Pharmacy

Incorporation of L-or D-Tic into position 7 of oxytocin (OT) and its deamino analogue ([Mpa 1 ]OT) resulted in four analogues, [L-Tic 7 ]OT (1), [D-Tic 7 ]OT (2), [Mpa 1 ,L-Tic 7 ]OT (3) and [Mpa 1 ,D-Tic 7 ]OT . Their biological properties were described by Fragiadaki et al. (Eur J Med Chem 42:799-806, 2007). Their NMR study (NO-ESY, TOCSY, 1 H-13 C HSQC spectra) is presented here. Analogues 1, 3 and 4 showed partial agonistic activity, analogue 2 was pure antagonist, suggesting that a cis conformation between residues 6 and 7 of the molecule does not result in antagonistic activity. However, the reduction in agonistic activity of analogues 1, 3 and 4 in comparison to oxytocin is consistent with the reduction of the trans conformation form. Binding affinity for the human oxytocin receptor with IC 50 value of 130, 730, 103, and 380 nM for peptides 1, 2, 3, and 4, respectively, showed lower affinity in the case of D analogues. Deamination slightly increased the affinity. The existence of both cis and trans configurations of the Cys 6 -D-Tic 7 bond is supported by observation of two sets of cross-peaks for 1 H and 13 C nuclei for most of the residues of the peptide not only in NOESY and TOCSY but also in 1 H-13 C HSQC spectra. The MS and HPLC indicate the presence of a single molecule/peptide, and NMR data thus suggest that this second set of peaks is due to the cis conformation.

We report the solid-phase synthesis and some pharmacological properties of twenty oxytocin (OT) analogues. Basic modifications at position 7 (introduction of a-aminoisobutyric acid [Aib], L-or D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid [L/D-Tic], L-a-t-butylglycine [Gly(Bu t )] and pipecolic acid [Pip]) were combined with D-Tyr(Et) 2 , L/D-( pEt)Phe 2 , D-Tic 2 , and Mpa 1 modifications and their various combinations in a total of 14 analogues. Additionally, two analogues having one more modification in position 3, i.e. Gly(Bu t ), and three analogues having glycine in position 9 substituted by D-Tic or Aib, were prepared. The analogues were tested for rat uterotonic activity in vitro, in the rat pressor assay and for binding affinity to human OT receptor. The analogue having the highest antioxytocic activity was [Mpa 1 , D-Tyr(Et) 2 , D-Tic 7 , Aib 9 ]OT having pA 2 ¼ 8.31 AE 0.19; this analogue was also selective.

We report the solid phase synthesis and some pharmacological properties of seventeen new oxytocin (OT) analogues. Basic modification at positions 8 and/or 9 (introduction of l-␣-t-butylglycine [Gly(Bu t )]) was combined with d-Cys 6 , d-Tyr(Et) 2 , Mpa 1 or Pen 1 modifications and their various combinations. We also present properties of two previously reported re-synthesized analogues ([Gly(Bu t ) 8 ]OT and [Mpa 1 , Gly(Bu t ) 8 ]OT). The analogues were tested for rat uterotonic activity in vitro, in the rat pressor assay and for binding affinity to human OTR.

Enhanced iodide ingestion is known to accelerate the incidence and severity of spontaneous autoimmune thyroiditis [iodide-accelerated spontaneous autoimmune thyroiditis (ISAT)] in NOD.H2(h4) mice. CD4+ cells are required for the development and maintenance of ISAT, but their target epitopes remain unknown. In this study, we show that the previously identified thyroglobulin (Tg) T cell epitope p2549-2560 containing thyroxine at position 2553 (T4p2553) induces thyroiditis as well as strong specific T and B cell responses in NOD.H2(h4) mice. In ISAT, activated CD4+ T cells specific for T4p2553 are detected before the disease onset in thyroid-draining cervical lymph nodes only in mice placed on an iodide-rich diet and not in age-matched controls. In addition, selective enrichment of CD4+ IFN-γ+ T4p2553-specific cells is observed among cervical lymph node cells and intrathyroidal lymphocytes. T4p2553 was equally detectable on dendritic cells obtained ex vivo from cervical lymph node cell...

Polymer-bound N-tritylhydrazines 4 were easily prepared by reacting polymeric tritylchlorides 3 with hydrazine. Subsequently, compounds 4 have been successfully applied to the solid phase synthesis of partially protected peptide hydrazides using 1-hydroxybenzotriazolyl esters of Fmoc-or Trt-amino acids. The synthesized peptide hydrazides can be quantitatively split off from the resins by mild acidic treatment, while the benzyl-and tert-butyl protecting groups remain unaffected.

Aib3,ThrS]OT, [Aib3,Thr(OMe)5]OT, [Aib3,OrnS]OT, [Thr(OMe)S,OrnS]OT and [Phe2,Thr(OMe)S,OrnS]OT were synthesized by solid-phase techniques. From the biological properties of these peptides, it seems that the simultaneous replacement of positions 3 and 5 of oxytocin with Aib and Thr(OMe) results in an analogue devoid of antagonistic activity in comparison with the singly substituted compounds. Simultaneous Orn s substitution does the same in the case of the Aib 3 analogue and even leads to agonistic activity in the case of the Thr(OMe) 5 analogue. Replacement of Tyr 2 by Phe 2, e.g. [Phe2,Thr(OMe)S,OrnS]OT, again favors the appearance of minor antagonistic potency.

It is becoming increasingly apparent that the coordination chemistry of oligopeptides with Cys-XY-Cys and His-XY-Cys sequences (X, Y= variable amino acid residues) is an important theme in transition metal and bioinorganic chemistry (1, 2). The ...

Thyroid hormone-binding (THB) Abs are frequently detected in autoimmune thyroid disorders but it is unknown whether they can exert immunoregulatory effects. We report that a THB mAb recognizing the 5 iodine atom of the outer phenolic ring of thyroxine (T4) can block T cell recognition of the pathogenic thyroglobulin (Tg) peptide (2549-2560) that contains T4 at aa position 2553 (T4(2553)). Following peptide binding to the MHC groove, the THB mAb inhibited activation of the A k-restricted, T4(2553)-specific, mouse T cell hybridoma clone 3.47, which does not recognize other T4-containing epitopes or noniodinated peptide analogues. Addition of the same THB mAb to T4(2553)-pulsed splenocytes largely inhibited specific activation of T4(2553)primed lymph node cells and significantly reduced their capacity to adoptively transfer thyroiditis to naive CBA/J mice. These data demonstrate that some THB Abs can block recognition of iodine-containing Tg epitopes by autoaggressive T cells and support the view that such Abs may influence the development or maintenance of thyroid disease.

Tg was purified from thyroid glands of female CD-1 mice (Charles River, Quebec, QC, Canada) after centrifugation of thyroid extract at 16,000 3 g, followed by passage through a Sepharose CL-4B column (Amersham Biosciences, Uppsala, Sweden), as previously described (10). F-moc L-thyroxine was produced as previously described (18, 19). The 12-mer Tg peptide T4p2553 (aa 2549-2560), STDD(T4)ASFSRAL, was synthesized at the University of Patras, Greece, and at Biosynthesis (Lewisville, TX) at .90% purity. The 19-mer OVA peptide (322-340), CISQAVHAAHAEI-NEAGRY, was synthesized at the Alberta Peptide Institute (Edmonton, AB, Canada) at .80% purity. Both peptides were blocked with an acetyl group at the N terminus and with an amide group at the C terminus. OVA was purchased from Sigma. CFA (with Mycobacterium butyricum) and IFA were purchased from Difco (Detroit, MI). Culture media and lymphocyte proliferation assays Inguinal, brachial, and axillary LNCs from Ag-primed mice were cultured in DMEM supplemented with 10% FCS (PAA Laboratories, Etobicoke, ON, Canada). In ISAT, thyroid-draining cLNCs or spleen cells were cultured in HL-1 medium (Lonza, Walkersville, MD). Media were supplemented with 100 U/ml penicillin/streptomycin, 2 mM glutamine (Life Technologies, Invitrogen, Grand Island, NY), and 5 3 10 25 M 2-ME (Sigma-Aldrich, St. Louis, MO). RPMI 1640 (Life Technologies, Invitrogen) with 10% FCS was used in activation assays of T cell hybridomas. Lymphocyte proliferation assays were performed by culturing 4 3 10 5 cells per well with Ag in 96-well flatbottom plates for 3-4 d at 37˚C. During the last 18 h, 1 mCi [ 3 H]thymidine (PerkinElmer, Boston, MA) was added to each well. Cells were harvested in a Classic Cell Harvester (Skatron Instruments), and radioactivity was measured in a LS6500 MultiPurpose Scintillation Counter. Stimulation index (S.I.) was defined as cpm in the presence of Ag/cpm in the absence of Ag. Generation and characterization of the KC1 hybridoma clone LNCs from T4p2553-primed NOD.H2 h4 mice were activated in vitro with 20 mM T4p2553 for 4 d and were subsequently fused with BW5147 a 2 b 2 cells (20), kindly provided by P. Marrack (National Jewish Medical and Research Center, Denver, CO), using polyethylene glycol 1500 (Roche Diagnostics, Mannheim, Germany). Screening for IL-2-secreting, peptidespecific hybrids was done using TA3 cells as APCs and the IL-2-dependent CTLL line, as previously described (21). Cloning was performed by limiting dilution at 0.3 cell per well. The IgG2a mAbs specific for A k (TIB 92) or influenza A nucleoprotein (NP) (HB 65) (American Type Culture Collection, Manassas, VA) were purified from culture supernatants, as previously described (22). Percent inhibition of KC1 activation by blocking mAbs was calculated as follows: [1 2 (cpm in the presence of mAb)/(cpm in the absence of mAb)] 3 100.