WO2011104627A1 - Metabolites of dop2-d-lys(dop2)-cyclo[cys-tyr-d-trp-lys-abu-cys]-thr-nh2 - Google Patents
Metabolites of dop2-d-lys(dop2)-cyclo[cys-tyr-d-trp-lys-abu-cys]-thr-nh2 Download PDFInfo
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- WO2011104627A1 WO2011104627A1 PCT/IB2011/000467 IB2011000467W WO2011104627A1 WO 2011104627 A1 WO2011104627 A1 WO 2011104627A1 IB 2011000467 W IB2011000467 W IB 2011000467W WO 2011104627 A1 WO2011104627 A1 WO 2011104627A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/655—Somatostatins
- C07K14/6555—Somatostatins at least 1 amino acid in D-form
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- Pop2 ⁇ P-Lys(Pop2)-cycio[C ⁇ ⁇ which may be designated as (pop2) 2 ⁇ D-Eys- ycio[Cys-Tyr-D-Trp-Lys-A -Cys]-T ⁇
- Tyr-D ⁇ Trp-Lys-Abi3-Cys]-Tlir-NH2 could be potentially metabolized in vitro into different amino-acid-contaming fragments as a result of proteolytic -cleavage in liver and kidney tissues.- However, the formation of non-peptidyi metabolites of Dop2-D- Lys(Dop2)-6yclo[CyS'Tyr-D'Trp-Lys-Ahu-eys]-Thx-NH2 remained to-be elucidated.
- the present inventors discovered non-peptidyi metabolites of Dop2-D- i Lys ⁇ op2)-cyclo[Cys-Tyr-D-Trp»Lys-Abu-Cys3-Tin--NH3 ⁇ 4 namely, the free N- termittal Dop2-OH.and D-Lys--(Dop2) 2 fragments, and confirmed their identities by LC-MS MS in plasma samples, from monkeys following subcutaneous (s.c.) administrations, FIG.
- M2 is. -Lys ⁇ Qp2)2-Cys-Tyr-D-Ti -Lys;
- M4 is- ' D-Lys-(Dop2)2-eyc!o[Cys-Tyr-D-Tip--Lys-Abxi-Cys3;
- MS is D"Lys «(D0p2)2-Cys- yr-i ) -Trp-Lys-Abu ⁇ Cys;
- M6 is J ⁇ Lys-(Dop2)2-cyclo[Cys-Tyr D-Tip ' Lys ⁇ Abu-Cys3-ThT;
- M7 is D-Lys ⁇ (Bop2)rCys.
- Dop2 is meant a compound having the structure of:
- Dpp2-Ofi may be represented by the following structure
- D-Lys-(Dop2 ⁇ 2 may be represented by the following structure:
- the dopamine agonistic moieties D-Lys-(Dop2) 2 and Dop2-OH were also formed in vitro by human liver S9 fractions in the presence of ADPH, as a result of extensive metabolism of Dop2-D-Lys(Dop2)-cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys]- Thr-NH 2 .
- Metabolite M6 was identified in plasma from rats of a subcutaneous toxicity study. The levels of circulating M6 were comparable to those of unchanged Dop2-D- Lys(Dop2)-cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 suggesting that M6 could account for a considerable fraction of the exposure to the drug-related material, Unchanged Dop2-D-Lys(Dop2)-cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-
- N3 ⁇ 4 was the main component in plasma from monkeys of a subcutaneous toxicity study. However, three additional metabolites could be detected corresponding to D- Lys-(Dop2) 2 , M3 and DOP2-OH, the latter in very low amounts (traces) and only after 1,5 hour post dose.
- the levels (pg/mf) of D-Lys-(Dop2) 2 in monkey plasma samples were 100 to 40 fold lower than the levels of unchanged Dop2-D-Lys(Dop2)- cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH 2 .
- Metabolite D-Lys-(Dop2) 2 was also detectable in plasma samples after a 3-day wash-out period between subsequent administrations. * Samples
- Human liver and monkey kidney S9 fractions were selected in these experiments since they were able to generate all of the peptidyl in vitro metabolites of Dop2-D ,ys(Dop2) ⁇ cyclo[Cys ⁇ Tyr-D ⁇ Trp-Lys-Abu ⁇ Cys]-Thr-NI-l2 metabolites, i.e., M1-M7.
- Rat kidney S9 fractions were used for in vitro generation of the ' C-labelled metabolite M6.
- TFA trifiuoroacetic acid
- Test incubations were performed over 30 minutes (human li ver S9), 60 minutes (rat kidney S9), and 120 minutes (human liver and: monkey kidney S9).
- a blank sample (without ⁇ ⁇ Dop2-D-Lys(Dop2) ⁇ cyclo[Cys-Tyr-D-T ⁇ -Lys-Abu ⁇ Cys] ⁇ Tlii-- NH 2 ) was prepared for each incubation time in all cases.
- the S9 protein concentrations were 4 mg/ml (human liver 89 fractions). 3 mg/ml (monkey kidney S9 fractions), and 1 mg/ml (rat kidney S9 fractions).
- Plasma samples from monkeys dosed with Dop2-D-Lys(Dop2)-cyclo[Cys- Tyr ⁇ D ⁇ Tq5-Lys-Abu-Cys]-T ' hr-NH2 were analysed.
- Tabic 2 summarizes the sampl analvzed.
- a pooled sample was prepared for each sampling time using plasma from 4 animals.
- Plasma samples from rats were analysed. Table 3 summarizes the samples analyzed.
- a pooled sample was prepared for the indicate ' sampling times using plasma from 3 animals.
- Frozen monkey plasma samples were thawed and kept in ice-water hath. Afterwards, a pooled plasma sample was prepared for each sampling time (0, 1.5, 5 and 24 hours) by mixing plasma from 4 different animals. After pooling, plasma proteins were precipitated by addition of one volume of aceionitrile containing 0.1 % TFA, and the samples were centriftiged at 20,000 x g for 15 minutes at 4°C. The aceionitrile content in the supernatants was evaporated under >3 ⁇ 4 stream, and the aqueous residue was injected to the LC-MS/MS system. Rat Plasma Samples.
- Rat plasma samples were processed individually. 100 ⁇ -aliquots of each lasma: sample were, mixed with LOO ⁇ . of aceionitrile for protein precipitation. After centrifligation, 800 ⁇ ! of 0,07% TritonTM X-1QQ were mixed with each supernatant, and the resulting , specimens were loaded into OasisTM HLP 10 rag plates (Waters Corp,,. Milfbrd, MA, USA) for solid phase .extraction. Elution was done with aceionitrile containing 0.5% formic acid (FA). The samples were evaporated under N2 stream, and the dry-extracts were obtained. individual samples were reconstituted m a solution consisting on waieracetoiHtriie (94.6) containing 1.15% FA and 0.03% TritonTM X ⁇ 100, and pool accordingly for LC-MS ' S analysis.
- Solvent A 50 niM A moniurn Formiaie pH 5.0
- Solvent B aceionitrile (0.05% FA)
- Solvent A water (0.1% FA, 0.01% TFA)
- Solvent B acetomtri]e :(0,l % PA, 0,01% TFA)
- Solvent A water -(0.1 % FA. 0.0.1 % IT A)
- Solvent B acetonitrile (0.1 % FA, 0.0:1 % TFA)
- MS analysis were perforated using eleeirospray ionization- (ESI) in posiiive-io zatiorvmode.
- ESI eleeirospray ionization-
- MRM method was created. Doubly and triply charged ion specie were monitored h these analyses.
- analyses were performed in IDA mode, using MRM information forDop2 ⁇ -Lys(Dop2)-cycloiCys-Tyr ⁇ D-Tip-Lys-Abu-Cys3-T r- N33 ⁇ 4 and metabolite M6 as survey for further ER scan acquisition.
- Thr-N% used in in vitro incubates and in monkey plasma.
- Thr-NH-2 used in in vitro incubates and in rat plasma.
- IDA is a software tool that helps select the best ionfs) to target for MS/MS data acquisition during an HPLC analysis.
- the benefits of IDA are the simultaneous collection of enhanced resolution MS and MS/MS dat to maximize the information acquired in a single-injection.
- control monkey S9 incubates confirmed the formation of the two metabolites M4 and M6, in addition to unchanged Dop2-D-Lys(Dop2)-cyclo[Cys-Tyr-D- ' I ⁇ -Lys-Abu-Cys]-Thr-NH 2 .
- Metabolite M4 Doubly charged monoisotopic molecular ion at m z 797,5 indicated a molecular mass of 1593. According to these data, metabolite M4 shows a loss of 100 mass units with respect to Dop2-D-Lys(Dop2)-cyclo[Cys- Tyr-D-Trp-Lys-Abu-Cysj-Thr-NHz, which matched up with the proposed Des-Thr-amide metabolite (molecule containing a disulphide bridge with the loss of the C-terrnmal amino acid: Thr-amide).
- Metabolite M6 Doubly charged monoisotopic molecular ion at m/z 848,0 indicated a molecular mass of 1694, According to these data, metabolite M6 shows an increase of 1 mass unit with respect to Dop2-D-Lys(Dop2)- cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Th.r-NH2, which matched u with the proposed Des-Amidated (acid) metabolite (molecule containing a disulphide bridge with the change of the C-terminal amide by a carbox lic acid).
- test monkey and human S9 incubates showed the formation of 5 metabolites (Ml, M2, M3, M5 and M7), in addition to unchanged Dop2-D ⁇ Lys(Dop2)-cyclo[Cys-Tyr-D-Trp- Lys-Abu-Cys]-Thr-NH 2 and metabolites M4 and M6,
- Metabolite M2 Doubly charged monoisotopic molecular ion at m z 704,5 indicated a molecular mass of 1407. According to these data, metabolite M2 shows a loss of 286 mass units with respect to Dop2-D-Lys(Dop2)-cyclo[Cys- Tyr-D-Trp-Lys-Abu-Cysj-Thr-N3 ⁇ 4 5 which matched up with a linear molecule resulting after the reduction of the disulphide bridge, and the loss of the three C-terminal ammo-acids: Abu, Cys and Thr-amide, Metabolite M 3; Doubly charged mOnoisotopic molecular ion at rn z 747,0 indicated a molecular mass of 1492.
- metabolite M3 shows a loss of 20.1 mass units with respect to D6p2-D ⁇ Lys(Dop2) ⁇ cyclG[Cys- Tyr-D.-T ⁇ -Lys-Abii-Cys]-Thr-NH2 S which matched u with a linear molecule resulting after the reduction of the disuiphide bridge, and the loss of the two : C- terminal amino acids: Cys and Thr-amide.
- Metabolite M5 Doubly charged monpisptopic- molecular ion at m/z 798.5 indicated a ttiolecHlar mass of 1595. According to these data, metabolite M5 shows a loss of 98 mass units with respect to Dop2-D-Lys(Dop2)-cyclo[Cys- Tyr-D-Trp-Lys-Abu-Cysj-Thr-i Hz, which matched, up with a linear molecule resulting after the reduction of the disulphide bridge, and the loss of the C- tefrmnal amino acid Thr-amide.
- Metabolite M7 Metabolite formed only in monkey kidney S9 fractions with singly charged monoisotopic molecular ion at m z 930,5 indicated a molecular mass of 929.5. According to these data, metabolite M7 shows a loss of 764 mass units with respect to Dop2-D-Lys(DDp2)-cyclo[:Cys-'Tyr-D-Trp-Lys- Abii-Cys3-Thr-NH2j which matched tip with a linear molecule resulting after the reduction of the disulphide bridge, and the loss of the six C-termifial amino acids: Tyr, D-T ' rp, Lys, Abu, Cys and Thr-amide.-
- Metabolite Ml Unidentified metabolite formed only by huma liver S9 fractions with doubly charged average molecular ion at m/z 949 indicated a molecular mass around 18.96, According to these data, metabolite Ml shows, an increase of around 203 mass units with respect to Dop2-D-Lys(Dop2)- cycio[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr ⁇ NH 2 .
- test 8 incubations suggest that extensive proteolytic hydrolysis of op2-D-Lys(Dop2)-eycto[Cys-Tyx-D-Trp-Lys-Abu ⁇ ys]-Tlir-NH2 is produced in vitro after reduction of the disulphide-bond, a process that would be mediated by a NADPH-dependent enzymatic reaction (test incubations with S9 fractions were performed in the presence ofNADPH cofac ' tor).
- D ⁇ I.,.ys ⁇ (Dop2)2 showed a protonated molecular ion at m z 827,6 and the doubly charged species at /z 414.3.
- the fragmentation pattern showed characteristic ions at m/z 561,4. 529.4 and 208.1.
- Dop2-OH showed a protonated molecular ion at m/z 359.28. Its fragmentation pattern showed characteristic ions at m z 208.1, 193.1 and 154.1.
- Precursor-Ion experiments were performed in samples corresponding to incubations of Dop2-D ⁇ Lys(Dop2)-cyelo[Cys-Tyr-D"Trp-LyS"Abu-Cys]-Tbr- H2 with S9 fractions from monkey kidney and human liver, In order to detect common fragments to be selected in further MRM analyses, ion at m z of 159 was selected as it provides a good signal to noise ratio for Dop2-D-Lys(Dop2)-eyclo[Cys-Tyr-D-Tqi- Lys-Abu-Cys]-Tk-Ni3 ⁇ 4.
- subcutaneous administration at the dose level of 12.5 mg/kg were pooled and analyzed by employing HPLC Method B (24 hour plasma samples, MRM transition 565.3 / 129.0), and also using the IDA method by employing HPLC Method C (8 hour plasma samples, MRM transition 565.3 / 129.0).
- M6 ER pattern showed also the isotopic profiles corresponding to the two molecule counterparts: i.e. unlabelled M6 (triply charged species at rn/z 565,7) and 14 C-M6 (triply charged species at m/z 571,0-),
- Monkey plasma samples corresponding to a daily subcutaneous administration at the dose level of 45 nig/kg were pooled (plasma from 4 animals at each blood sampling time) and analyzed by MRM following HPLC Method B according to the. transitions listed in Table 4. MRM chromatograms were obtained for the following samples:, . pre-dose, 1.5 hours, 5 hours and 24 hours. Unchanged Dop2-D-Lys(Dop2)- cyclo[Cys « Tyr l p-Lys-Abu-Cysj-Tlu-NH 2 was the main component in all samples except for pre-dose sample.
- a semi-quantitative evaluation of Dop2, D-Lys-(Dop2)2 and unchanged Dop2- D-Lys(Dop2)-cyGlo[CyS"Tyr ⁇ D-Trp-Lys-Abu*Cys]-Thr-NH2 was performed by measuring the LC-MS/MS response (MRM mode) in a sample containing the same amount of the three standards (1 ⁇ /ml).
- Dop2, D-Ly ; s-(Dop2)2 response in terms of peak height was found to be approximately 20 fold higher than Dop2-D-Lys(Dop2)» vyclo[Gys-Tyr-D-Trp-LyS"Abu ⁇ Cys]-Tlii-NH 2 using the transitions described in Table 4, which were the same used in monkey plasma analyses.
- Membranes were prepared by homogenizing cells expressing the human recombinant dopamine-2 receptor in 20 ml of ice-cold 50 mM Tris-HCl with a 'Brinkman Polytron (setting 6, 15 seconds).
- Buffer was added to obtain a final volume of 40 ml, and the homogenate was centrifuged in a Sorval SS-34 rotor at 39,000 g for 10 minutes at 0 ⁇ 4°C, The resulting supernatant was decanted -and -discarded. The pellet was rehotrsogerhzed m iee-cold buffer, pre-iiieiibated afc 37°C for 10 minutes, diluted, and centrifuged as before. The - ' final, pellet was resuspended in buffer and held on ice for the receptor binding assay ,
- Membranes were prepared ' by homogenizing cells expressing the human recombinant 5-hydroxy-tryptamine 2B (h5 ⁇ HT2B) receptor in 20 ml of ice-cold 50 mM Tris-HCl with a Brinkman Polytron (setting 6, 15 seconds). Buffer was added to obtain a final volume of 40 nil, and the homogenate was centrifuged in .a Sorval SS-34 rotor at 39,000 g for 10 min at 0-4-°C. The resulting supernatant was decanted and discarded. The pellet was rehomogemzed in ice-cold buffer, pre-irtcubated at 37°C for 10 minutes, diluted, and centrifuged as before. The final pellet was resuspended in buffer and held on ice for the receptor binding assay.
- h5 ⁇ HT2B 5-hydroxy-tryptamine 2B
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Abstract
The present invention relates to metabolites of Dop2-D-Lys(Dop2)-cycIo[Cys- Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2.
Description
Metabolites of Dop2-D-Lys(Dop2)-cydo[Cys-<Tyr.D-Trp-Lys-AbiJ-Cys]-TIir~NH2
Pop2~P-Lys(Pop2)-cycio[C^^ which may be designated as (pop2)2^D-Eys- ycio[Cys-Tyr-D-Trp-Lys-A -Cys]-T^ Is a chimeric molecule consisting- of two dopamine agonist moieties covalently linked to the N-tenninus of a cyclic analogue, which is disclosed in WO 2004/091490, the content of which, is incorporated herein in its entirety, and which may be represented by the following structure:
The present inventors had hypothesized that DGp2-D~Lys(Dop2)~cycio[Gys^
Tyr-D~Trp-Lys-Abi3-Cys]-Tlir-NH2 could be potentially metabolized in vitro into different amino-acid-contaming fragments as a result of proteolytic -cleavage in liver and kidney tissues.- However, the formation of non-peptidyi metabolites of Dop2-D- Lys(Dop2)-6yclo[CyS'Tyr-D'Trp-Lys-Ahu-eys]-Thx-NH2 remained to-be elucidated.
The present inventors discovered non-peptidyi metabolites of Dop2-D-i Lys^op2)-cyclo[Cys-Tyr-D-Trp»Lys-Abu-Cys3-Tin--NH¾ namely, the free N- termittal Dop2-OH.and D-Lys--(Dop2)2 fragments, and confirmed their identities by LC-MS MS in plasma samples, from monkeys following subcutaneous (s.c.) administrations, FIG. 1 Indicates the free N-terminal Dop2-OH and D-Lys-(Dop2)2 fragments, together with ail of the peptidyi in vitro metabolites of Dop2-D- Lys(I)op2)-cyclo[Cys-Tyr--P-I^"Lys-Abu-Cys]-l¾r-NH2.
In vitro transformation of Dop2-D-Lys(Dop2)-cyclo[Cys-Tyr-D-Trp-Lys-
Abu-Cysj-Thr-NHj using liver and kidney S9 fractions showed a NADPH-dependent pattern. In. the absence of NADPH, two main metabolites were formed probably by peptidases through hydrolysis of the C-terminai residues amide (M6) and Thr-amide ( 4), both conserving the disuiphide bond structure. In die presence of NADPH, extensive proteolytic hydrolysis of Dop2-P"Lys(Pop2)-cyclo[Cys-Tyr-D"Trp-Lys- Abu-Cys]-Thr-NH2 was produced subsequent to the reduction of the disulphide-bond, & process that would be mediated by a NADPH-dependent enzymatic reaction. The main metabolites and their proposed chemical modifications are shown in Table 1.
* Amino acid and NF¾ losses mean that the peptide is in the earhoxylic acid form.
The -metabolites M2 and M7, as shown in FIG. 1. aad indicated in Table l^m be designated as follows;
M2 is. -Lys^Qp2)2-Cys-Tyr-D-Ti -Lys;
M3 i s D-Ly s -(Dop2)2-Cys-Tyr- -Trp-Ly s- Ab u;
M4 is-'D-Lys-(Dop2)2-eyc!o[Cys-Tyr-D-Tip--Lys-Abxi-Cys3;
MS is D"Lys«(D0p2)2-Cys- yr-i)-Trp-Lys-Abu~Cys;
M6 is J^Lys-(Dop2)2-cyclo[Cys-Tyr D-Tip'Lys~Abu-Cys3-ThT; and
M7 is D-Lys~(Bop2)rCys.
By "Dop2" is meant a compound having the structure of:
Dpp2-Ofi may be represented By the following structure
D-Lys-(Dop2}2 may be represented by the following structure:
Metabolite M6 was identified in plasma from rats of a subcutaneous toxicity study. The levels of circulating M6 were comparable to those of unchanged Dop2-D- Lys(Dop2)-cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 suggesting that M6 could account for a considerable fraction of the exposure to the drug-related material, Unchanged Dop2-D-Lys(Dop2)-cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-
N¾ was the main component in plasma from monkeys of a subcutaneous toxicity study. However, three additional metabolites could be detected corresponding to D- Lys-(Dop2)2, M3 and DOP2-OH, the latter in very low amounts (traces) and only after 1,5 hour post dose. The levels (pg/mf) of D-Lys-(Dop2)2 in monkey plasma samples were 100 to 40 fold lower than the levels of unchanged Dop2-D-Lys(Dop2)- cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2. Metabolite D-Lys-(Dop2)2 was also detectable in plasma samples after a 3-day wash-out period between subsequent administrations. * Samples
In Vitro Incubates of Dop2-D-LysfDop2')-cycIo|'Cys-Tyr-D-Trp-Lys-Abu-Cys']-Thr- NH2 with S9 Fractions from Human Liver. Monkey Kidney and Rat Kidney
Human liver and monkey kidney S9 fractions were selected in these experiments since they were able to generate all of the peptidyl in vitro metabolites of Dop2-D ,ys(Dop2)~cyclo[Cys~Tyr-D~Trp-Lys-Abu~Cys]-Thr-NI-l2 metabolites, i.e., M1-M7. Rat kidney S9 fractions were used for in vitro generation of the ' C-labelled metabolite M6.
S9 fractions were incubated with 25 μΜ Dop2-D-Lys(Dop2)-cyclo[Cys-T r- D-Tip-Lys-Ab -Cysi-Tbr-NH2 (or wC-I beled DQp2"D-Lys(Dop2)^eycIo[Cys-ly^D- Trp-Lys-Ajb -Cysj-Thr-NH'? for rat kidney S9 incubations) at 3 C in S O mM
Tris-HGl buffer pH 7,4 containing 5 rnM MgC¾, in the presence of a NADPH- generating system consisting of 4 mM D-glucose 6-phosphaie, 2 U/mi glucose diphosphate dehydrogenase, and 1 mM β-NADP, The incubations were performed in the presence .(test samples) or absence (control samples) of β-NADP, and the reactions v/ere started by the addition of β-ΝΑΒΡ (of equivalent volume of buffer), and "were -quenched at the specified times by the addi tion of one volume of acetoiiitrilc containing 0.1 % trifiuoroacetic acid (TFA) .
Test incubations were performed over 30 minutes (human li ver S9), 60 minutes (rat kidney S9), and 120 minutes (human liver and: monkey kidney S9). A blank sample (without ^^ Dop2-D-Lys(Dop2)~cyclo[Cys-Tyr-D-T^-Lys-Abu~Cys]~Tlii-- NH2) was prepared for each incubation time in all cases.
The S9 protein concentrations were 4 mg/ml (human liver 89 fractions). 3 mg/ml (monkey kidney S9 fractions), and 1 mg/ml (rat kidney S9 fractions).
Moriksv Plasma Samples
Plasma samples from monkeys dosed with Dop2-D-Lys(Dop2)-cyclo[Cys- Tyr~D~Tq5-Lys-Abu-Cys]-T'hr-NH2 were analysed. Tabic 2 summarizes the sampl analvzed.
* A pooled sample was prepared for each sampling time using plasma from 4 animals.
T; Sampling time
Rat Plasma Samples
Plasma samples from rats were analysed. Table 3 summarizes the samples analyzed.
* A pooled sample was prepared for the indicate ' sampling times using plasma from 3 animals.
T: Sampling time
* Sampl f reparation tor Analysis
In P¾ro,focubates.with S.9 fractions
For analysis, all the samples generated were centriftiged at approximately
20,000 g .for 15 minutes at 4°C. After ceni ifti atioa, 100 ttl-alkraois of the superaatants were diluted with 250 μί of 0.1% acetic acid and injected to the liquid chromatography- tandem mass spectrometry (LC-MS/MS) system,
Monkey Plasma Samples
Frozen monkey plasma samples were thawed and kept in ice-water hath. Afterwards, a pooled plasma sample was prepared for each sampling time (0, 1.5, 5 and 24 hours) by mixing plasma from 4 different animals. After pooling, plasma proteins were precipitated by addition of one volume of aceionitrile containing 0.1 % TFA, and the samples were centriftiged at 20,000 x g for 15 minutes at 4°C. The aceionitrile content in the supernatants was evaporated under >¾ stream, and the aqueous residue was injected to the LC-MS/MS system. Rat Plasma Samples.
Rat plasma samples were processed individually. 100 μΐ-aliquots of each lasma: sample were, mixed with LOO μί. of aceionitrile for protein precipitation. After centrifligation, 800 μ! of 0,07% Triton™ X-1QQ were mixed with each supernatant, and the resulting, specimens were loaded into Oasis™ HLP 10 rag plates (Waters Corp,,. Milfbrd, MA, USA) for solid phase .extraction. Elution was done with aceionitrile containing 0.5% formic acid (FA). The samples were evaporated under N2 stream, and the dry-extracts were obtained.
individual samples were reconstituted m a solution consisting on waieracetoiHtriie (94.6) containing 1.15% FA and 0.03% Triton™ X·· 100, and pool accordingly for LC-MS 'S analysis.
• High Perfermaaee Μ^^ά€ί^&^Άί^§ΧΆΡ . (H£¾£I -¾t^ysi¾
HPLC Conditions - Method. A
The following HPLC method was used for in vitro samples (S9 fractions):
Column: Luna CIS (150 x 4,6 ram, 5 μπι. Phenomenex)
Solvent A: 50 niM A moniurn Formiaie pH 5.0
Solvent B: aceionitrile (0.05% FA)
Column Temperature: 50 °C
Flow rats: 1 ra!/aiin
HPLC Conditions --Method B
The following HPLC method was used, fer plasma samples:
Column: Symmetry 300 CI S (50 x 2.1 mm, 3.5 μχη, Waters)
Guard column: Symmetry 300 CI 8 (TO x 2.1 mm, 3.5 μηι, Waters)
Solvent A: water (0.1% FA, 0.01% TFA)
Solvent B: acetomtri]e :(0,l % PA, 0,01% TFA)
Column Temperature: 50 "C
Flow rate; 0.8 mlhmn
Gradient:
The following HPLC method was used for selected rat plasma samples; Column: Symmetry 300 CI 8 (100 x 2.1 mm, 3.5 run. Waters) Guard column: Symmetry 300 CIS (.1.0 x 2.1 'mm, 3,5 μτη, Waters)
Solvent A: water -(0.1 % FA. 0.0.1 % IT A)
Solvent B: acetonitrile (0.1 % FA, 0.0:1 % TFA)
Column Temperature: 50 fJC
Flow rate: 0,4 ml/mm.
Gradient:
MS analysis were perforated using eleeirospray ionization- (ESI) in posiiive-io zatiorvmode.
Enhanced esoiiition Scan and Enhanced Product Ion Analysis
The strategy followed for the analysis of these samples was based on the application of IDA (Information Dependent Acquisition) experiments, Enhanced Multiply Charged scan (EMC) was used as survey scan, If a particular ion in the ehromaxogram met. the criteria defined in the IDA method, then an Enhanced Resolution scan (ER) and an Enhanced Product ion sca (ΕΡΪ) were triggered;.
Precursor Ion Analysis
The strategy defined above was modified by substituting the survey scan. In this case, a Precursor Ion scan was used as such. Again, if a particular ion met the IDA criteria, ER and EPI scans were triggered.
Multiple -Reaction Monitoring IMRM)
After examination o the fragmentation patterns- of the different .metabolites, MRM method was created. Doubly and triply charged ion specie were monitored h these analyses.
For some rat plasma samples, analyses were performed in IDA mode, using MRM information forDop2^-Lys(Dop2)-cycloiCys-Tyr~D-Tip-Lys-Abu-Cys3-T r- N3¾ and metabolite M6 as survey for further ER scan acquisition.
a: Transition for Dop2-D-Lys(Oop2)-cyd0[Cys~Tyr-D~Trp-Lys»Ab«-Cys]
Thr-N% used in in vitro incubates and in monkey plasma.
b; Transition for Dop2rD-Lys(Dop2)*cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys3
Thr-NH-2 used in in vitro incubates and in rat plasma.
c and d: Two transitions were monitored for M7: a) 465.8 / 159.0 and b) 310:8 / 509.4.
(M + 2H+)/2;-'njolecular ion corresponding to the dubly charged species (M + 3H !)/3 : molecular ion corresponding to the triply charged species
Incubates of Dop2-D-Lys(Dop2)-cycio[Cys-Tyr~D-Trp~Lys~Abu-Cys]~Thr- NI¾ with monkey kidney and human liver S9 tractions were used to characterise the main in vitro metabolites of Dop2-D~Lys(Dop2)~cyclo{Cys~Tvr~D-Tfp~Lys~Abu- Cys3-Thf-N¾.
The initial strategy followed for the analysis of these samples -was based; on. the application of IDA experiments. IDA is a software tool that helps select the best ionfs) to target for MS/MS data acquisition during an HPLC analysis, The benefits of IDA are the simultaneous collection of enhanced resolution MS and MS/MS dat to maximize the information acquired in a single-injection.
For in vitro -incubates of Dop2'iD-Lys{Dop2)-cyclo[Cys-Tyr-D~Trp--Lys-Ab CySj~Thr-NH2, the HPLC effluent (See HPLC Method A) was analyzed in IDA mode on the mass spectrometer. This mode -completes a. survey scan (enhanced multiply charged scan EMC) and then performs an enhanced resolution (ER) scan and an enhanced product-ion (EPI) scan on the most intense peaks from the survey scan.
The analysis of control monkey S9 incubates (incubated without NADPH cofacior) confirmed the formation of the two metabolites M4 and M6, in addition to unchanged Dop2-D-Lys(Dop2)-cyclo[Cys-Tyr-D-'I^-Lys-Abu-Cys]-Thr-NH2.
Unchanged Dop2-D-Lys(Dop2)-cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr- NH2: Doubly charged monoisotopic molecular ion at the mass-charge ratio (m/z) 847,5 confired the target molecular mass of 1693.
Metabolite M4: Doubly charged monoisotopic molecular ion at m z 797,5 indicated a molecular mass of 1593. According to these data, metabolite M4 shows a loss of 100 mass units with respect to Dop2-D-Lys(Dop2)-cyclo[Cys- Tyr-D-Trp-Lys-Abu-Cysj-Thr-NHz, which matched up with the proposed Des-Thr-amide metabolite (molecule containing a disulphide bridge with the loss of the C-terrnmal amino acid: Thr-amide).
Metabolite M6: Doubly charged monoisotopic molecular ion at m/z 848,0 indicated a molecular mass of 1694, According to these data, metabolite M6 shows an increase of 1 mass unit with respect to Dop2-D-Lys(Dop2)- cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Th.r-NH2, which matched u with the proposed Des-Amidated (acid) metabolite (molecule containing a disulphide bridge with the change of the C-terminal amide by a carbox lic acid).
For the unchanged Dop2~D-Lys(Dop2)-cyclo[Cys~Tyr-D-Trp-Lys-Abu-Cys]- Thr-M¾ and metabolites M4 and M6, it was not possible to obtain foil sequence coverage by MS/MS due to the presence of the disulphide bond, although the MS MS spectra showed common ions coming from the dopaminergic moieties (m/z 265,2 and 341).
The results from control S9 incubations suggest that Dop2-D-Lys(Dop2)- cyclo[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr-NH2 would be mainly metabolised by peptidases through hydrolysis of the C-terminal amide and the Thr residue, but preserving the disulphide-bond structure in the remaining moiety (M4 and M6).
Test Incubates (with NADPH")
The analysis of test monkey and human S9 incubates (incubated in the presence of NADPH cofacior) showed the formation of 5 metabolites (Ml, M2, M3, M5 and M7), in addition to unchanged Dop2-D~Lys(Dop2)-cyclo[Cys-Tyr-D-Trp- Lys-Abu-Cys]-Thr-NH2 and metabolites M4 and M6,
Metabolite M2: Doubly charged monoisotopic molecular ion at m z 704,5 indicated a molecular mass of 1407. According to these data, metabolite M2 shows a loss of 286 mass units with respect to Dop2-D-Lys(Dop2)-cyclo[Cys- Tyr-D-Trp-Lys-Abu-Cysj-Thr-N¾5 which matched up with a linear molecule resulting after the reduction of the disulphide bridge, and the loss of the three C-terminal ammo-acids: Abu, Cys and Thr-amide,
Metabolite M 3; Doubly charged mOnoisotopic molecular ion at rn z 747,0 indicated a molecular mass of 1492. According to these -data, metabolite M3 shows a loss of 20.1 mass units with respect to D6p2-D~Lys(Dop2)~cyclG[Cys- Tyr-D.-T^-Lys-Abii-Cys]-Thr-NH2S which matched u with a linear molecule resulting after the reduction of the disuiphide bridge, and the loss of the two: C- terminal amino acids: Cys and Thr-amide.
Metabolite M5: Doubly charged monpisptopic- molecular ion at m/z 798.5 indicated a ttiolecHlar mass of 1595. According to these data, metabolite M5 shows a loss of 98 mass units with respect to Dop2-D-Lys(Dop2)-cyclo[Cys- Tyr-D-Trp-Lys-Abu-Cysj-Thr-i Hz, which matched, up with a linear molecule resulting after the reduction of the disulphide bridge, and the loss of the C- tefrmnal amino acid Thr-amide.
Metabolite M7: Metabolite formed only in monkey kidney S9 fractions with singly charged monoisotopic molecular ion at m z 930,5 indicated a molecular mass of 929.5. According to these data, metabolite M7 shows a loss of 764 mass units with respect to Dop2-D-Lys(DDp2)-cyclo[:Cys-'Tyr-D-Trp-Lys- Abii-Cys3-Thr-NH2j which matched tip with a linear molecule resulting after the reduction of the disulphide bridge, and the loss of the six C-termifial amino acids: Tyr, D-T'rp, Lys, Abu, Cys and Thr-amide.-
Metabolite Ml : Unidentified metabolite formed only by huma liver S9 fractions with doubly charged average molecular ion at m/z 949 indicated a molecular mass around 18.96, According to these data, metabolite Ml shows, an increase of around 203 mass units with respect to Dop2-D-Lys(Dop2)- cycio[Cys-Tyr-D-Trp-Lys-Abu-Cys]-Thr~NH2.
The corresponding EPI spectra of M2, M35 MS, M7 and Ml were obtained. For MS, ίϊ-series up to S ion (Tyr Lys) were observed in the MS/MS spectrum, together wit the remaining common ions coming from the dopaminergic moieties. In the case of M3. an intense doubly charged M ion corresponding to the amide bond cleavage between Lys and Aib residues Was assigned, ^-series ions at m/z 781.5, 1047.5 and 1:233.5 were also observed 'Full sequence coverage based on the a-series was observed for M2 (m/z 781.5, 1047.5 and 1233,5). For M7, the cleavage of the amide bond between Lys and Cys was observed, at m/z 809.6. The presence of ions corresponding to Dop2 and to the fragmentation of P-Lys-(Dop2)2 was also observed although, showing low intensity.
The results from test 8 incubations suggest that extensive proteolytic hydrolysis of op2-D-Lys(Dop2)-eycto[Cys-Tyx-D-Trp-Lys-Abu^ys]-Tlir-NH2 is produced in vitro after reduction of the disulphide-bond, a process that would be mediated by a NADPH-dependent enzymatic reaction (test incubations with S9 fractions were performed in the presence ofNADPH cofac'tor).
Table 5: IDA Experiments in Incubates with S9 Fractions. Molecular Mass of Metabolites and Proposed Modification in Doo2T.)-i,vsfDoD2 -cvcloiCvs-
* Amino acid and N¾ loses mean that the peptide is in the carhoxylie acid form,
* See FIG. 1 showing the proposed modifications for Dop2-D-Lys(Dop¾-cyclo{Cys- Tyr"D~Trp-Lys--Abu--Cys]--Thr-i<iH2 metabolites.
(M+H1 : molecular ion coiTespoiiding to the singly charged species
(M + 2H*)>2: molecular ion -corresponding' to the d biy charged species
The ER and EPl spectra of D-Lys-(Dop2)2 and Dop2-OH reference standards were obtained. These compounds were analysed using the HPLC Method A,
D~I.,.ys~(Dop2)2 showed a protonated molecular ion at m z 827,6 and the doubly charged species at /z 414.3. The fragmentation pattern showed characteristic ions at m/z 561,4. 529.4 and 208.1.
Dop2-OH showed a protonated molecular ion at m/z 359.28. Its fragmentation pattern showed characteristic ions at m z 208.1, 193.1 and 154.1.
* rec rsor-^
Lys AbH-Cysi"Thf"Ni¾ Metabolites In Ine bates with S9 r etiOTis;
Selec tion of Fragments for MRM Analysis
Precursor-Ion experiments were performed in samples corresponding to incubations of Dop2-D~Lys(Dop2)-cyelo[Cys-Tyr-D"Trp-LyS"Abu-Cys]-Tbr- H2 with S9 fractions from monkey kidney and human liver, In order to detect common fragments to be selected in further MRM analyses, ion at m z of 159 was selected as it provides a good signal to noise ratio for Dop2-D-Lys(Dop2)-eyclo[Cys-Tyr-D-Tqi- Lys-Abu-Cys]-Tk-Ni¾. Product Ion Spectrum in the range 50-500 arnus of the doubly charged parent ion at a collision energy of 50 eV was obtained. In the Product Ion Spectra .corresponding to the different metabolites, the intensity of these ions may vary depending on the col lision energy or type of analyzer used.
Incubates of Dop2-D-Lys(Dop2)-cyclo CyS"Tyr~D-T3^~Lys-Abii~Cys]-Thr- N¾ with S9 fractions were analyzed in IDA mode in the mass spectrometer using -the precursor-ion scan as survey scan. Ions meeting the IDA criteria underwent ER and EM scans.
The results of this set of analyses confirmed the presence of the common fragment ion at m z 159,0 for unchanged DQp2-D-Lys(Dop2)-cyclo[CyS"Tyr-D^Trp- Lys-Abu-Cys]-Thr- H2 and metabolitss Ml, M2, M3, M4, MS, M6 and M7. For * •DQf£-D-Lys(Dop2)~cyc!p[Cys^ and M6, the triply charged species were detected in the ER scan (m z 565.4 for Dop2-D-Lys(Dop2}- c eiofC s-I^^ r -Lys-A u-G sj-Tlir-NHz and m z 565,8 for M6,
.Samples
In vitro incubates of Dop2-P~Lys(Dop2 cyclo[Cys-Tyr-D-Trp-Lys-Abu- Cys]-Thr-N¾ with human liver S9 fraction, and plasma samples from dosed rats -and monkeys were analysed by LC-MS/ S in MRM mode monitoring the transitions described in Table 4.
Incubates of Dop2 LysfDop2')°cycio C^
Human Liver S9 Fractions
MRM profile corresponding to an incubate of Dop2-D~Lys(Dop2)~cyc3o[Gys-
Tyr-D-Trp-Lys-Ab -Cysj-Thr-NHa with human liver S9 fractions, in the presence of NADPH, was obtained. The sample was analysed using the HPLC Method A.
The results of these analyses confirmed that the MRM method was suitable to detect the peptide-like in vitro human metabolites (metabolites Ml to M6).
Surprisingly, the presence of the compounds D«Lys-(Dpp2)2 and Dop2-OFI was also detected in these samples, indicating that I)-Lys~(Dop2)? and Dop2-OH are effectivel formed in vitro by human liver S9 fractions as a resuit'of extensive metabolism of Dop2-D-Lys(Dop2)-cyclo[Gys-Tyr-D»Trp-LyS"Abu-Cys]-Thr-Ni:i2,
The analysis of rat plasma samples showed the presence of a circulating unknown compound that appeared, in the RM profiles in addition to Dop2-D- Lys(Dop2)-cyeio[Cys~Tyr-D-Tip-Lys-Abu--Cys]~Tlir--NH2. This compound showed a LC-MS/MS response comparable to unchanged Dop2-D-Lys(Dop2)-cyclo[Cys~Tyr- D-Tip-LyS"Abu--Cys]-Tlir-NH at blood sampling times > 8 hours, appearing also at earlier sampling times, suggesting that it could account for a considerable fraction of the exposure to the drug-related material.
In order to identify this compound, rat plasma samples following a
subcutaneous administration at the dose level of 12.5 mg/kg were pooled and analyzed by employing HPLC Method B (24 hour plasma samples, MRM transition 565.3 / 129.0), and also using the IDA method by employing HPLC Method C (8 hour
plasma samples, MRM transition 565.3 / 129.0). For Dop2-D~L,ys(DGp2)-Gyclo[Cys~ Tyr-D-Trp-Lys-Abtt-Cys]-Thr-MH¾ the characteristic isotopic profile of the triply charged species was obtained (molecular ion at rn/z 565.3), while a triply charged ion of m/z 565,6 (mass increment of 0,3) was detected for the other peak which corresponded to a molecular mass of 694, According to these data, this compound would show an increase of 1 mass unit with respect to Dop2-P-Lys(Dop2)-cyclo[Cys- Ty»'l Trp-Lys-Ahu-Cys]-T¾r- H2, which matched up with the proposed
modification described for metabolite M6,
To confirm this hypothesis, a sample corresponding to ! "C-!abcllcd Dop2- -
Lys(Eiop2)-cyclo C3 s-Tyr-D-Trp-Lys-Abu-Cysj»Thr-NH2 incubated with rat kidney S9 traction in the absence of NADPH was analyzed using the same conditions as for the 8 hour rat plasma sample. The ER spectra for "C-labeiled Dop2~D~Lys(Dop2)~ eyclo[Cys~Tyr-D~Trp-Lys-Abu-Cysj~Thr-NH2 showed the two characteristic species: unlabeled ,]¾ 2TD-Lys(D0 2)^ (triply charged monoisotopic molecular ion at m z 565.3), and i C-labeHed Dop2-B- Lys(Dop2)-cydo[Cys-TyTd (triply charged
monoisotopic molecular ion atm/z 571,0), which correspond to an average shift of 16 mass units in Dop2-D-Lys(Dop2)-cyclo[Cys-Tyr-D-Trp-Lys-Ahu-Cys3-T¾r-NH2 due to the content of C- 14 atoms, in agreement, M6 ER pattern showed also the isotopic profiles corresponding to the two molecule counterparts: i.e. unlabelled M6 (triply charged species at rn/z 565,7) and 14C-M6 (triply charged species at m/z 571,0-),
Monkey Plasma Samples; Detection of Metabolites M3, D-Lys-(.Dop2)? and Bop2~ OH.
Monkey plasma samples corresponding to a daily subcutaneous administration at the dose level of 45 nig/kg, were pooled (plasma from 4 animals at each blood sampling time) and analyzed by MRM following HPLC Method B according to the. transitions listed in Table 4. MRM chromatograms were obtained for the following samples:, .pre-dose, 1.5 hours, 5 hours and 24 hours. Unchanged Dop2-D-Lys(Dop2)- cyclo[Cys«Tyr l p-Lys-Abu-Cysj-Tlu-NH2 was the main component in all samples except for pre-dose sample. Three additional metabolites could be detected corresponding to D-Lys~(Dop2)2} M3 and Dop2~OH; the latter in very low amoimts (traces) and only i the 1 ,5 hour sample. Metabolite D~Lys-(Dop2j2 was also detectable in plasma samples after a 3-day wash-out period between subsequent administrations. Metabolites Ml, M2. M4. M5 and M6 were not detected in. the analysed ro.on.key plasma samples,
Semi-Quantitative Approach to DQp2-D-Lys(Dop2)-eycl.o[Cys-Tyr-D-T p LysrA Cys j-Thr-M¾ Monkey Plasma. Metabolites
A semi-quantitative evaluation of Dop2, D-Lys-(Dop2)2 and unchanged Dop2- D-Lys(Dop2)-cyGlo[CyS"Tyr~D-Trp-Lys-Abu*Cys]-Thr-NH2 was performed by measuring the LC-MS/MS response (MRM mode) in a sample containing the same amount of the three standards (1 μ/ml). Dop2, D-Ly;s-(Dop2)2 response in terms of peak height was found to be approximately 20 fold higher than Dop2-D-Lys(Dop2)» vyclo[Gys-Tyr-D-Trp-LyS"Abu^Cys]-Tlii-NH2 using the transitions described in Table 4, which were the same used in monkey plasma analyses.
Membranes were prepared by homogenizing cells expressing the human recombinant dopamine-2 receptor in 20 ml of ice-cold 50 mM Tris-HCl with a 'Brinkman Polytron (setting 6, 15 seconds). Buffer was added to obtain a final volume of 40 ml, and the homogenate was centrifuged in a Sorval SS-34 rotor at 39,000 g for 10 minutes at 0~4°C, The resulting supernatant was decanted -and -discarded. The pellet was rehotrsogerhzed m iee-cold buffer, pre-iiieiibated afc 37°C for 10 minutes, diluted, and centrifuged as before. The -'final, pellet was resuspended in buffer and held on ice for the receptor binding assay ,
For assay, aliquots of the washed membrane preparations, and test compounds were incubated for 15 minutes (37°C) with 0.25 nM [¾]spiperone (16.5 CjVrnmol, New England Nuclear, Boston, MA, USA) in 50 raM Tris HQ,. 20 mM NaCl, 5 raM C1, 2.rnM C&CI2, 1 mM MgClj. The final assay 'volume was 1,0 ml, The incubations wer terminated by rapid filtration through GF B filters using a Brandel -filtration -manifold. Each tube and filter were then washed three times with 5 -ml aliquots of ice-cold 'buffer. Specific binding was defined as the total radioligand bound minus that bound in the presence of 1000 nM (+) butaelamoL
* 5jS,T¾B R djg¾gand Binding Assay
Membranes were prepared' by homogenizing cells expressing the human recombinant 5-hydroxy-tryptamine 2B (h5~HT2B) receptor in 20 ml of ice-cold 50 mM Tris-HCl with a Brinkman Polytron (setting 6, 15 seconds). Buffer was added to obtain a final volume of 40 nil, and the homogenate was centrifuged in .a Sorval SS-34 rotor at 39,000 g for 10 min at 0-4-°C. The resulting supernatant was decanted and discarded. The pellet was rehomogemzed in ice-cold buffer, pre-irtcubated at 37°C for 10 minutes, diluted, and centrifuged as before. The final pellet was resuspended in buffer and held on ice for the receptor binding assay.
For assay, aliquots of the washed membrane preparations and test compounds were incubated for 30 mm (37°'C) with 1.5 riM [3H]LSD (63.3 Ci/rnmaL Perkitt- Blmer, Boston, MA, USA) in 50 mM Tris-HCl, containing 2.5 mM Mgt¾, and 0.1% BSA. The final assay volume was 1.0 ml. The incubations were terminated by rapid filtration through GF/C filters (pre-soaked in 0.5% ΡΕΪ/0.!% BSA) using a Brandel filtration manifold. Each tube and filter were then washed three- times with 5-ml aliquots of ice-cold buffer. Specific binding was defined as -the: total radioligand bound minus that bound in the presence of 1000 nM cinanserin.
The resulting in vitro binding data are shown in Table 6.
Claims
What is claimed is:
1. A compound having the formula:
D-Lys-(Dcfp2)2;
Dop2-OH;
D"Lys-(Dop2)2-Cys;
D-Lys~(Dop2)?-cyclc Cys-Tyr-D-Trp-Lys~A u-Cys]--Thr;
D-Lys*(Dop2)2-cyclo[Cys-Tyr-D~Trp-Lys-Abtt-Cys3.;
D-Lys-(Dop2)2 ^s-Tyr-I>-Trp-Lys-Abu-Cys;
D .,y3-(DGp2] Cys-Tyr-D-T^J-Lys-Abu; or
D- Lys-(Dop2) j-Cy s~Tyr-D-Tr p- Lys ;
or a pharmaceutically acceptable salt thereof,
2. A pharmaceutical composition comprising the compound according claim ί and a pharmaceutically acceptable carrier, diluent or excipiest.
1, substantially pure compound having the formula:
D~Lys-(Dop2)2;
Dop2-OH;
.D-Lys-(Dop2)2~Gys;
D^Ly8-(Dop2)2rcyc[ofC ¾-TyT-D-Trp-Ly8-Ab¾-Cys]-Tlir;
D-Lys-{D0p2)2-cyclo[Cys-Tyr- -Tro-Lys-Abu-Cys];
B-Lysv03op2)2-Cy.s-TyrrD-Trp-Lys-Abu-Cys;
D-Lys-(D0p2)2-Gy8-Tyr-B-Trp-Lys-Abu; or
D-I,ys-(Dop2)2-Cys-Tyr-D-Tip-Lys;
or a pharmaceutically acceptable salt thereof.
4. A. pharmaceutical composition comprising the substantially pure compound according to claim 3 and a pharmaceutically acceptable carrier, diluent or excipient
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8952128B2 (en) | 2012-11-01 | 2015-02-10 | Ipsen Pharma S.A.S. | Somatostatin-dopamine chimeric analogs |
| US9777039B2 (en) | 2012-11-01 | 2017-10-03 | Ipsen Pharma S.A.S. | Somatostatin analogs and dimers thereof |
| GB2590341A (en) * | 2019-10-04 | 2021-06-23 | Tiburio Therapeutics Inc | Storage stable somatostatin-dopamine chimeric compounds and salt forms thereof |
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|---|---|---|---|---|
| WO2002100888A1 (en) * | 2001-06-08 | 2002-12-19 | Societe De Conseils De Recherches Et D'applications Scientifiques S.A.S. | Somatostatin-dopamine chimeric analogs |
| WO2004091490A2 (en) | 2003-04-11 | 2004-10-28 | Societe De Conseils De Recherches Et D'applications Scientifiques S.A.S. | Somatostatin-dopamine chimeric analogs |
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| WO2002100888A1 (en) * | 2001-06-08 | 2002-12-19 | Societe De Conseils De Recherches Et D'applications Scientifiques S.A.S. | Somatostatin-dopamine chimeric analogs |
| WO2004091490A2 (en) | 2003-04-11 | 2004-10-28 | Societe De Conseils De Recherches Et D'applications Scientifiques S.A.S. | Somatostatin-dopamine chimeric analogs |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US8952128B2 (en) | 2012-11-01 | 2015-02-10 | Ipsen Pharma S.A.S. | Somatostatin-dopamine chimeric analogs |
| US9603942B2 (en) | 2012-11-01 | 2017-03-28 | Ipsen Pharma S.A.S. | Somatostatin-dopamine chimeric analogs |
| US9731027B2 (en) | 2012-11-01 | 2017-08-15 | Ipsen Pharma S.A.S. | Somatostatin-dopamine chimeric analogs |
| US9777039B2 (en) | 2012-11-01 | 2017-10-03 | Ipsen Pharma S.A.S. | Somatostatin analogs and dimers thereof |
| GB2590341A (en) * | 2019-10-04 | 2021-06-23 | Tiburio Therapeutics Inc | Storage stable somatostatin-dopamine chimeric compounds and salt forms thereof |
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