfonylureas, pioglitazone and basal insulin. Use of Adlyxin improved hemoglobin A1c levels (a measure of blood sugar levels) in these trials.

In addition, more than 6,000 patients with type 2 diabetes at risk for atherosclerotic cardiovascular disease were treated with either Adlyxin or a placebo in a cardiovascular outcomes trial. Use of Adlyxin did not increase the risk of cardiovascular adverse events in these patients.

Adlyxin should not be used to treat people with type 1 diabetes or patients with increased ketones in their blood or urine (diabetic ketoacidosis).

The most common side effects associated with Adlyxin are nausea, vomiting, headache, diarrhea and dizziness. Hypoglycemia in patients treated with both Adlyxin and other antidiabetic drugs such as sulfonylurea and/or basal insulin is another common side effect. In addition, severe hypersensitivity reactions, including anaphylaxis, were reported in clinical trials of Adlyxin.

The FDA is requiring the following post-marketing studies for Adlyxin:

  • Clinical studies to evaluate dosing, efficacy and safety in pediatric patients.
  • A study evaluating the immunogenicity of lixisenatide.

Adlyxin is manufactured by Sanofi-Aventis U.S. LLC, of Bridgewater, New Jersey.




lixisenatide;Lixisenatide|Lixisenatide Acetate;Lixisenatide Acetate
CAS: 320367-13-3
MF: C215H347N61O65S
MW: 4858.53

C215 H347 N61 O65 S

L-Lysinamide, L-histidylglycyl-L-α-glutamylglycyl-L-threonyl-L-phenylalanyl-L-threonyl-L-seryl-L-α-aspartyl-L-leucyl-L-seryl-L-lysyl-L-glutaminyl-L-methionyl-L-α-glutamyl-L-α-glutamyl-L-α-glutamyl-L-alanyl-L-valyl-L-arginyl-L-leucyl-L-phenylalanyl-L-isoleucyl-L-α-glutamyl-L-tryptophyl-L-leucyl-L-lysyl-L-asparaginylglycylglycyl-L-prolyl-L-seryl-L-serylglycyl-L-alanyl-L-prolyl-L-prolyl-L-seryl-L-lysyl-L-lysyl-L-lysyl-L-lysyl-L-lysyl-







827033-10-3; Lixisenatide [INN]; UNII-74O62BB01U; DesPro36Exendin-4(1-39)-Lys6-NH2;   DesPro36Exendin-4(1-39)-Lys6-NH2
Molecular Formula: C215H347N61O65S
Molecular Weight: 4858.49038 g/mol
IUPAC Condensed


from PubChem


from PubChem


from PubChem


from PubChem


Sanofi (formerly sanofi-aventis, formerly Aventis), under license from Zealand Pharma, has developed and launched lixisenatide

Lixisenatide (trade name Lyxumia) is a once-daily injectable GLP-1 receptor agonist for the treatment of diabetes, discovered by Zealand Pharma A/S of Denmark and licensed and developed by Sanofi.[1] Lixisenatide was accepted for review by the US FDA on February 19, 2013, and approved by the European Commission on February 1, 2013.[2] On September 12, 2013, Sanofi delayed the approval process in the US, citing internal data from a cardiovascular risk study. The drug will likely be resubmitted for approval in 2015.

Lixisenatide is a once-daily injectable GLP-1 receptor agonist discovered by Zealand Pharma A/S of Denmark and licensed and developed by Sanofi. As of September 2010 it is in clinical trials for diabetes. Lixisenatide was accepted for review by the US FDA on February 19, 2013, and approved by the European Commission on February 1, 2013. The drug will likely be resubmitted for approval in 2015.

Mechanism of action

GLP-1 is a naturally-occurring peptide that is released within minutes of eating a meal. It is known to suppress glucagon secretion from pancreatic alpha cells and stimulate insulin secretion by pancreatic beta cells. GLP-1 receptor agonists are used as an add-on treatment for type 2 diabetes and their use is endorsed by the European Association for the Study of Diabetes, the American Diabetes Association, the American Association of Clinical Endocrinologists and the American College of Endocrinology.

Physical and chemical properties

Lixisenatixe has been described as “des-38-proline-exendin-4 (Heloderma suspectum)-(1–39)-peptidylpenta-L-lysyl-L-lysinamide”, meaning it is derived from the first 39 amino acids in the sequence of the peptide exendin-4, found in the Gila monster (Heloderma suspectum), omitting proline at position 38 and adding six lysine residues. Its complete sequence is:[3]



US 20110313131



CN 105713082

The title method comprises the steps of: (1) coupling Fmoc-Lys(Boc)-OH and resin to obtain Fmoc-Lys(Boc)-resin, (2) protecting amino acid with Fmoc, conducting solid-phase synthesis to obtain lixisenatide wholly protected 20-44-peptide resin, (3) conducting solid-phase synthesis to obtain wholly protected 15-19-peptide resin, (4) coupling the wholly protected 20-44-peptide resin and wholly protected 15-19-peptide resin, (5) coupling other amino acids till solid-phase synthesis finishes, (6) cracking lixisenatide peptide resin to obtain crude peptide, and (7) purifying through RP-HPLC.  The method improves crude peptide purity and purifn. yield.




利西拉, the English name: Lixisenatide, is a polypeptide containing 44 amino acids, the structural formula is as follows: peptide sequence as follows:

Figure CN104211801AD00031

H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Al a-Val-Arg-Leu-Phe-IIe-Glu -Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pr O-Ser-Lys-Lys-Lys-Lys-Lys-Lys-NH 2 Li Xila to (Lixisenatide ) by Sanofi-Aventis developed once a day subcutaneously with glucagon-like peptide -I (GLP-I) receptor agonists, for the treatment of type II diabetes, on February 1, 2013 Sanofi Lee Division -Aventis of exenatide is approved EMEA, for the adjuvant treatment of poorly stable dose of basal insulin (or metformin) in the treatment of type II diabetes to improve HbAlc and postprandial blood glucose levels.

CN201210030151. 2 used in a pure solid phase sequential coupling method synthetic peptides. The method amino resin as the carrier, using conventional coupling sequence, the final cut to give Li Xila.

 US6528486 patent for the compound, synthetic methods mentioned it to phase condensation method Fmoc / tBu strategy.

The [0005] W02005058954 synthesis method including the gradual condensation process Fmoc / tBu strategy, Boc strategy of gradual condensation methods and genetic engineering.

The  W02001004156 synthesis method for the gradual condensation process Fmoc / tBu strategy.

 Since Li Xila abroad mostly used to synthesize Fmoc solid phase synthesis method, a gradual shrinking gradually synthesis step more, resulting in more types of product impurities, US 20130284912 Special Report polypeptide impurity: Di-Ser33- Leisy pull and Di-Ala35- Li Xila come, Di-Ser 33- Li Xila come and Di-Ala35- Li Xila to atmosphere amino acid sequence as follows: Di-Ser33- Li Xila to the amino acid sequence: H-His -Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Al a-Val-Arg-Leu-Phe-IIe-Glu-Trp- Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Ser-Gly-Ala-Pr 〇-Pr〇-Ser-Lys_Lys_Lys_Lys_Lys_LyS-NH2 Di-Ala35- Li Xila to the amino acid sequence: H-His-Gly- Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Al a-Val-Arg-Leu-Phe-IIe-Glu-Trp-Leu-Lys -Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Ala-Pr 〇-Pr〇-Ser-Lys_Lys_Lys_Lys_Lys_LyS-NH2 toxicity of these impurities are impurities larger, and very difficult to separate from the main peak , the presence of the impurities seriously affect 利西拉 to content and the use of safety. Hence the need to find an effective way to remove it and to reach the high standard level of 0.1% or less. The present inventors have found that this impurity is difficult to remove by means of the prior art, although there are ways to remove part of, but removal is not ideal, it is difficult to achieve high quality standards is likely to cause 利西拉 level while reducing their yield.

In summary, the existing Li Xila to the solid phase synthesis, low yield of the synthesis, impurities, in particular, are not well controlled impurity Di-Ser 33- Li Xila come and Di-Ala35 – Li Xila to, does not apply to industrial production

Example i ^ a: Preparation 利西拉 to fine peptide acetate Weigh 利西拉 above 44. 70g to 45L crude peptide was dissolved in water, purified by C18 column, the first purification conditions: mobile phase: A phase: 0 I% TFA; B phase: acetonitrile; gradient program was: 15% B, 60 minutes to 60% B; detection wavelength 220 nm; peak fraction collection purposes. The second purification conditions: mobile phase was: A phase: 0 3% HAC; B phase: acetonitrile; gradient program was: 10% B, 60 minutes to 60% B; detection wavelength 220 nm; peak fraction collection purposes. Desalting conditions: Mobile phase: A phase: an aqueous solution of 20 mmol / L ammonium acetate: acetonitrile = 95: 5; B phase: water: acetonitrile = 95: 5; C phase: 0.03% aqueous solution of acetic acid: acetonitrile = 95 : 5; D phase: 0.03% aqueous solution of acetic acid: acetonitrile = 50: 50; gradient program: mobile phase A isocratic for 15 minutes, convert isocratic mobile phase B for 10 minutes, is converted into the flow Phase C isocratic 10 minutes, converted into a mobile phase D isocratic 25 minutes; detection wavelength 220 nm; peak fraction collection purposes; rotary evaporation concentrated and lyophilized to give Li Xila acetate fine peptide 22. 65g which HPLC spectrum shown in Figure 5, HPLC purity of 99.75% (area normalization method), Di-Ser33- Li Xila come to 0.03% (area normalization method), Di-Ala35- Li Xila to the content of 0.05% (area normalization method). Purification total yield of 51%, total yield 41%. Its mass spectrum as shown in Figure 6, [M + H] + = 4858. 691, 利西拉 precise molecular weight to the theoretical: 4857.53, the sample mass is consistent with the theoretical molecular weight.


CN 103709243


Example 2: Preparation 利西拉 to crude peptide

利西拉 [0116] Example 24 was prepared to be placed 125.4g peptide resin cleavage reaction to 10ml / g resin ratio added lysis reagent (TFA: thioanisole: EDT: TIS: water = 86: 5 : 5: 3: 1 (V / V)), stirred at room temperature 2.5h. The reaction was purified by frit funnel filtration, the filtrate was collected, the resin was washed 3 times and then a small amount of TFA, the combined filtrates concentrated under reduced pressure. Frozen precipitation in anhydrous ether was added, washed three times with anhydrous diethyl ether, and dried in vacuo to give a white solid powder, i.e. Li Xila to crude peptide 47.lg, by weight of the crude peptide yield 97.2%, HPLC purity 63.8% 0

利西拉 to crude peptide preparation: 27 patients [0117] Example

利西拉 [0118] The Example 25 was prepared to be placed 123.7g peptide resin cleavage reaction to 10ml / g resin ratio added lysis reagent (TFA: thioanisole: EDT: TIS: water = 86: 5 : 5: 3: 1 (V / V)), stirred at room temperature 2.5h. The reaction was purified by frit funnel filtration, the filtrate was collected, the resin was washed 3 times and then a small amount of TFA, the combined filtrates concentrated under reduced pressure. Frozen precipitation in anhydrous ether was added, washed three times with anhydrous diethyl ether, and dried in vacuo to give a white solid powder, i.e. Li Xila to crude peptide 46.9g, yield the crude peptide by weight 96.5%, HPLC purity 64.2% 0

28 Example 2: Preparation 利西拉 to fine peptide acetate

 Example weighed 26 to 27 after 利西拉 to any 30.0g crude peptide was dissolved in 3000ml of water using Waters2545RP-HPLC system, wavelength 230nm, 50 X 250mm column of reverse phase C18 column, 0.2% TFA conventional / acetonitrile mobile phase were fractionated peaks of fractions, refined peptide purity greater than 98.5%. The fine peptide solution using Waters2545RP-HPLC system, 50 X 250mm column was C18 reverse phase column, 0.1% acetic acid / acetonitrile mobile phase transfer salt, the purpose of peak fractions were collected, concentrated by rotary evaporation and lyophilized to give Li Xila acetate fine salt peptide> 9.0g, RP-HPLC purity ≥98.5%. Purification Yield ≥30%, total yield ≥29.0%.


CN 102875663


Example 9

[0239] The crude peptide Li Xila to 4000g (including Li Xila to 1139g) was dissolved with purified water 100L, collected by filtration and the filtrate set aside.

[0240] purification chromatographic conditions:

[0241] HPLC Model: Novasep LC450

 Column: 450X250mm, built-phenyl silane bonded silica gel as stationary phase filler, the filler particle size of 10 μ m0

 flow rate: 5000ml / min.

The detection wavelength: 280nm.

 Mobile phase A phase: 10% 30mM D- 30mM sodium tartrate and disodium hydrogenphosphate in methanol / 90% aqueous (v / v), adjusted to pH 2.5 with phosphoric acid.

[0246] Mobile phase A phase preparation process: Weigh 1280g 2070g D- sodium tartrate and disodium hydrogenphosphate, after an appropriate amount of purified water was dissolved through 0.45 μ m membrane filter, the filtrate collected all 300L tank, added 30L chromatographically pure After methanol was added to the 300L scale purification of water, adjusted to pH 2.5 with phosphoric acid. Repeat preparation run.

[0247] The mobile phase B phase: HPLC grade acetonitrile.

Figure CN102875663BD00132

[0249] sample volume: 250.0g (6250ml).

[0250] Purification: column equilibration the sample so that after 5 minutes, run a gradient purification, monitoring and staging purposes peak fractions were collected. The collected fractions (chromatographic conditions purity testing to the same conditions as above 利西拉 determination to area normalization method measured) purity test, the purity of greater than or equal to 98% of the fractions after removing most of the acetonitrile in turn salt; purity of 70% or more less than 98% of the fraction recovered after removal of most of the acetonitrile and the purification procedure is repeated, again collected purity greater than or equal to 98% of the fraction after removal of most of the acetonitrile are also used to turn salt; purity of less than 70 % of fractions by waste disposal.

[0251] points and 16 injections, repeat the above operation.

[0252] turn salt chromatographic conditions:

[0253] HPLC Model: Novasep LC450

[0254] Column: 450 X 250mm, built-C8 reversed-phase chromatography packing, the particle size of the filler is 10 μ m.

[0255] flow rate: 5000ml / min.

[0256] The detection wavelength: 280nm.

[0257] Mobile phase A phase: 0.2% acetic acid (v / v) solution.

[0258] The mobile phase B phase: HPLC grade acetonitrile.

[0259] gradient

Figure CN102875663BD00141

[0260] sample volume: 2500ml.

[0261] Purification: The column equilibration the sample for 5 minutes, run a gradient purification, monitoring and collecting the target peak fractions. The purpose of the peak fractions were concentrated by rotary evaporation under reduced pressure to 9000ml after lyophilization.

[0262] After the freeze-dried to give a white powder refined peptide 704g. Purity of 98.39%, the impurity content of less than 0.5%. Purification yield 61.8% (in crude Li Xila to content), total yield of 17.6%.


CN 102558338


Preparation of Fmoc-Lys (Boc) -Lys (Boc) -Lys (Boc) -Lys (Boc) -Rink Amide-MBHAResin:

[0096] To the resulting Fmoc-Lys (Boc) -Lys (Boc) -Lys (Boc) -RinkAmide-MBHAResin mouth of a 20% strength piperidine / DMF solution for 10 minutes, the reaction was drained, washed with DMF Resin 6 (50ml * 6). Weigh Fmoc-Lys (Boc) -〇H3.52g, H0Bt1.01g, HBTU2.84g, TMP1.98ml, DMF50ml added to dissolve slowly with stirring under ice-cooling for 3 minutes, at room temperature for 2 hours, the reaction Ninhydrin detection method completed, pumping off the reaction solution, DMF the resin was washed twice (50mlX2), DCM the resin was washed twice (50mlX2), to give Fmoc-Lys (B oc) -Lys (Boc) -Lys (Boc) -Lys (Boc) -RinkAmide-MBHAResin. As used in the above operation Fmoc-Lys (Boc) -OH: HOBt: HBTU: TMP ratio is 1: 1: 1: 2, wherein Fmoc-Lys (Boc) -OH is the number of moles of Fmoc-RinkAmide-MBHAResin number of moles 3 times.

[0097] Li Xila fully protected side chain was prepared to -Rink Amide-MBHA Resin:

[0098] To the resulting Fmoc-Lys (Boc) -Lys (Boc) -Lys (Boc) -Lys (Boc) -RinkAmide-MBHA Resin added 20% piperidine / DMF solution for 10 minutes, drained reaction solution, washed 6 times with DMF. Weigh Jie 111〇 (3-1 ^ 8 billion (3) -0 13.528, 1 (»Shu 1.018,01 (:!! 1.391111 added 50,111,101 ^ dissolve slowly stirring for 3 minutes in an ice bath, poured into the solid phase resin is mixed with the reaction column, at room temperature for 2 hours, the reaction Ninhydrin detection method is completed, the reaction solution was deprived, DMF the resin was washed twice (50ml X 2), DCM the resin was washed twice (50ml X 2), to give Fmoc-Lys ( Boc) -Lys (Boc) -Lys (Boc) -Lys (Boc) -Lys (Boc) -Rink Amide-MBHAResin above operation used by the Fmoc-Lys (Boc) -〇H:. HOBt: DIC ratio is 1: 1: L2, which Fmoc-Lys (Boc) is three times the number of moles -〇H Fmoc-Rink Amide-MBHA Resin moles of repeat after the coupling step, followed by the completion of the 39 lysine to first. connecting protected amino acids histidine, followed by addition of 20% piperidine / DMF solution for 10 minutes, the reaction was drained, DMF the resin was washed six times (50ml X 6), DCM the resin was washed six times (50ml X 6 ), MeOH contraction of the resin three times with MeOH 50ml, each contraction 5min. After the resin was dried in vacuo to give a full side-chain protected peptide resin to the Li Xila 27. 5g, weight resin 17. 5g.

[0099] Li Xila to crude peptide preparation:

[0100] Weigh side chains fully protected Li Xila to -Rink Amide-MBHA Resin 27. 5 grams, into a round bottom flask.Configuration 275 ml lysis buffer, wherein trifluoroacetic acid: thioanisole: ethanedithiol: anisole, phenol = 93: 4: 1: 1.5: 2 (volume ratio). Lysate in the refrigerator after the pre-freeze 1 hour before Sheng Youli put to Silas to -Rink Amide-MBHA Resin round bottom flask, stirred at room temperature for 2 hours. The reaction mixture was filtered, the resin was washed with 20ml TFA and the combined filtrate.

[0101] The volume of the filtrate was slowly poured into 2,750 ml of diethyl ether frozen (frozen advance ether), a white precipitate appears, at 3000 rpm / centrifuged 5 minutes, the resulting solid was washed twice with ether, then the solid was dried under vacuum to give Li Xila trifluoroacetate crude peptide to 15. 3g.

[0102] Li Xila to large scale production of fine peptide:

[0103] Sample Preparation: The crude peptide was dissolved in water, the sample was completely dissolved by membrane filtration, the filtrate was collected for use.

[0104] Purification conditions: Column: octadecyl silane bonded silica gel as stationary phase column, the column diameter and length: 300_X250mm. Mobile phase: A phase: 35mm〇l / L phosphoric acid solution adjusted with triethylamine to pH 6. 7; B phase: acetonitrile, flow rate: 2200ml / min, Gradient: B%: 12% ~32%, detection wavelength: 280nm . The injection volume was 75g. Purification process: the column with 50% acetonitrile rinse clean after balance sample, sample amount is 75g. Linear gradient 120min, the purpose of collecting peaks will be collected 利西拉 solution was concentrated by rotary evaporation under reduced pressure to about 80mg / ml and reserve the water temperature exceeds 40 ° C without conditions.

[0105] turn salt: turn salt conditions: Column: octadecyl silane bonded silica gel as stationary phase column, the column diameter and length: 300mmX250mm. Mobile phase: A phase: mass concentration of 0.2% aqueous acetic acid; B phase: HPLC grade acetonitrile, flow rate: 2200ml / min, detection wavelength: 280nm. Gradient: B%: 6% ~36%. The injection volume was 48-60g. Salt transfer process: the column with 50% acetonitrile rinse clean after the sample, the sample volume is 1600ml sample solution. Linear gradient 90min, the purpose of collecting peaks collected Li Xila to solutions were concentrated by rotary evaporation to about 80ml / g after go to the appropriate size vials, then freeze-dried to obtain the purity of greater than 99.5% The Li Xila come.

Old post


Sanofi Provides Update on Lixisenatide New Drug Application in U.S.

Paris, France – September 12, 2013 – Sanofi (EURONEXT: SAN and NYSE: SNY) announced today its decision to withdraw the lixisenatide New Drug Application (NDA) in the U.S., which included early interim results from the ongoing ELIXA cardiovascular (CV) outcomes study. The company plans to resubmit the NDA in 2015, after completion of the ELIXA CV study.

The decision to withdraw the lixisenatide application follows discussions with the U.S. Food and Drug Administration (FDA) regarding its proposed process for the review of interim data. Sanofi believes that potential public disclosure of early interim data, even with safeguards, could potentially compromise the integrity of the ongoing ELIXA study. Sanofi’s decision is not related to safety issues or deficiencies in the NDA………………………read all at



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WO2014077802A1 * 13 Nov 2012 22 May 2014 Ipsen Pharma S.A.S. Purification method of a glp-1 analogue
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  1.  Christensen, M; Knop, FK; Holst, JJ; Vilsboll, T (2009). “Lixisenatide, a novel GLP-1 receptor agonist for the treatment of type 2 diabetes mellitus”. IDrugs : the investigational drugs journal 12 (8): 503–13. PMID 19629885.
  2.  “Sanofi New Drug Application for Lixisenatide Accepted for Review by FDA”. Newsire. 19 February 2013.
  3.  “International Nonproprietary Names for Pharmaceutical Substances (INN). Recommended INN: List 61” (PDF). WHO Drug Information 23 (1): 66f. 2009.
Clinical data
Trade names Lyxumia
License data
Routes of
Subcutaneous injection
Legal status
Legal status
  • UK: POM (Prescription only)
CAS Number 827033-10-3
ATC code A10BX10 (WHO)
PubChem CID 16139342
ChemSpider 17295846
Chemical data
Formula C215H347N61O65S
Molar mass 4858.49 g/mol

///////FDA 2016, SANOFI, FDA,  approves , Adlyxin, lixisenatide, type 2 diabetes, Sanofi-Aventis U.S. LLC, Bridgewater, New Jersey, Lyxumia,  利西拉, PEPTIDE, 




Mifamurtide (Mepact) мифамуртид , ميفامورتيد , 米法莫肽 ,



Mifamurtide (Mepact)

  • MF C59H109N6O19P
  • MW 1237.499
CGP-19835, MFCD09954133, MTP-cephalin, Mtp-PE
Muramyl tripeptide phosphatidylethanolamine
N-Acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine 2-(1′,2′-dipalmitoyl-sn-glycero-3′-hydroxyphosphoryloxy)ethylamide
(2R,5S,8R,13S,22R)-2-{[(3R,4R,5S,6R)-3-Acetamido-2,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-yl]oxy}-8-carbamoyl-19-hydroxy-5,13-dimethyl-19-oxido-3,6,11,14,25-pentaoxo-18,20,24-trioxa-4,7,12 ;,15-tetraaza-19λ5-phosphatetracontan-22-yl hexadecanoate
83461-56-7  CAS
838853-48-8 (mifamurtide sodium · xH2O)

Mifamurtide (trade name Mepact, marketed by Takeda) is a drug against osteosarcoma, a kind of bone cancer mainly affecting children and young adults, which is lethal in about a third of cases. The drug was approved in Europe in March 2009.

ChemSpider 2D Image | Mifamurtide | C59H109N6O19P


The drug was invented by Ciba-Geigy (now Novartis) in the early 1980s and sold to Jenner Biotherapies in the 1990s. In 2003,IDM Pharma bought the rights and developed it further.[1] IDM Pharma was acquired by Takeda along with mifamurtide in June 2009.[2]

Mifamurtide had already been granted orphan drug status by the U.S. Food and Drug Administration (FDA) in 2001, and theEuropean Medicines Agency (EMA) followed in 2004. It was approved in the 27 European Union member states plus Iceland, Liechtenstein, and Norway by a centralized marketing authorization in March 2009. The drug was denied approval by the FDA in 2007.[3][4] Mifamurtide has been licensed by the EMA since March, 2009.[5]


Mifamurtide is indicated for the treatment of high-grade, nonmetastasizing, resectable osteosarcoma following complete surgical removal in children, adolescents, and young adults, aged two to 30 years.[1][6][7] Osteosarcoma is diagnosed in about 1,000 individuals in Europe and the USA per year, most under the age of 30.[8] The drug is used in combination with postoperative, multiagent chemotherapy to kill remaining cancer cells and improve a patient’s chance of overall survival.[6]

In a phase-III clinical trial in about 800 newly diagnosed osteosarcoma patients, mifamurtide was combined with the chemotherapeutic agents doxorubicin and methotrexate, with or without cisplatin and ifosfamide. The mortality could be lowered by 30% versus chemotherapy plus placebo. Six years after the treatment, 78% of patients were still alive. This equals an absolute risk reduction of 8% .[1]

Adverse effects

In a clinical study, mifamurtide was given to 332 subjects (half of whom were under age of 16) and most side effects were found to be mild to moderate in nature. Most patients experience fewer adverse events with subsequent administration.[9][10]Common side effects include fever (about 90%), vomiting, fatigue and tachycardia (about 50%), infections, anaemia, anorexia, headache, diarrhoea and constipation(>10%).[1][11]


After application of the liposomal infusion, the drug is cleared from the plasma within minutes and is concentrated in lung, liver, spleen, nasopharynx, and thyroid. The terminal half-life is 18 hours. In patients receiving a second treatment after 11–12 weeks, no accumulation effects were observed.[12]


Mifamurtide is a fully synthetic derivative of muramyl dipeptide (MDP), the smallest naturally occurring immune stimulatory component of cell walls from Mycobacterium species. It has similar immunostimulatory effects as natural MDP with the advantage of a longer half-life in plasma.

NOD2 is a pattern recognition receptor which is found in several kinds of white blood cells, mainly monocytes and macrophages. It recognises muramyl dipeptide, a component of the cell wall of bacteria. Mifamurtide simulates a bacterial infection by binding to NOD2, activating white cells. This results in an increased production of TNF-α, interleukin 1,interleukin 6, interleukin 8, interleukin 12, and other cytokines, as well as ICAM-1. The activated white cells attack cancer cells, but not, at least in vitro, other cells.[13]


Consequently, the combination of mifamurtide with these types of drugs is contraindicated. However, mifamurtide can be coadministered with low doses of NSAIDs. No evidence suggests mifamurtide interacts with the studied chemotherapeutics, or with the cytochrome P450 system.[14]


Scheme of a liposome formed by phospholipids in an aqueous solution

Mifamurtide is muramyl tripeptide phosphatidylethanolamine (MTP-PE), a synthetic analogue of muramyl dipeptide. The side chains of the molecule give it a longer elimination half-life than the natural substance. The substance is applied encapsulated into liposomes (L-MTP-PE). Being a phospholipid, it accumulates in the lipid bilayer of the liposomes in the infusion.[15]


One method of synthesis (shown first) is based on N,N’-dicyclohexylcarbodiimide (DCC) assisted esterification of N-acetylmuramyl-L-alanyl-DisoglutaminylL-alanine with N-hydroxysuccinimide, followed by a condensation with 2-aminoethyl-2,3-dipalmitoylglycerylphosphoric acid in triethylamine (Et3N).[16] A different approach (shown second) uses N-acetylmuramyl-L-alanyl-D-isoglutamine, hydroxysuccinimide and alanyl-2-aminoethyl-2,3-dipalmitoylglycerylphosphoric acid;[17] that is, the alanine is introduced in the second step instead of the first.

Mifamurtide synthesis.png Mifamurtide synthesis2.png


Mifamurtide is an anticancer agent for the treatment of osteosarcoma, the most common primary malignancy of bone tissue mainly affecting children and adolescents.10

The drug was invented by Ciba-Geigy (now Novartis) in the early 1980s and the agent was subsequently licensed to Jenner Biotherapies in the 1990s.

IDM Pharma bought the rights to the drug from Jenner in April 2003.78 In March 2009, mifamurtide was approved in the 27 European Union member states plus Iceland, Liechtenstein and Norway via a centralized marketing authorization.

After the approval, IDM Pharma was acquired by Takeda, which began launching mifamurtide, as Mepact, in February 2010.

Mifamurtide, a fully synthetic lipophilic derivative of muramyl dipeptide (MDP), is muramyl tripeptide phosphatidylethanolamine (MTP-PE), which is formulated as a liposomal infusion.79 Being a phospholipid, mifamurtide accumulates in the lipid bilayer of the liposomes upon infusion.

After application of the liposomal infusion, the drug is cleared from the plasma within minutes. However, it is concentrated in lung, liver, spleen, nasopharynx and thyroid, and the terminal half-life is 18 h, which is longer than the natural substance.

Two synthetic routes have been reported,80,81 and Scheme 16 describes the more processamenable route.

Commercially available 1,2-dipalmitoyl-sn-glycero- 3-phosphoethanolamine (110) was coupled with N-Boc-L-alanine (111) by means of N-hydroxysuccinimide (112), DCC in DMF to give amide 113, which was followed by hydrogenolysis of the CBZ group to give the corresponding L-alanyl-phosphoric acid 114.

Next, commercially available N-acetylmuramoyl-L-alanyl-Disoglutamine (115) was subjected to hydroxybenzotriazole (HOBT) and DIC in DMF to provide the corresponding succinimide ester 116 which was condensed with compound 114 to provide mifamurtide (IX).

No yields were provided for these transformations.


79. Prous, J. R.; Castaner, J. Drugs Future 1989, 14, 220.
80. Baschang, G.; Tarcsay, L.; Hartmann, A.; Stanek, J. EP 0027258 A1, 1980.
81. Brundish, D. E.; Wade, R. J. Labelled Compd. Radiopharm. 1985, 22, 29.


mifamurtide, the English called mifamurtide, formula C59Hltl9N6O19P, primarily for the treatment of non-metastatic

Resectable osteosarcoma (a rare but the main cause of death for children and young people osteoma), having the formula as follows:

Figure CN103408635AD00051

mifamurtide by certain stimuli such as macrophages and other white blood cells to kill tumor cells. Currently, mifamurtide listed injections into spherical liposome vesicles are muramyl tripeptide (MTP). This lipid trigger macrophages to consume mifamurtide. Once consumed mifamurtide, MTP-stimulated macrophages, in particular we will look for tumors in the liver, spleen and lung macrophages and kill it.

 mifamurtide injection approved for marketing based on the results of phase III clinical study. Taiwan’s National Cancer Institute Cooperative Group (NCI) established by the Children’s Oncology Group (COG) study, complete treatment of this product in patients with osteosarcoma largest research project in the book of about 800 cases. Evaluation of mifamurtide and 3-4 adjuvant chemotherapy (cis molybdenum, doxorubicin, methotrexate, cyclophosphamide with or the same as) the results of combination therapy. Studies have shown that mifamurtide used in combination with chemotherapy can reduce the mortality rate of about 30%, 78% of treated patients survived more than six years.

Shortcomings disclosed the full liquid phase synthesis technology route mifamurtide, but all-liquid phase synthesis: [0006] Currently, mifamurtide universal rely wholly liquid phase synthesis, relevant literature (220 Drugs Futl989, 14, (3)) that the synthesis requires intermediate purification steps cumbersome, time-consuming, and the total yield of the whole liquid phase synthesis is less than 30%, which has been the main factors affecting the productivity of mifamurtide

A method for logging meter synthetic peptide, characterized in that it comprises the following steps: Step 1, under the effect of coupling agent, an amino group, and Fmoc-D-Glu on the amino resin (OPG) -OH main chain carboxyl acylation, a compound of formula I; Step 2, Fmoc removal of the protecting group the compound of formula I, under the effect of coupling with Fmoc-L-Ala-OH acylation, a compound of formula 2; step 3, Fmoc removal of the protecting group the compound of formula 2, in the role of a coupling agent, with a compound of formula 3 for acylation, a compound of formula 4; step 4, PG protecting group removing compound of formula 4, the coupling the role of agent, and HL-Ala-OPG acylation, a compound of formula 5; Step 5, PG protecting group removal compound of formula 5, under the effect of coupling agent, and an amino acid performed on brain phospholipids reaction of a compound of formula 6, and then the resin was added Lysates deaminated compound of formula 7; Step 6, the compound of formula 7 to obtain the removal of benzyl mifamurtide;

Figure CN103408635AC00021
Figure CN103408635AC00031

Wherein Fmoc is the amino protecting group; wherein PG is a carboxy-protecting group for Allyl or Dmab; Resin as the amino resin.

Example: Synthesis of mifamurtide crude peptide

 Example 11 to give the formula hydrogenolysis at atmospheric pressure to 16 hours Example 7 was added to 7.42 g compound 250ml single neck flask, dried 150ml of methanol was added to dissolve 0.4 g of 10% palladium on carbon.Completion of the reaction, palladium-carbon was filtered off, the filtrate was concentrated by rotary evaporation to 65ml, is mifamurtide crude peptide solution. Mifamurtide synthetic crude peptide: 15 [0173] Example

 Example 12 to give the formula hydrogenolysis at atmospheric pressure to 16 hours Example 7 was added to 4.21 g compound 150ml single neck flask, dried 85ml of methanol was added to dissolve 0.2 g of 10% palladium on carbon.Completion of the reaction, palladium-carbon was filtered off, the filtrate was concentrated by rotary evaporation to 37ml, is mifamurtide crude peptide solution.

16 [0175] Example 2: Preparation of mifamurtide

 The embodiment 14 of crude peptide solution obtained in Example 65ml, IOOOml round bottom flask was added, under magnetic stirring, 650ml of anhydrous diethyl ether was added dropwise. Upon completion, at room temperature for crystallization. After filtration and drying the filter cake, the filter cake was again dissolved in 65ml of methanol. This methanol solution was added IOOOml round bottom flask, under magnetic stirring, 650ml of anhydrous diethyl ether was added dropwise. Upon completion, at room temperature for crystallization. Filtered cake was dried in vacuo to give mifamurtide 5.62g, yield 86.5%, purity 99.4%, total yield 74.5%

Preparation of mifamurtide of: 17 Example

 The embodiment of the crude peptide solution obtained in Example 15, 37ml, 500ml round bottom flask was added, under magnetic stirring, 370ml of anhydrous diethyl ether was added dropwise. Upon completion, at room temperature for crystallization. After filtration and drying the filter cake, the filter cake was again dissolved in 37ml of methanol. This solution was added to methanol 500ml round bottom flask, under magnetic stirring, 370ml of anhydrous diethyl ether was added dropwise. Upon completion, at room temperature for crystallization. Filtered, the filter cake was dried under vacuum to give · mifamurtide 3.16g, yield 85.8%, purity 99.5%, 72.2% overall yield.


  1.  “Mifamurtide: CGP 19835, CGP 19835A, L-MTP-PE, liposomal MTP-PE, MLV 19835A, MTP-PE, muramyltripeptide phosphatidylethanolamine”. Drugs in R&D 9 (2): 131–5. 2008. doi:10.2165/00126839-200809020-00007. PMID 18298131.
  2.  “First Treatment to Improve Survival in 20 Years Now Available for Patients With Osteosarcoma (Bone Cancer)”. Takeda. November 2009. Retrieved 23 March 2010.
  3.  “IDM Pharma’s MEPACT (Mifamurtide, L-MTP-PE) Receives Approval in Europe for Treatment of Patients with Non-Metastatic, Resectable Osteosarcoma”. PR Newswire. 2009-03-09. Retrieved 2009-11-12.
  4.  “IDM Pharma receives not approvable letter for Mifamurtide for treatment of osteosarcoma”. The Medical News. 2007-08-28. Retrieved 2009-11-12.
  5.  Mepact for Healthcare Professionals, retrieved 2009-11-12
  6. ^ Jump up to:a b EMA (2009-03-06). “Mepact: Product Information. Annex I: Summary of Product Characteristics” (PDF). p. 2. Retrieved 2009-11-12.
  7.  EMA (2009-05-06). “Mepact: European Public Assessment Report. Summary for the public” (PDF). p. 1. Retrieved 2009-11-12.
  8.  Meyers, P. A. (2009). “Muramyl tripeptide (mifamurtide) for the treatment of osteosarcoma”. Expert Review of Anticancer Therapy 9 (8): 1035–1049.doi:10.1586/era.09.69. PMID 19671023.
  9.  Meyers, P. A.; Schwartz, C. L.; Krailo, M. D.; Healey, J. H.; Bernstein, M. L.; Betcher, D.; Ferguson, W. S.; Gebhardt, M. C.; Goorin, A. M.; Harris, M.; Kleinerman, E.; Link, M. P.; Nadel, H.; Nieder, M.; Siegal, G. P.; Weiner, M. A.; Wells, R. J.; Womer, R. B.; Grier, H. E.; Children’s Oncology, G. (2008). “Osteosarcoma: the Addition of Muramyl Tripeptide to Chemotherapy Improves Overall Survival–A Report from the Children’s Oncology Group”.Journal of Clinical Oncology 26 (4): 633–638. doi:10.1200/JCO.2008.14.0095.PMID 18235123.
  10.  Meyers, P. A.; Schwartz, C. L.; Krailo, M.; Kleinerman, E. S.; Betcher, D.; Bernstein, M. L.; Conrad, E.; Ferguson, W.; Gebhardt, M.; Goorin, A. M.; Harris, M. B.; Healey, J.; Huvos, A.; Link, M.; Montebello, J.; Nadel, H.; Nieder, M.; Sato, J.; Siegal, G.; Weiner, M.; Wells, R.; Wold, L.; Womer, R.; Grier, H. (2005). “Osteosarcoma: A Randomized, Prospective Trial of the Addition of Ifosfamide and/or Muramyl Tripeptide to Cisplatin, Doxorubicin, and High-Dose Methotrexate”. Journal of Clinical Oncology 23 (9): 2004–2011. doi:10.1200/JCO.2005.06.031. PMID 15774791.
  11. (EMA 2009, pp. 5–7)
  12.  (EMA 2009, p. 8)
  13.  (EMA 2009, pp. 7–8)
  14. (EMA 2009, p. 4)
  15.  Fidler, I. J. (1982). “Efficacy of liposomes containing a lipophilic muramyl dipeptide derivative for activating the tumoricidal properties of alveolar macrophages in vivo”. Journal of Immunotherapy 1 (1): 43–55.
  16.  Prous, J. R.; Castaner, J. (1989). “ENV 2-3/MTP-PE”. Drugs Fut. 14 (3): 220.
  17.  Brundish, D. E.; Wade, R. (1985). “Synthesis of N-[2-3H]acetyl-D-muramyl-L-alanyl-D-iso-glutaminyl-L-alanyl-2-(1′,2′-dipalmitoyl-sn-glycero-3′-phosphoryl)ethylamide of high specific radioactivity”. J Label Compd Radiopharm 22 (1): 29–35. doi:10.1002/jlcr.2580220105.
CN1055736A * Jan 28, 1986 Oct 30, 1991 E·R·斯奎布父子公司 Process for preparing 4,4-dialkyl-2-azetidinones
CN101709079A * Dec 22, 2009 May 19, 2010 江苏诺泰制药技术有限公司 Synthesis method of romurtide
US4323560 * Oct 6, 1980 Apr 6, 1982 Ciba-Geigy Corporation Novel phosphorylmuramyl peptides and processes for the manufacture thereof
1 * PROUS, J. ET AL: “ENV 2-3/MTP-PE“, 《DRUGS FUT》, vol. 14, no. 3, 31 March 1989 (1989-03-31), pages 220
2 * 黄胜炎: “抗肿瘤药新品与研发进展“, 《上海医药》, vol. 30, no. 9, 30 September 2009 (2009-09-30), pages 412 – 414
Systematic (IUPAC) name
2-[(N-{(2R)-[(2-acetamido-2,3-dideoxy-D-glucopyranos-3-yl)oxy]-propanoyl}-L-alanyl-D-isoglutaminyl-L-alanyl)amino]ethyl (2R)-2,3-bis(hexadecanoyloxy)propyl hydrogen phosphate
Clinical data
License data
  • not investigated
Routes of
intravenous liposomal infusion over one hour
Legal status
Legal status
  • ℞ (Prescription only)
Pharmacokinetic data
Bioavailability N/A
Biological half-life minutes (in plasma)
18 hrs (terminal)
CAS Number 83461-56-7 Yes
838853-48-8 (mifamurtide sodium · xH2O)
ATC code L03AX15 (WHO)
PubChem CID 11672602
ChemSpider 9847332
KEGG D06619 Yes
Chemical data
Formula C59H109N6O19P
Molar mass 1237.499 g/mol

//////////83461-56-7,  838853-48-8,  CGP-19835,  Mepact,  MFCD09954133,  Mifamurtide,  mifamurtide sodium,  MTP-cephalin,  Mtp-PE,  Muramyl tripeptide, phosphatidylethanolamine,  PEPTIDE,  мифамуртид,  ميفامورتيد,  米法莫肽


Pidotimod, 匹多莫德 , пидотимод , بيدوتيمود ,




(4R)-3-[(2S)-5-oxopyrrolidine-2-carbonyl]-1,3-thiazolidine-4-carboxylic acid

CAS 121808-62-6

Thymodolic acid, Pidotimod, Timodolic acid, PGT/1A, Axil, Onaka, Pigitil, Polimod

(4R)-3-(5-oxo-L-prolyl)-l ,3-thiazolidine-4-carboxylic acid,  ITI 231723.

3-(L-pyroglutamyl)-L-thiazolidine-4-carboxylic acid

  • 4-Thiazolidinecarboxylic acid, 3-[(5-oxo-2-pyrrolidinyl)carbonyl]-, [R-(R*,S*)]-
  • (4R)-3-[[(2S)-5-Oxo-2-pyrrolidinyl]carbonyl]-4-thiazolidinecarboxylic acid
  • Adimod
  • Axil (pharmaceutical)
  • Pigitil
Thymodolic acid
Timodolic acid
Pidotimod; 121808-62-6; (R)-3-((S)-5-Oxopyrrolidine-2-carbonyl)thiazolidine-4-carboxylic acid; Pidotomod; PGT/1A; Pidotimod [INN];
Molecular Formula: C9H12N2O4S
Molecular Weight: 244.26758 g/mol

Stefano Poli, Corona Lucio Del


Pidotimod is an immunostimulant.[1]


Pidotimod, whose chemical name is (4R)-3-(5-oxo-L-prolyl)-l ,3-thiazolidine-4-carboxylic acid, was first disclosed in ITI 231723. It is a synthetic peptide-like molecule provided with an in vitro and in vivo immunomodulating action (Giagulli et al., International Immunopharmacology, 9, 2009, 1366-1373). The immune system assists in maintaining a homeostatic balance between the human body and all foreign substances. An abnormality in this balance may cause a defective or aberrant response towards non-self substances, as well as loss of tolerance toward self-antigens, in such cases, the immune system imbalance exhibits clinically as signs of disease.

Pidotimod has been shown to induce dendritic cell maturation and up-regulate the expression of HLA-DR and co-stimulatory molecules CD83 and CD86, which are integral to communication with adaptive immunity cells. Pidotimod has also been shown to stimulate dendritic cells to release pro-inflammatory molecules such as MCP-1 and TNF-a cytokines, and to inhibit thymocyte apoptosis caused by a variety of apoptosis-inducing molecules. Pidotimod exerts a protective action against infectious processes, although not through direct antimicrobial or antiviral action. Rather, pidotimod stimulates both innate and acquired immunity by enhancing humoral and cell-mediated immunity mechanisms.

Pidotimod, which may be administered as solid or liquid forms, for example, via an oral route, has been shown to increase natural resistance to viral or bacterial infections in animal models. Efficacy demonstrated in patients includes respiratory, urinary and genital infections, in particular recurrent respiratory infections in pediatric patients, respiratory infections in asthmatic patients and chronic obstructive pulmonary disease in adults and elderly patients.

Besides exhibiting activity to illnesses characterized by immune defects, pidotimod has been reported to be of benefit in to patients with other kinds of diseases, not directly related to immune defects, including gastroenterology diseases such as ulcerative colitis and irritable bowel syndrome, and dermatological diseases such as psoriasis and atopic dermatitis where symptoms relating to these diseases have been attenuated. In gastroenterology diseases pidotimod may be administered either by oral or by rectal route. Oral route or topical application, for example in creams or gels containing pidotimod, may be used to treat dermal conditions.

Further use of pidotimod includes treatment of inflammatory diseases, in particular those characterized by an aberrant activation of the non-canonical NF-kB pathway. Diseases implicated by such activation include allergic diseases, autoimmune diseases, and numerous other inflammatory diseases. Allergic diseases include allergic rhinitis, allergic conjunctivitis, contact dermatitis, eczema and allergic vasculitis.

Autoimmune diseases include alopecia areata, ankylosing spondylitis, autoimmune cardiomyopathy, autoimmune connective tissue diseases, autoimmune enteropathy, autoimmune hepatitis, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, celiac disease, chronic fatigue syndrome, cystic fibrosis, hashimoto’s thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IGA nephropathy, juvenile idiopathic arthritis for juvenile rheumatoid arthritis, or Still’s disease) Kawasaki’s disease, lichen planus, lupus erythematosus, rheumatoid arthritis, rheumatic fever, Sj5gren’s syndrome, spondyloarthropathy, temporal arteritis (or giant cell arteritis), urticarial vasculitis, and vitiligo.

Other inflammatory diseases include Alzheimer’s disease, atherosclerosis, chronic liver diseases, chronic nephropathy, gastritis, glomerulonephritis, hydradenitis suppurativa, hypogammaglobulinemia, interstitial cystitis, lichen sclerosus, liver steatosis, metabolic syndrome, obesity, Parkinson’s disease, pemphigus vulgaris, post-ischemic inflammation, raynaud phenomenon, restless leg syndrome, retroperitoneal fibrosis, and thrombocytopenia.




CN 104926922

Synthesis pidotimod

A method for producing pidotimod, characterized in that: comprising the steps of: a) L- thiazolidine-4-carboxylic acid: L- cysteine formaldehyde solution was added dropwise, stirred at room temperature, filtered to give L- thiazolidine-4-carboxylic acid; (2) metal ion load type cation exchange resin preparation: strongly acidic with hydrochloric acid cation exchange resin is converted to the hydrogen form, the hydrogen form strong acid cation exchange resin was added a solution of a metal ion compound In, 40 ~ 80 ° C for 1 to 6 hours, cooled to room temperature, and dried to obtain a supported metal ion cation exchange resin; (3) Synthesis of pidotimod: the step (1) of L- thiazolidine – 4- carboxylic acid, in step (2) of the load as a catalyst metal ion type cation exchange resin, L- pyroglutamic acid and N, N- dimethylformamide mixed, 40 ~ 80 ° C for 1 to 4 hours, filtered to give a white solid, the white solid was acidified with hydrochloric acid, to give the finished pidotimod.


Figure CN104926922AD00042

In four flask IOg L- thiazolidine-4-carboxylic acid, 11. 3g g L- pyroglutamic acid, 320mL N, N- dimethylformamide, 12g modified resin, 70 ° C the reaction 2 hours. Filtration, the reaction mixture by rotary evaporation, after removal of part of the solvent, placed in an ice bath to cool, the precipitated solid was suction filtered to give a white solid, this white solid was acidified with 37% hydrochloric acid, was allowed to stand at KTC, crystallization, filtration, a white product 14. 4g, a yield of 78.3%. Measured melting point 192 ~ 194 ° C, [a] 25D = – 150 ° (literature values mp: 192 ~ 194 ° C, [a] 25D = – 150 °).The whole preparation reaction pidotimod total yield of 64%. By HPLC, pidotimod content of 98.5%.


Zhang, Yi; Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 2009, 877(24), PG 2566-2570




Example 14 – Preparation of Pidotimod

Pidotimod was prepared following Example 1 of EP0422566 Al .







A solution of 16.78 g (0.084 mole) of ethyl L-thiazolidine-4-carboxylate hydrochloride in 33 ml of water is treated with 16.78 g of potassium carbonate and extracted with 40 ml of ethyl acetate. The organic phase is dried over sodium sulfate, filtered and diluted to 85 ml with ethyl acetate. The solution is stirred and cooled to 0-5°C, then 19.2 g (0.093 mole) of dicyclohexylcarbodiimide dissolved in 20 ml of ethyl acetate and 12 g (0.093 mole) of L-pyroglutamic acid are added thereto. The reaction mixture is stirred for 1 hour at 0-5°C, then 12 hours at room temperature, dicyclohexylurea is filtered, the filtrate is evaporated under vacuum and the oily residue, consisting in ethyl 3-(L-pyroglutamyl)-L-thiazolidine-4-carboxylate is taken up into 25 ml of water. 3.73 g of sodium hydroxide dissolved in 13.3 ml of water are dropped into the resulting solution. After 30 minutes, the reaction mixture is acidified with concentrated hydrochloric acid at 0-5°C, kept for 2 hours at 5°C, then filtered washing with little cool water and dried to obtain 17.8 g (87.6%) of 3-(L-pyroglutamyl)-L-thiazolidine-4-carboxylic acid, m.p. 193-194°C.


23 g (0.1 mol) of L-N-t-butoxycarbonylpyroglutamic acid (E. Schröder and E. Klinger, Ann. Chem., 673, 1964, 202) and 16.1 g (0.1 mol) of ethyl L-thiazolidine-4-carboxylate are dissolved in 150 ml of THF, to the solution stirred at 0-5°C, 21 g (0.105 mol) of dicyclohexylcarbodiimide are added and the slurry is stirred for 15 hours at room temperature. The dicyclohexylurea is filtered, the wear filtrate is evaporated u.v. and the oily residue is kept in 40 ml of water. In the solution 6.6 g of potassium hydroxyde in a little water are dropped in 30′ at 15-20°C, the pH is adjusted to 2 with hydrochloric acid at 0-5°C and after 2 hours the precipitated L-pyroglutamyl-L-thiazolidine-4-carboxylic acid is filtered and dried, giving 88%, mp. 193-4°.


Drugs Fut 1991,16(12),1096

Liebigs Ann Chem 1964,673

The synthesis of pidotimod has been carried out using N-tert-butoxycarbonyl-L-pyroglutamic acid as starting material, in order to avoid the formation of diketopiperazine derivatives. L-Glutamic acid (I) was condensed with di-tert-butyl dicarbonate by means of triethylamine in DMF to give N-(tert-butoxycarbonyl)-L-glutamic acid (II), which is dissolved in THF and treated with dicyclohexylcarbodiimide (DCC) to obtain N-(tert-butoxycarbonyl)-L-glutamic anhydride (III). The treatment of anhydride (III) with dicyclohexylamine in THF-ethyl ether affords the dicyclohexylamine salt of N-(tert-butoxycarbonyl)-L-pyroglutamic acid (IV), which by acidification with aqueous citric acid yields the corresponding free acid (V). The condensation of equimolecular amounts of N-(tert-butoxycarbonyl)-L-pyroglutamic acid (V) with L-thiazolidine-4-carboxylic acid ethyl ester (VIII) by means of DCC in methylene chloride gives the coupled ester (IX), which is hydrolyzed with aqueous NaOH, and the corresponding sodium salt acidified to yield the N-tert-butoxycarbonyl derivative (X). Finally, this compound is deprotected with trifluoroacetic acid to obtain crystalline pidotimod (XI). The intermediate thiazolidine (VIII) has been obtained as follows: Esterification of L-thiazolidine-4-carboxylic acid (VI) with ethanol by means of SOCl2 gives the corresponding ethyl ester hydrochloride (VII), which by treatment with K2CO3 in water yields the free ester (VIII).



Arzneim-Forsch Drug Res 1994,44(12a),1402

Two new related routes for the synthesis of pidotimod have been reported: 1) The condensation of L-pyroglutamic acid (I) with L-thiazolidine-4-carboxylic acid ethyl ester (II) by means of dicyclohexylcarbodiimide (DCC) in methylene chloride gives the corresponding dipeptide ethyl ester (III), which is saponified with aqueous 1N NaOH. 2) By condensation of the activated ester L-pyroglutamic acid pentachlorophenyl ester (IV) with L-thiazolidine-4-carboxylic acid (V) by means of triethylamine in DMF.



Novel crystalline, amorphous and solid forms of di-pidotimod benzathine (designated as Forms M and H), their hydrates, processes for their preparation and compositions comprising them are claimed. Also claimed is their use for treating viral or bacterial infections, respiratory, urinary and/or genital infections, ulcerative colitis, irritable bowel syndrome, psoriasis and atopic dermatitis

Example 14 – Preparation of Pidotimod

Pidotimod was prepared following Example 1 of EP0422566 Al .


Figure 17 is a Ή solution-state NMR spectrum of Form H


CN 104447947

Indian Pat. Appl. (2014), IN 2013MU00181 A

WO 2014111957

CN 103897025


CN1557303A * Jan 16, 2004 Dec 29, 2004 太阳石(唐山)药业有限公司 Use of Pidotimod in preparation of hepatitis B treating medicine
EP0382180A2 * Feb 7, 1990 Aug 16, 1990 POLI INDUSTRIA CHIMICA S.p.A. Derivatives of thiazolidine-4-carboxylic acid, its preparation and pharmaceutical compositions containing it
IT1231723B Title not available
1 * DATABASE CA [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; DUAN, RUOZHU ET AL: “Application and prospects of immunostimulants“, XP002722997, retrieved from STN Database accession no. 2006:478774
2 * DATABASE CA [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; LI, YIPING ET AL: “Effects of pidotimod on immune function of patients with chronic hepatitis C“, XP002722996, retrieved from STN Database accession no. 2007:598452
3 * DATABASE CA [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; WU, RONGRONG ET AL: “Application of immunomodulatory drugs in treatment of chronic hepatitis B“, XP002722995, retrieved from STN Database accession no. 2010:125278
4 * DATABASE MEDLINE [Online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; March 2002 (2002-03), VARGAS CORREA JORGE B ET AL: “[Pidotimod in recurring respiratory infection in children with allergic rhinitis, asthma, or both conditions].“, XP002722994, Database accession no. NLM12092522 & VARGAS CORREA JORGE B ET AL: REVISTA ALERGIA MEXICO (TECAMACHALCO, PUEBLA, MEXICO : 1993) 2002 MAR-APR, vol. 49, no. 2, March 2002 (2002-03), pages 27-32, XP8168769, ISSN: 0002-5151
5 * GOURGIOTIS DIMITRIOS ET AL: “Immune modulator pidotimod decreases the in vitro expression of CD30 in peripheral blood mononuclear cells of atopic asthmatic and normal children“, JOURNAL OF ASTHMA, ASTHMA PUBLICATIONS SOCIETY, OSSINING, NY, US, vol. 41, no. 3, 1 January 2004 (2004-01-01), pages 285-287, XP008164025, ISSN: 0277-0903, DOI: 10.1081/JAS-120026085
6 * XIN JIN ET AL: “Sublingual Surprise: A New Variant of Oral Lichen Planus“, THE AMERICAN JOURNAL OF MEDICINE, vol. 127, no. 1, 1 January 2014 (2014-01-01), pages 28-30, XP055112640, ISSN: 0002-9343, DOI: 10.1016/j.amjmed.2013.10.002


  1.  Du XF, Jiang CZ, Wu CF, Won EK, Choung SY (September 2008). “Synergistic immunostimulating activity of pidotimod and red ginseng acidic polysaccharide against cyclophosphamide-induced immunosuppression”. Archives of pharmacal research 31 (9): 1153–9.doi:10.1007/s12272-001-1282-6. PMID 18806958.
Systematic (IUPAC) name
(4R)-3-(5-oxo-L-prolyl)-1,3-thiazolidine-4-carboxylic acid
Clinical data
AHFS/ International Drug Names
ATC code L03AX05 (WHO)
PubChem CID 65944
ChemSpider 59348 Yes
UNII 785363R681 Yes
KEGG D07261 Yes
Synonyms (4R)-3-[(2S)-5-oxopyrrolidine-2-carbonyl]-1,3-thiazolidine-4-carboxylic acid
Chemical data
Formula C9H12N2O4S
Molar mass 244.26758 g/mol

//////////////Pidotimod, Thymodolic acid, Pidotimod, Timodolic acid, PGT/1A, Axil, Onaka, Pigitil, Polimod, H-Pyr-Thz-OH,  121808-62-6, ITI 231723, peptide, QA-7522, SMR000466390, Thymodolic acid, Timodolic acid, UNII:785363R681, 匹多莫德 , пидотимод ,  بيدوتيمود ,




Mastoparan, Peptide (H-INLKALAALAKKIL-NH2)

IUPAC Condensed



PEPTIDE1{I.N.L.K.A.L.A.A.L.A.K.K.[*N[C@H](C(=O)*)C(C)CC |$_R1;;;;;_R2;;;;$|].L.[am]}$$$$

Ile – Asn – Leu – Lys – Ala – Leu – Ala – Ala – Leu – Ala – Lys – Lys – Ile – Leu -NH2
Mastoparan; Mast cell degranulating peptide (Vespula lewisii); NSC351907; CAS 72093-21-1;
Molecular Formula: C70H131N19O15
Molecular Weight: 1478.90744 g/mol
  • 18: PN: WO0181408 SEQID: 37 claimed protein
  • 18: PN: WO2010069074 SEQID: 16 claimed protein
  • L-Leucinamide, L-isoleucyl-L-asparaginyl-L-leucyl-L-lysyl-L-alanyl-L-leucyl-L-alanyl-L-alanyl-L-leucyl-L-alanyl-L-lysyl-L-lysyl-L-isoleucyl-
  • Mastoparan 1
  • NSC 351907


Mastoparan (Vespula lewisii) has been shown to cause an increase in the production of Arachidonic Acid (sc-200770) catalyzed by PLA2 from porcine pancreas and bee venom. This compound also displays toxicity by regulating G proteins via mimicking of G-protein-coupled receptors. Additionally, Mastoparan has been reported as a stimulator of insulin release by pancreatic islets, which acts through GTP-binding proteins and PLA2. In other experiments, this agent has demonstrated the ability to cause exocytosis of rat peritoneal mast cells and also stimulate the accumulation of inositol phosphates in hepatocytes. Additionally, Mastoparan has been noted to act as a mitogen in Swiss 3T3 cells and stimulate pertussis toxin-sensitive Arachidonate release without phosphoinositide breakdown. Mastoparan (Vespula lewisii) is an inhibitor of CaM. Mastoparan (Vespula lewisii) is an activator of Heterotrimeric G Protein and PLA2.
Technical Information
Physical State: Solid
Derived from: Synthetic. Originally isolated from wasp venom (Vespula lewisii)
Solubility: Soluble in water (2.6 mg/ml), and 100% ethanol.
Storage: Store at -20° C
Refractive Index: n20D 1.53
IC50: Na+,K+-ATPase: IC50 = 7.5 µM

Mastoparan is a peptide toxin from wasp venom. It has the chemical structure Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-Leu-NH2.[2]

The net effect of mastoparan’s mode of action depends on cell type, but seemingly always involves exocytosis. In mast cells, this takes the form of histamine secretion, while in platelets and chromaffin cells release serotonin and catecholamines are found, respectively. Mastoparan activity in the anterior pituitary gland leads to prolactin release.

In the case of histamine secretion, the effect of mastoparan takes place via its interference with G protein activity. By stimulating theGTPase activity of certain subunits, mastoparan shortens the lifespan of active G protein. At the same time, it promotes dissociation of any bound GDP from the protein, enhancing GTP binding. In effect, the GTP turnover of G proteins is greatly increased by mastoparan. These properties of the toxin follow from the fact that it structurally resembles activated G protein receptors when placed in a phospholipid environment. The resultant G protein-mediated signaling cascade leads to intracellular IP3 release and the resultant influx of Ca2+.

In an experimental study conducted by Tsutomu Higashijima and his counterparts, mastoparan was compared to melittin, which is found in bee venom.[2] Mainly, the structure and reaction to phosphate was studied in each toxin. Using Circular Dichroism (CD), it was found that when mastoparan was exposed to methanol, an alpha helical form existed. It was concluded that strong intramolecular hydrogen bonding occurred. Also, two negative bands were present on the CD spectrum. In an aqueous environment, mastoparan took on a nonhelical, unordered form. In this case, only one negative band was observed on the CD spectrum. Adding phosphate buffer to mastoparan resulted in no effect.

Melittin produced a different conformational change than mastoparan. In an aqueous solution, melittin went from a nonhelical form to an alpha helix when phosphate was added to the solution. The binding of melittin to the membrane was believed to result fromelectrostatic interactions, not hydrophobic interactions.

Infections caused by multidrug resistant bacteria are currently an important problem worldwide. Taking into account data recently published by the WHO, lower respiratory infections are the third cause of death in the world with around 3.2 million deaths per year, this number being higher compared to that related to AIDS or diabetes mellitus [1]. It is therefore important to solve this issue, although the perspectives for the future are not very optimistic. During the last 30 years an enormous increase has been observed of superbugs isolated in the clinical setting, especially from the group called ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) which show high resistance to all the antibacterial agents available [2]. We will focus on Acinetobacter baumannii, the pathogen colloquially called “iraquibacter” for its emergence in the Iraq war. It is a Gram-negative cocobacillus and normally affects people with a compromised immune system, such as patients in the intensive care unit (ICU) [3] and [4]. Together with Escherichia coliand P. aeruginosa, A. baumannii are the most common cause of nosocomial infections among Gram-negative bacilli. The options to treat infections caused by this pathogen are diminishing since pan-drug resistant strains (strains resistant to all the antibacterial agents) have been isolated in several hospitals [5]. The last option to treat these infections is colistin, which has been used in spite of its nephrotoxic effects [6]. The evolution of the resistance of A. baumannii clinical isolates has been established by comparing studies performed over different years, with the percentage of resistance to imipenem being 3% in 1993 increasing up to 70% in 2007. The same effect was observed with quinolones, with an increase from 30 to 97% over the same period of time[7]. In Spain the same evolution has been observed with carbapenems; in 2001 the percentage of resistance was around 45%, rising to more than 80% 10 years later [8]. Taking this scenario into account, there is an urgent need for new options to fight against this pathogen. One possible option is the use of antimicrobial peptides (AMPs) [9],[10] and [11], and especially peptides isolated from a natural source [12]. One of the main drawbacks of using peptides as antimicrobial agents is the low stability or half-life in human serum due to the action of peptidases and proteases present in the human body[13], however there are several ways to increase their stability, such as using fluorinated peptides [14] and [15]. One way to circumvent this effect is to study the susceptible points of the peptide and try to enhance the stability by protecting the most protease labile amide bonds, while at the same time maintaining the activity of the original compound. Another point regarding the use of antimicrobial peptides is the mechanism of action. There are several mechanisms of action for the antimicrobial peptides, although the global positive charge of most of the peptides leads to a mechanism of action involving the membrane of the bacteria [16]. AMPs has the ability to defeat bacteria creating pores into the membrane [17], also acting as detergents [18], or by the carpet mechanism [19]. We have previously reported the activity of different peptides against colistin-susceptible and colistin-resistant A. baumannii clinical isolates, showing that mastoparan, a wasp generated peptide (H-INLKALAALAKKIL-NH2), has good in vitro activity against both colistin-susceptible and colistin-resistant A. baumannii [20]. Therefore, the aim of this manuscript was to study the stability of mastoparan and some of its analogues as well as elucidate the mechanism of action of these peptides.


Volume 101, 28 August 2015, Pages 34–40

Research paper

Sequence-activity relationship, and mechanism of action of mastoparan analogues against extended-drug resistantAcinetobacter baumannii

  • a ISGlobal, Barcelona Ctr. Int. Health Res. (CRESIB), Hospital Clínic – Universitat de Barcelona, Barcelona, Spain
  • b Biomedical Institute of Seville (IBiS), University Hospital Virgen del Rocío/CSIC/University of Seville, Seville, Spain
  • c Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain
  • d Department of Clinical Microbiology, CDB, Hospital Clinic, School of Medicine, University of Barcelona, Barcelona, Spain
  • e Department of Organic Chemistry, University of Barcelona, Barcelona, Spain



•The most susceptible position of mastoparan is the peptide bond between isoleucine and asparagine.
•The positive charge present in the N-terminal play an important role in the antimicrobial activity of the peptides.
•Mastoparan and its enantiomer version exhibit a mechanism of action related to the membrane disruption of bacteria.
•Three of the mastoparan analogues synthesized have good activity against highly resistant Acinetobacter baumannii.
•Two of the active analogues showed a significant increase in the human serum stability compared to mastoparan.


The treatment of some infectious diseases can currently be very challenging since the spread of multi-, extended- or pan-resistant bacteria has considerably increased over time. On the other hand, the number of new antibiotics approved by the FDA has decreased drastically over the last 30 years. The main objective of this study was to investigate the activity of wasp peptides, specifically mastoparan and some of its derivatives against extended-resistant Acinetobacter baumannii. We optimized the stability of mastoparan in human serum since the specie obtained after the action of the enzymes present in human serum is not active. Thus, 10 derivatives of mastoparan were synthetized. Mastoparan analogues (guanidilated at the N-terminal, enantiomeric version and mastoparan with an extra positive charge at the C-terminal) showed the same activity against Acinetobacter baumannii as the original peptide (2.7 μM) and maintained their stability to more than 24 h in the presence of human serum compared to the original compound. The mechanism of action of all the peptides was carried out using a leakage assay. It was shown that mastoparan and the abovementioned analogues were those that released more carboxyfluorescein. In addition, the effect of mastoparan and its enantiomer against A. baumannii was studied using transmission electron microscopy (TEM). These results suggested that several analogues of mastoparan could be good candidates in the battle against highly resistant A. baumannii infections since they showed good activity and high stability.

Graphical abstract

Image for unlabelled figure


  1.  PDB: 2CZP; Todokoro Y, Yumen I, Fukushima K, Kang SW, Park JS, Kohno T, Wakamatsu K, Akutsu H, Fujiwara T (August 2006). “Structure of Tightly Membrane-Bound Mastoparan-X, a G-Protein-Activating Peptide, Determined by Solid-State NMR”. Biophys. J. 91 (4): 1368–79. doi:10.1529/biophysj.106.082735. PMC 1518647. PMID 16714348.
  2.  Higashijima T, Uzu S, Nakajima T, Ross EM (May 1988). “Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins)”. J. Biol. Chem. 263 (14): 6491–4. PMID 3129426.


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Solution structure of mastoparan from Vespa simillima xanthoptera.[1]
Symbol Mastoparan_2
Pfam PF08251
InterPro IPR013214
TCDB 1.C.32
OPM superfamily 160
OPM protein 2czp

///////Peptide, Antimicrobial peptide, Mastoparan, Acinetobacter baumannii,  NSC351907,  72093-21-1, NSC 351907




Difelikefalin, CR-845; MR-13A-9; MR-13A9

4-amino-1- (D-phenylalanyl-D-phenylalanyl-D-leucyl-D-lysyl) piperidine-4-carboxylic acid

Phase III

C36H53N7O6, 679.40573

Originator Ferring Pharmaceuticals
Developer Cara Therapeutics
Class Analgesic drugs (peptides)
Mechanism Of Action Opioid kappa receptor agonists
Who Atc Codes D04A-X (Other antipruritics), N02A (Opioids)
Ephmra Codes D4A (Anti-Pruritics, Including Topical Antihistamines, Anaesthetics, etc), N2A (Narcotics)
Indication Pain, Osteoarthritis, Pruritus

A kappa opioid receptor agonist potentially for treatment of post-operative pain and uremic pruritus.

Difelikefalin, also known CR845, is a novel and potent kappa opioid receptor agonist. CR845 exhibit low P450 CYP inhibition and low penetration into the brain. CR845 may be useful in the prophylaxis and treatment of pain and inflammation associated with a variety of diseases and conditions .

No. CAS 1024828-77-0

2D chemical structure of 1024828-77-0

Difelikefalin ( INN ) (Developmental Code Names CR845 , FE-202845 ), Also Known As D -Phe- D -Phe- D -Leu- D -Lys- [Ganma- (4-N-Piperidinyl) Amino Carboxylic Acid] (As The Acetate Salt ), Is An Analgesic Opioid Peptide [2] Acting As A Peripherally-Specific , Highly Selective Agonist Of The kappa-Opioid Receptor (KOR). [1] [3] [4] [5] It Is Under Development By Cara Therapeutics As An Intravenous Agent For The Treatment Of Postoperative Pain . [1] [3] [5] An Oral Formulation Has Also Been Developed. [5] Due To Its Peripheral Selectivity, Difelikefalin Lacks The Central Side Effects Like Sedation , Dysphoria , And Hallucinations Of Previous KOR-Acting Analgesics Such As Pentazocine And Phenazocine . [1] [3] In Addition To Use As An Analgesic, Difelikefalin Is Also Being Investigated For The Treatment Of Pruritus (Itching). [1] [3] [4 ] Difelikefalin Has Completed Phase II Clinical Trials For Postoperative Pain And Has Demonstrated Significant And “Robust” Clinical Efficacy, Along With Being Safe And Well-Tolerated. [3] [5] It Is Also In Phase II Clinical Trials For Uremic Pruritus In Hemodialysis Patients. [4]

Difelikefalin Acts As An Analgesic By Activating KORs On Peripheral Nerve Terminals And KORs Expressed By Certain Immune System Cells . [1] Activation Of KORs On Peripheral Nerve Terminals Results In The Inhibition Of Ion Channels Responsible For Afferent Nerve Activity , Causing Reduced Transmission Of Pain Signals , While Activation Of KORs Expressed By Immune System Cells Results In Reduced Release Of Proinflammatory , Nerve-Sensitizing Mediators (Eg, Prostaglandins ). [1]


WO 2015198505

κ opioid receptor agonists are known to be useful as therapeutic agents for various pain. Among, kappa opioid receptor agonist with high selectivity for peripheral kappa opioid receptors, are expected as a medicament which does not cause the central side effects. Such as peripherally selective κ opioid receptor agonist, a synthetic pentapeptide has been reported (Patent Documents 1 and 2).
 The following formula among the synthetic pentapeptide (A)
[Formula 1] Being Represented By Compounds Are Useful As Pain Therapeutics. The Preparation Of This Compound, Solid Phase Peptide Synthesis Methods In Patent Documents 1 And 2 Have Been Described.

Document 1 Patent: Kohyo 2010-510966 JP
Patent Document 2: Japanese Unexamined Patent Publication No. 2013-241447
 Compound (1) or a salt thereof and compound (A), for example as shown in the following reaction formula, 4-aminopiperidine-4-carboxylic acid, D- lysine (D-Lys), D- leucine (D-Leu) , it can be prepared by D- phenylalanine (D-Phe) and D- phenylalanine (D-Phe) sequentially solution phase peptide synthesis methods condensation.
[Of 4]

The present invention will next to examples will be described in further detail.
1 (1) Synthesis of Cbz-D-Lys (Boc) -α-Boc-Pic-OMe (3)
to the four-necked flask of 2L, α-Boc-Pic- OMe · HCl [α-Boc-4 – aminopiperidine-4-carboxylic acid methyl hydrochloride] were charged (2) 43.7g (148mmol), was suspended in EtOAc 656mL (15v / w). To the suspension of 1-hydroxybenzotriazole (HOBt) 27.2g (178mmol), while cooling with Cbz-D-Lys (Boc) -OH 59.2g (156mmol) was added an ice-bath 1-ethyl -3 – (3-dimethylcarbamoyl amino propyl) was added to the carbodiimide · HCl (EDC · HCl) 34.1g (178mmol). After 20 minutes, stirring was heated 12 hours at room temperature. After completion of the reaction, it was added and the organic layer was 1 N HCl 218 mL of (5.0v / w). NaHCO to the resulting organic layer 3 Aq. 218ML (5.0V / W), Et 3 N 33.0 g of (326Mmol) was stirred for 30 minutes, and the mixture was separated. The organic layer HCl 218ML 1N (5.0V / W), NaHCO 3 Aq. 218mL (5.0v / w), NaClaq . Was washed successively with 218ML (5.0V / W), Na 2 SO 4 dried addition of 8.74g (0.2w / w). Subjected to vacuum filtration, was concentrated under reduced pressure resulting filtrate by an evaporator, and pump up in the vacuum pump, the Cbz-D-Lys (Boc) -α-Boc-Pic-OMe (3) 88.9g as a white solid obtained (96.5% yield, HPLC purity 96.5%).

(2) D-Lys (Boc) Synthesis Of -Arufa-Boc-Pic-OMe (4)
In An Eggplant-Shaped Flask Of 2L, Cbz-D-Lys (Boc) -Arufa-Boc-Pic-OMe (3) 88.3g (142mmol) were charged, it was added and dissolved 441mL (5.0v / w) the EtOAc. The 5% Pd / C to the reaction solution 17.7g (0.2w / w) was added, After three nitrogen substitution reduced pressure Atmosphere, Was Performed Three Times A Hydrogen Substituent. The Reaction Solution Was 18 Hours With Vigorous Stirring At Room Temperature To Remove The Pd / C And After The Completion Of The Reaction Vacuum Filtration. NaHCO The Resulting Filtrate 3 Aq. 441ML And (5.0V / W) Were Added For Liquid Separation, And The Organic Layer Was Extracted By The Addition Of EtOAc 200ML (2.3V / W) In The Aqueous Layer. NaHCO The Combined Organic Layer 3 Aq. 441ML And (5.0V / W) Were Added for liquid separation, and the organic layer was extracted addition of EtOAc 200mL (2.3v / w) in the aqueous layer. NaClaq the combined organic layers. 441mL and (5.0v / w) is added to liquid separation, was extracted by the addition EtOAc 200ML Of (2.3V / W) In The Aqueous Layer. The Combined Organic Layer On The Na 2 SO 4 Dried Addition Of 17.7 g of (0.2W / W), Then The Filtrate Was Concentrated Under Reduced Pressure Obtained Subjected To Vacuum Filtration By an evaporator, and pump up in the vacuum pump, D-Lys (Boc) -α-Boc-Pic- OMe (4) to give 62.7g (90.5% yield, HPLC purity 93.6%).
(3) Cbz-D-Leu -D-Lys (Boc) -α-Boc-Pic-OMe synthesis of (5)
in the four-necked flask of 2L, D-Lys (Boc) -α-Boc-Pic-OMe (4) was charged 57.7 g (120 mmol), was suspended in EtOAc 576mL (10v / w). HOBt 19.3g (126mmol) to this suspension, was added EDC · HCl 24.2g (126mmol) while cooling in an ice bath added Cbz-D-Leu-OH 33.4g (126mmol). After 20 minutes, after stirring the temperature was raised 5 hours at room temperature, further the EDC · HCl and stirred 1.15 g (6.00 mmol) was added 16 h. After completion of the reaction, it was added liquid separation 1N HCl 576mL (10v / w) . NaHCO to the resulting organic layer 3 Aq. 576ML (10V / W), Et 3 N 24.3 g of (240Mmol) was stirred for 30 minutes, and the mixture was separated. The organic layer HCl 576ML 1N (10V / W), NaHCO 3 Aq. 576mL (10v / w), NaClaq . Was washed successively with 576ML (10V / W), Na 2 SO 4 dried addition of 11.5g (0.2w / w). After the filtrate was concentrated under reduced pressure obtained subjected to vacuum filtration by an evaporator, and pump up in the vacuum pump, the Cbz-D-Leu-D- Lys (Boc) -α-Boc-Pic-OMe (5) 85.8g It was obtained as a white solid (98.7% yield, HPLC purity 96.9%).
(4) D-Leu-D -Lys (Boc) -α-Boc-Pic-OMe synthesis of (6)
in an eggplant-shaped flask of 1L, Cbz-D-Leu- D-Lys (Boc) -α-Boc-Pic -OMe the (5) 91.9g (125mmol) were charged, was added and dissolved 459mL (5.0v / w) the EtOAc. The 5% Pd / C to the reaction solution 18.4g (0.2w / w) was added, After three nitrogen substitution reduced pressure atmosphere, was performed three times a hydrogen substituent. The reaction solution was subjected to 8 hours with vigorous stirring at room temperature to remove the Pd / C and after the completion of the reaction vacuum filtration. NaHCO the resulting filtrate 3 Aq. 200mL (2.2v / w) were added to separate liquid, NaHCO to the organic layer 3 Aq. 200mL (2.2v / w), NaClaq . It was sequentially added washed 200mL (2.2v / w). To the resulting organic layer Na 2 SO 4 dried added 18.4g (0.2w / w), to the filtrate concentrated under reduced pressure obtained subjected to vacuum filtration by an evaporator, and a pump-up with a vacuum pump. The resulting amorphous solid was dissolved adding EtOAc 200mL (2.2v / w), was crystallized by the addition of heptane 50mL (1.8v / w). Was filtered off precipitated crystals by vacuum filtration, the crystals were washed with a mixed solvent of EtOAc 120mL (1.3v / w), heptane 50mL (0.3v / w). The resulting crystal 46.1g to added to and dissolved EtOAc 480mL (5.2v / w), was crystallized added to the cyclohexane 660mL (7.2v / w). Was filtered off under reduced pressure filtered to precipitate crystals, cyclohexane 120mL (1.3v / w), and washed with a mixed solvent of EtOAc 20mL (0.2v / w), and 30 ° C. vacuum dried, D-Leu- as a white solid D-Lys (Boc) -α- Boc-Pic-OMe (6) to give 36.6 g (48.7% yield, HPLC purity 99.9%).
(5) Synthesis of Cbz-D-Phe-D- Leu-D-Lys (Boc) -α-Boc-Pic-OMe (7)
to the four-necked flask of 1L, D-Leu-D- Lys (Boc) -α-Boc-Pic-OMe with (6) 35.8g (59.6mmol) was charged, it was suspended in EtOAc 358mL (10v / w). To this suspension HOBt 9.59g (62.6mmol), Cbz- D-Phe-OH 18.7g was cooled in an ice bath is added (62.6mmol) while EDC · HCl 12.0g (62.6mmol) It was added. After 20 minutes, a further EDC · HCl After stirring the temperature was raised 16 hours was added 3.09 g (16.1 mmol) to room temperature. After completion of the reaction, it was added and the organic layer was 1N HCl 358mL of (10v / w). NaHCO to the resulting organic layer 3 Aq. 358ML (10V / W), Et 3 N 12.1 g of (119Mmol) was stirred for 30 minutes, and the mixture was separated. The organic layer HCl 358ML 1N (10V / W), NaHCO 3 Aq. 358mL (10v / w), NaClaq . Was washed successively with 358ML (10V / W), Na 2 SO 4 dried addition of 7.16g (0.2w / w). After the filtrate was concentrated under reduced pressure obtained subjected to vacuum filtration by an evaporator, and pump up in the vacuum pump, Cbz-D-Phe-D -Leu-D-Lys (Boc) -α-Boc-Pic-OMe (7) was obtained 52.5g as a white solid (yield quant, HPLC purity 97.6%).
(6) D-Phe-D -Leu-D-Lys (Boc) synthesis of -α-Boc-Pic-OMe ( 8)
in an eggplant-shaped flask of 2L, Cbz-D-Phe- D-Leu-D-Lys ( Boc) -α-Boc-Pic- OMe (7) the 46.9g (53.3mmol) were charged, the 840ML EtOAc (18V / W), H 2 added to and dissolved O 93.8mL (2.0v / w) It was. The 5% Pd / C to the reaction mixture 9.38g (0.2w / w) was added, After three nitrogen substitution reduced pressure atmosphere, was performed three times a hydrogen substituent. The reaction solution was subjected to 10 hours with vigorous stirring at room temperature to remove the Pd / C and after the completion of the reaction vacuum filtration. NaHCO the resulting filtrate 3 Aq. 235mL (5.0v / w) were added to separate liquid, NaHCO to the organic layer 3 Aq. 235mL (5.0v / w), NaClaq . It was added sequentially cleaning 235mL (5.0v / w). To the resulting organic layer Na 2 SO 4 dried addition of 9.38g (0.2w / w), then the filtrate was concentrated under reduced pressure obtained subjected to vacuum filtration by an evaporator, pump up with a vacuum pump to D-Phe -D-Leu-D-Lys ( Boc) -α-Boc-Pic-OMe (7) was obtained 39.7g (yield quant, HPLC purity 97.3%).
351mL was suspended in (10v / w). To this suspension HOBt 7.92g (51.7mmol), Boc-D-Phe-OH HCl HCl
(8) D-Phe-D -Phe-D-Leu-D-Lys-Pic-OMe Synthesis Of Hydrochloric Acid Salt (1)
In An Eggplant-Shaped Flask Of 20ML Boc-D-Phe-D -Phe-D- Leu-D- lys (Boc) -α -Boc- Pic-OMe (9) and 2.00gg, IPA 3.3mL (1.65v / w), was suspended by addition of PhMe 10mL (5v / w). It was stirred at room temperature for 19 hours by addition of 6N HCl / IPA 6.7mL (3.35v / w). The precipitated solid was filtered off by vacuum filtration and dried under reduced pressure to a white solid of D-Phe-D-Phe- D- Leu-D-Lys-Pic- OMe 1.59ghydrochloride (1) (yield: 99 .0%, HPLC purity 98.2%) was obtained.
(9) D-Phe-D -Phe-D-Leu-D-Lys-Pic-OMe Purification Of The Hydrochloric Acid Salt (1)
In An Eggplant-Shaped Flask Of 20ML-D-Phe-D- Phe D-Leu -D-Lys- pic-OMe hydrochloride crude crystals (1) were charged 200mg, EtOH: MeCN = 1: after stirring for 1 hour then heated in a mixed solvent 4.0 mL (20v / w) was added 40 ° C. of 5 , further at room temperature for 2 was time stirring slurry. Was filtered off by vacuum filtration, the resulting solid was dried under reduced pressure a white solid ((1) Purification crystals) was obtained 161 mg (80% yield, HPLC purity 99.2% ).
(10) D-Phe-D -Phe-D-Leu-D-Lys-Pic Synthesis (Using Purified
(1)) Of (A) To A Round-Bottomed Flask Of 10ML D-Phe-D-Phe-D- -D-Lys Leu-Pic-OMe Hydrochloride Salt (1) Was Charged With Purified Crystal 38.5Mg (0.0488Mmol), H 2 Was Added And Dissolved O 0.2ML (5.2V / W). 1.5H Was Stirred Dropwise 1N NaOH 197MyuL (0.197mmol) at room temperature. After completion of the reaction, concentrated under reduced pressure by an evaporator added 1N HCl 48.8μL (0.0488mmol), to obtain a D-Phe-D-Phe- D-Leu-D-Lys- Pic (A) (yield: quant , HPLC purity 99.7%).

D-Phe-D-Phe- D-Leu-D-Lys-Pic-OMe (1) physical properties 1 H NMR (400 MHz, 1M DCl) [delta] ppm by: 0.85-1.02 (yd,. 6 H), 1.34-1.63 ( m, 5 H), 1.65-2.12 ( m, 5 H), 2.23-2.45 (m, 2 H), 2.96-3.12 (m, 4 H), 3.19 (ddt, J = 5.0 & 5.0 & 10.0 Hz), 3.33-3.62 (m, 1 H), 3.68-3.82 (m, 1 H), 3.82-3.95 (m, 4 H), 3.95-4.18 (m, 1 H), 4.25-4.37 (m, 2 H), 4.61-4.77 (M, 2 H), 7.21-7.44 (M, 10 H) 13 C NMR (400MHz, 1M DCl) Deruta Ppm: 21.8, 22.5, 24.8, 27.0, 30.5, 30.8, 31.0, 31.2, 31.7, 37.2 , 37.8, 38.4, 39.0, 39.8, 40.4, 40.6, 41.8, 42.3, 49.8, 50.2, 52.2, 52.6, 54.6, 55.2, 57.7, 57.9, 127.6, 128.4, 129.2, 129.6, 129.7, 129.8 dp 209.5 ℃

Example 2
(Trifluoroacetic Acid (TFA)
Use) (1) D-Phe-D-Phe-D-Leu-D-Lys-Pic-OMe TFA Synthesis Of Salt (1)
TFA 18ML Eggplant Flask Of 50ML (18V / W) , 1- Dodecanethiol 1.6ML (1.6V / W), Triisopropylsilane 0.2ML (0.2V / W), H 2 Sequentially Added Stirring The O 0.2ML (0.2V / W) Did. The Solution To The Boc-D-Phe- D- Phe-D-Leu-D -Lys (Boc) -α-Boc-Pic-OMe the (9) 1.00g (1.01mmol) was added in small portions with a spatula. After completion of the reaction, concentrated under reduced pressure by an evaporator, it was added dropwise the resulting residue in IPE 20mL (20v / w). The precipitated solid was filtered off, the resulting solid was obtained and dried under reduced pressure to D-Phe-D-Phe- D-Leu -D-Lys-Pic-OMe · TFA salt as a white solid (1) (Osamu rate 93.0%, HPLC purity 95.2%).
(2) D-Phe-D -Phe-D-Leu-D-Lys-Pic synthesis of (A)
to a round-bottomed flask of 10mL D-Phe-D-Phe -D-Leu-D-Lys-Pic-OMe TFA were charged salt (1) 83mg (0.0843mmol), was added and dissolved H2O 431μL (5.2v / w). Was 12h stirring dropwise 1N NaOH 345μL (0.345mmol) at room temperature. After completion of the reaction, concentrated under reduced pressure by an evaporator added 1N HCl 84.3μL (0.0843mmol), to obtain a D-Phe-D-Phe- D-Leu-D-Lys-Pic (A) ( yield: quant, HPLC purity 95.4%).
3 (HCl / EtOAc
Use) (1) In An Eggplant-Shaped Flask Of 30ML Boc-D-Phe-D -Phe-D-Leu-D-Lys (Boc) -Arufa-Boc-Pic-OMe (9) 1. It was charged with 00g (1.01mmol ), was added and dissolved EtOAc7.0mL (7.0v / w). 4N HCl / EtOAc 5.0mL (5.0v / w) was added after 24h stirring at room temperature, the precipitated solid was filtered off by vacuum filtration, washed with EtOAc 2mL (2.0v / w). The resulting solid D-Phe-D-Phe- D-Leu-D-Lys-Pic-OMe hydrochloride (1) was obtained 781mg of a white solid was dried under reduced pressure (the 96.7% yield, HPLC purity 95.4%).
(2) D-Phe-D -Phe-D-Leu-D-Lys-Pic (A) Synthesis of
eggplant flask of 10mL D-Phe-D-Phe -D-Leu-D-Lys-Pic-OMe hydrochloride were charged salt (1) 90 mg (0.112 mmol), H 2 was added and dissolved O 0.47mL (5.2v / w). Was 12h stirring dropwise 1N NaOH 459μL (0.459mmol) at room temperature. After completion of the reaction, concentrated under reduced pressure by an evaporator added 1N HCl 0.112μL (0.112mmol), was obtained D-Phe-D-Phe- D-Leu-D-Lys-Pic (A) ( yield: quant, HPLC purity 93.1%).
4 Example
Compound (1) Of The Compound By Hydrolysis Synthesis Of (The A) (Compound (1) Without
Purification) Eggplant Flask 10ML D-Phe-D-Phe -D-Leu-D-Lys-Pic-OMe (1) Charged Hydrochloride Were (Without Pre-Step Purification) 114.5Mg (0.142Mmol), H 2 Was Added And Dissolved O 595MyuL (5.2V / W). Was 14H Stirring Dropwise 1N NaOH 586MyuL (0.586Mmol) At Room Temperature. After Completion Of the reaction, concentrated under reduced pressure by an evaporator added 1N HCl 0.15μL (0.150mmol), was obtained D-Phe-D-Phe- D-Leu-D-Lys-Pic (A) (yield: quant, HPLC purity 95.2 %).
Example 1 Comparative
Path Not Via The Compound (1) (Using Whole Guard Boc-D-Phe-D-Phe-D-Leu-D-Lys (Boc) -Alpha-Boc-Pic-OMe
(A)) (1) D–Boc Phe- D-Phe-D-Leu-D-Lys (Boc) -Arufa-Boc-Pic-OH Synthesis Of
Eggplant Flask Of 30ML Boc-D-Phe-D -Phe-D-Leu-D- Lys (Boc) -α- Boc-Pic -OMe (9) were charged 1.00g (1.00mmol), was added and dissolved MeOH 5.0mL (5.0v / w). After stirring for four days by the addition of 1N NaOH 1.1 mL (1.10mmol) at room temperature, further MeOH 5.0mL (5.0v / w), 1N NaOH 2.0mL the (2.0mmol) at 35 ℃ in addition 3h and the mixture was stirred. After completion of the reaction, 1 N HCl 6.1 mL was added, After distilling off the solvent was concentrated under reduced pressure was separated and the organic layer was added EtOAc 5.0mL (5.0mL) .NaClaq. 5.0mL (5.0v / w) Wash the organic layer was added, the organic layer as a white solid was concentrated under reduced pressure to Boc-D-Phe-D- Phe-D-Leu-D-Lys (Boc) – α-Boc-Pic-OH 975.1mg (99.3% yield, HPLC purity 80.8% )
(2) D-Phe-D -Phe-D-Leu-D-Lys-Pic synthesis of (A)
to a round-bottomed flask of 20mL Boc-D-Phe-D -Phe-D-Leu-D-Lys (Boc) It was charged -α-Boc-Pic-OH ( 10) 959mg (0.978mmol), was added and dissolved EtOAc 4.9mL (5.0v / w). And 4h stirring at room temperature was added dropwise 4N HCl / EtOAc 4.9mL (5.0mL) at room temperature. After completion of the reaction, it was filtered under reduced pressure, a white solid as to give D-Phe-D-Phe- D-Leu-D-Lys-Pic the (A) (96.4% yield, HPLC purity 79.2%) .
 If not via the compound of the present invention (1), the purity of the compound obtained (A) was less than 80%.


  1.  S. Sinatra Raymond; Jonathan S. Jahr;. J. Michael Watkins-Pitchford (14 October 2010) The Essence Of Analgesia And Analgesics …. Cambridge University Press Pp 490-491 ISBN  978-1-139-49198-3 .
  2.  A Janecka, Perlikowska R, Gach K, Wyrebska A, Fichna J (2010) “Development Of Opioid Peptide Analogs For Pain Relief”.. Curr Pharm Des… 16 (9):. 1126-35 Doi : 10.2174 / 138161210790963869 . PMID  20030621 .
  3. Apfelbaum Jeffrey (8 September 2014). Ambulatory Anesthesia, An Issue Of Anesthesiology Clinics, . Elsevier Health Sciences. Pp. 190-. ISBN  978-0-323-29934-3 .
  4.  Cowan Alan;. Gil Yosipovitch (10 April 2015) Pharmacology Of Itch …. Springer Pp 307- ISBN  978-3-662-44605-8 .
  5.  Allerton Charlotte (2013). Pain Therapeutics: Current And Future Treatment Paradigms …. Royal Society Of Chemistry Pp 56- ISBN  978-1-84973-645-9 .


1: Cowan A, Kehner GB, Inan S. Targeting Itch With Ligands Selective For kappa Opioid
. Receptors Handb Exp Pharmacol 2015; 226:.. 291-314 Doi:
.. 10.1007 / 978-3-662-44605-8_16 Review PubMed PMID: 25861786.

Systematic (IUPAC) Name
Amino–4 1- ( D -Phenylalanyl- D -Phenylalanyl- D -Leucyl- D -Lysyl) Piperidine-4-Carboxylic Acid
Clinical data
Of Routes
Pharmacokinetic Data
Bioavailability Pasento 100 ( IV ) [1]
Metabolism Metabolized Not [1]
Biological half-life Hours 2 [1]
Excretion As Unchanged Excreted
Drug Via Bile And Urine [1]
CAS Number 1024828-77-0
ATC code None
ChemSpider 44208824
Chemical data
Formula C 36 H 53 N 7 O 6
Molar mass 679.85 g / mol

///// Difelikefalin,  CR845 , FE-202845,  Phase III, PEPTIDES

CC (C) C [C @ H] (C (= O) N [C @ H] (CCCCN) C (= O) N1CCC (CC1) (C (= O) O) N) NC (= O) [ C @@ H] (Cc2ccccc2) NC (= O) [C @@ H] (Cc3ccccc3) N




Elpamotide str drawn bt worlddrugtracker


L-Arginyl-L-phenylalanyl-L-valyl-L-prolyl-L-alpha-aspartylglycyl-L-asparaginyl-L-arginyl-L-isoleucine human soluble (Vascular Endothelial Growth Factor Receptor) VEGFR2-(169-177)-peptide

MF C47 H76 N16 O13
Molecular Weight, 1073.2164
L-Isoleucine, L-arginyl-L-phenylalanyl-L-valyl-L-prolyl-L-α-aspartylglycyl-L-asparaginyl-L-arginyl-
  • 10: PN: WO2008099908 SEQID: 10 claimed protein
  • 14: PN: WO2009028150 SEQID: 1 claimed protein
  • 18: PN: JP2013176368 SEQID: 18 claimed protein
  • 1: PN: WO2009028150 SEQID: 1 claimed protein
  • 2: PN: WO2010027107 TABLE: 1 claimed sequence
  • 6: PN: WO2013133405 SEQID: 6 claimed protein
  • 8: PN: US8574586 SEQID: 8 unclaimed protein
  • 8: PN: WO2004024766 SEQID: 8 claimed sequence
  • 8: PN: WO2010143435 SEQID: 8 claimed protein

Phase III

A neoangiogenesis antagonist potentially for the treatment of pancreatic cancer and biliary cancer.


CAS No.673478-49-4, UNII: S68632MB2G

Company OncoTherapy Science Inc.
Description Angiogenesis inhibitor that incorporates the KDR169 epitope of vascular endothelial growth factor (VEGF) receptor 2 (KDR/Flk-1; VEGFR-2)
Molecular Target Vascular endothelial growth factor (VEGF) receptor 2 (VEGFR-2) (KDR/Flk-1)
Mechanism of Action Angiogenesis inhibitor; Vaccine
Therapeutic Modality Preventive vaccine: Peptide vaccine
  • Originator OncoTherapy Science
  • Class Cancer vaccines; Peptide vaccines
  • Mechanism of Action Cytotoxic T lymphocyte stimulants
  • 16 Jun 2015 No recent reports on development identified – Phase-II/III for Pancreatic cancer (Combination therapy) and Phase-II for Biliary cancer in Japan (SC)
  • 09 Jan 2015 Otsuka Pharmaceutical announces termination of its license agreement with Fuso Pharmaceutical for elpamotide in Japan
  • 01 Feb 2013 OncoTherapy Science and Fuso Pharmaceutical Industries complete a Phase-II trial in unresectable advanced Biliary cancer and recurrent Biliary cancer (combination therapy) in Japan (UMIN000002500)


Elpamotide str drawn bt worlddrugtracker

Elpamotide , credit kegg

Elpamotide is a neoangiogenesis inhibitor in phase II clinical trials at OncoTherapy Science for the treatment of inoperable advanced or recurrent biliary cancer. Phase III clinical trials was also ongoing at the company for the treatment of pancreas cancer, but recent progress report for this indication are not available at present.

Consisting of VEGF-R2 protein, elpamotide is a neovascular inhibitor with a totally novel mechanism of action. Its antitumor effect is thought to work by inducing strong immunoreaction against new blood vessels which provide blood flow to tumors. The drug candidate only act against blood vessels involved in tumor growth and is associated with few adverse effects.

Gemcitabine is a key drug for the treatment of pancreatic cancer; however, with its limitation in clinical benefits, the development of another potent therapeutic is necessary. Vascular endothelial growth factor receptor 2 is an essential target for tumor angiogenesis, and we have conducted a phase I clinical trial using gemcitabine and vascular endothelial growth factor receptor 2 peptide (elpamotide). Based on the promising results of this phase I trial, a multicenter, randomized, placebo-controlled, double-blind phase II/III clinical trial has been carried out for pancreatic cancer. The eligibility criteria included locally advanced or metastatic pancreatic cancer. Patients were assigned to either the Active group (elpamotide + gemcitabine) or Placebo group (placebo + gemcitabine) in a 2:1 ratio by the dynamic allocation method. The primary endpoint was overall survival. The Harrington-Fleming test was applied to the statistical analysis in this study to evaluate the time-lagged effect of immunotherapy appropriately. A total of 153 patients (Active group, n = 100; Placebo group, n = 53) were included in the analysis. No statistically significant differences were found between the two groups in the prolongation of overall survival (Harrington-Fleming P-value, 0.918; log-rank P-value, 0.897; hazard ratio, 0.87, 95% confidence interval [CI], 0.486-1.557). Median survival time was 8.36 months (95% CI, 7.46-10.18) for the Active group and 8.54 months (95% CI, 7.33-10.84) for the Placebo group. The toxicity observed in both groups was manageable. Combination therapy of elpamotide with gemcitabine was well tolerated. Despite the lack of benefit in overall survival, subgroup analysis suggested that the patients who experienced severe injection site reaction, such as ulceration and erosion, might have better survival

The vaccine candidate was originally developed by OncoTherapy Science. In January 2010, Fuso Pharmaceutical, which was granted the exclusive rights to manufacture and commercialize elpamotide in Japan from OncoTherapy Science, sublicensed the manufacturing and commercialization rights to Otsuka Pharmaceutical. In 2015, the license agreement between Fuso Pharmaceutical and OncoTherapy Science, and the license agreement between Fuso Pharmaceutical and Otsuka Pharmaceutical terminated.

WO 2010143435

US 8574586

WO 2012044577

WO 2010027107

WO 2013133405

WO 2009028150

WO 2008099908

WO 2004024766



The injectable formulation containing peptides, because peptides are unstable to heat, it is impossible to carry out terminal sterilization by autoclaving. Therefore, in order to achieve sterilization, sterile filtration step is essential. Sterile filtration step is carried out by passing through the 0.22 .mu.m following membrane filter typically absolute bore is guaranteed. Therefore, in the stage of pre-filtration, it is necessary to prepare a peptide solution in which the peptide is completely dissolved. However, peptides, since the solubility characteristics by its amino acid sequence differs, it is necessary to select an appropriate solvent depending on the solubility characteristics of the peptide. In particular, it is difficult to completely dissolve the highly hydrophobic peptide in a polar solvent, it requires a great deal of effort on the choice of solvent. It is also possible to increase the solubility by changing the pH, or depart from the proper pH range as an injectable formulation, in many cases the peptide may become unstable.
 In recent years, not only one type of peptide, the peptide vaccine formulation containing multiple kinds of peptides as an active ingredient has been noted. Such a peptide vaccine formulation is especially considered to be advantageous for the treatment of cancer.
 The peptide vaccine formulation for the treatment of cancer, to induce a specific immune response to the cancer cells, containing the T cell epitope peptides of the tumor-specific antigen as an active ingredient (e.g., Patent Document 1). Tumor-specific antigens these T-cell epitope peptide is derived, by exhaustive expression analysis using clinical samples of cancer patients, for each type of cancer, specifically overexpressed in cancer cells, only rarely expressed in normal cells It never is one which has been identified as an antigen (e.g., Patent Document 2). However, even in tumor-specific antigens identified in this way, by a variety of having the cancer cells, in all patients and all cancer cells, not necessarily the same as being highly expressed. That is, there may be a case in which the cancer in different patients can be an antigen that is highly expressed cancer in a patient not so expressed. Further, even in the same patient, in the cellular level, cancer cells are known to be a heterogeneous population of cells (non-patent document 1), another even antigens expressed in certain cancer cells in cancer cells may be the case that do not express. Therefore, in one type of T-cell epitope peptide vaccine formulations containing only, there is a possibility that the patient can not be obtained a sufficient antitumor effect is present. Further, even in patients obtained an anti-tumor effect, the cancer cells can not kill may be present. On the other hand, if the vaccine preparation comprising a plurality of T-cell epitope peptide, it is likely that the cancer cells express any antigen. Therefore, it is possible to obtain an anti-tumor effect in a wider patient, the lower the possibility that cancer cells can not kill exists.
 The effect of the vaccine formulation containing multiple types of T-cell epitope peptide as described above, the higher the more kinds of T-cell epitope peptides formulated. However, if try to include an effective amount of a plurality of types of T cell peptide, because the peptide content of the per unit amount is increased, to completely dissolve the entire peptide becomes more difficult. Further, because it would plurality of peptides having different properties coexist, it becomes more difficult to maintain all of the peptide stability.
 For example, in European Patent Publication No. 2111867 (Patent Document 3), freeze-dried preparation of the vaccine formulation for the treatment of cancer comprising a plurality of T-cell epitope peptides have been disclosed. This freeze-dried preparation, in the preparation of peptide solution before freeze drying, each peptide depending on its solubility properties, are dissolved in a suitable solvent for each peptide. Furthermore, when mixing the peptide solution prepared in order to prevent the precipitation of the peptide, it is described that mixing the peptide solution in determined order. Thus, to select a suitable solvent for each peptide, possible to consider the order of mixing each peptide solution is laborious as the type of peptide increases.

In order to avoid difficulties in the formulation preparation, as described above, a vaccine formulation comprising one type of T-cell epitope peptides, methods for multiple types administered to the same patient is also contemplated. However, when administering plural kinds of vaccine preparation, it is necessary to vaccination of a plurality of locations of the body, burden on a patient is increased. Also peptide vaccine formulation, the DTH (Delayed Type Hypersensitivity) skin reactions are often caused called reaction after inoculation. Occurrence of skin reactions at a plurality of positions of the body, increases the discomfort of the patient. Therefore, in order to reduce the burden of patients in vaccination is preferably a vaccine formulation comprising a plurality of T-cell epitope peptide. Further, even when the plurality of kinds administering the vaccine formulation comprising a single type of epitope peptides, when manufacturing each peptide formulation is required the task of selecting an appropriate solvent for each peptide.

Patent Document 1: International Publication No. WO 2008/102557
Patent Document 2: International Publication No. 2004/031413 Patent
Patent Document 3: The European Patent Publication No. 2111867

///////////Elpamotide, Phase III,  A neoangiogenesis antagonist, pancreatic cancer and biliary cancer, OTS-102, OncoTherapy Science Inc, peptide


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