Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.

Ht31: the first protein kinase A anchoring protein to integrate protein kinase A and Rho signaling.

In an attempt to isolate protein kinase A anchoring proteins (AKAPs) involved in vasopressin-mediated water reabsorbtion, the complete sequence of the human AKAP Ht31 was determined and a partial cDNA of its rat orthologue (Rt31) was cloned. The Ht31 cDNA includes the estrogen receptor cofactor Brx and the RhoA GDP/GTP exchange factor proto-lymphoid blast crisis (Lbc) sequences. The Ht31 gene was assigned to chromosome 15 (region q24-q25). It encodes Ht31 and the smaller splice variants Brx and proto-Lbc. A protein of the predicted size of Ht31 (309 kDa) was detected in human mammary carcinoma and HeLa cells. Anti-Ht31/Rt31 antibodies immunoprecipitated RhoA from primary cultured rat renal inner medullary collecting duct cells, indicating an interaction between the AKAP and RhoA in vivo. These results suggest that Ht31/Rt31 represent a new type of AKAP, containing both an anchoring and a catalytic domain, which appears to be capable of modulating the activity of an interacting partner. Ht31/Rt31 have the potential to integrate Rho and protein kinase A signaling pathways, and thus, are prime candidates to regulate vasopressin-mediated water reabsorbtion.

Cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically.

Evidence that multiple, probably non-endocytic mechanisms are involved in the uptake into mammalian cells of the alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) is presented. Extensive cellular uptake of N-terminally GC-elongated derivatives of I, conjugated by disufide bridges to differently charged peptides, indicated that I-like model peptides might serve as vectors for intracellular delivery of polar bioactive compounds. The mode of the cellular internalization of I comprising energy-, temperature-, pH- and ion-dependent as well as -independent processes suggests analogy to that displayed by small unstructured peptides reported previously (Oehlke et al., Biochim. Biophys. Acta 1330 (1997) 50-60). The uptake behavior of I also showed analogy to that of several protein-derived helical peptide sequences, recently found to be capable of efficiently carrying tagged oligonucleotides and peptides directly into the cytosol of mammalian cells (Derossi et al., J. Biol. Chem. 269 (1994) 10444-10450; Lin et al., J. Biol. Chem. 270 (1995) 14255-14258; Fawell et al., Proc. Natl. Acad. Sci. USA 91 (1994) 664-668; Chaloin et al., Biochemistry 36 (1997) 11179-11187; Vives et al., J. Biol. Chem., 272 (1997) 16010-16017).

Spin trapping using 2,2-dimethyl-2H-imidazole-1-oxides.

The ability of novel cyclic nitrones, 4-substituted 2,2-dimethyl-2H-imidazole-1-oxides (IMO's) to trap a variety of short-lived free radicals has been investigated using ESR spectroscopy. IMO's scavenge oxygen-, carbon- and sulfur-derived free radicals to give persistent nitroxides. Compared to the spin trap 5,5-dimethyl-pyrroline-1-oxide, a higher lifetime of hydroxyl radical adducts and a higher selectivity related to the trapping of carbon-centered radicals was found. A reaction between IMO's and superoxide was not observed. ESR parameters of 4-carboxyl-2,2-dimethyl-2H-imidazole-1-oxide (CIMO) spin adducts are highly sensitive to the structure of the trapped radical, e.g., different spectra were detected with radicals derived from Na2SO3 and NaHSO3. From the data obtained, a successful application of these new spin traps in biological systems can be expected.

[Potential cardiotonics. 14. Synthesis and in vitro positive inotropic actions of 4-morpholino-5-(4-pyridinyl)pyridine derivatives]. / Potentielle Kardiotonika. 14. Mitteilung: Synthese und positiv inotrope Wirkungen in vitro von 2-Morpholino-5-(pyrid-4-yl)pyridin-Derivaten.

By the reaction of 2-chloro-5-(4-pyridinyl)pyridines 1-6 with morpholine as well as by derivation of the 2-morpholino-pyridine-3-carboxamide 8 the 3-substituted 2-morpholino-5-(4-pyridinyl)pyridines 7-14 were prepared. The evaluation for positive inotropic properties in spontaneously beating isolated guinea pig atria gave for the 3-cyano derivative 7 (AWD 122-14) the best activity. The potency is comparable to that of milrinone and is due to partial by inhibition of phosphodiesterase III (PDE III).

[Potential cardiotonic agents. 8. 2-acyloxyalkylamino-, 2-acyloxyalkoxy-, and 2-acylaminoalkylamino-3-cyan-5-(pyrid-4-yl) pyridine]. / Potentielle Kardiotonika. 8. Mitteilung: 2-Acyloxyalkylamino-, 2-Acyloxyalkoxy- und 2-Acylaminoalkylamino-3-cyan-5-(pyrid-4-yl) pyridine.

By acylation of our previously described cardiotonic active 2-hydroxyalkylamino, 2-hydroxyalkoxy, 2-aminoalkyl-amino and 2-piperazino substituted 3-cyano-5-(4-pyridinyl) pyridines with acetic anhydride, propionic anhydride or aroyl and heteroaroyl chlorides, respectively, the corresponding in position 2 O- or N-acylated 3-cyano-5-(4-pyridinyl)pyridines were prepared. Cardiovascular activity of the obtained derivatives is discussed in comparison with that of the parent compounds.

[Potential cardiotonics. 7. Synthesis and cardiovascular effects of 5-(4-pyridinyl)-, 6-methyl-4-(4-pyridinyl) and 6-methyl-5-phenyl-substituted 3-cyano-2-aminoalkylamino-pyridines]. / Potentielle Kardiotonika. 7. Mitteilung5: Darstellung und kardiovaskuläre Eigenschaften von 5-(pyrid-4-yl)-, 6-methyl-5-(pyrid-4-yl)- und 6-methyl-5-phenyl-substituierten 2-Aminoalkylamino-3-cyan-pyridinen.

The title compounds were synthesized by treating of 2-chloro-pyridines 1-3 with the appropriate aminoalkylamines or piperazines. In isolated guinea pig atria some compounds showed greater positive inotropic activity than amrinone. Heart rate was decreased or remained unchanged. In anesthetized dogs some derivatives exerted a dose-dependent increase in myocardial contractility and, additionally, a decrease in blood pressure.

[Potential cardiotonic agents. 6. Synthesis and cardiovascular properties of 5-(4-pyridinyl)-, 6-methyl-5-(4-pyridinyl)- and 6-methyl-5-phenyl-substituted 3-cyano-2-oxaalkyoxy-pyridines]. / Potentielle Kardiotonika. 6. Mitteilung: Darstellung und kardiovaskuläre Eigenschaften von 5-(pyrid-4-yl)-, 6-methyl-5-(pyrid-4-yl)- und 6-methyl-5-phenylsubstituierten 3-Cyan-2-oxaalkoxy-pyridinen.

The headline compounds were prepared by the reaction of 2-chloropyridines 1-3 with the appropriate alcohols in presence of potassium hydroxide and the sodium alkoxides, respectively. Especially some of the 3-cyano-2-hydroxyalkoxy-5-(4-pyridinyl)pyridines showed remarkable positive inotropic potency and, additionally, a vasodilator activity. In spontaneously beating isolated guinea pig atria they had a greater activity than amrinone.

[Potential cardiotonic agents. 2. Synthesis and pharmacologic properties of 5-(pyri-4-yl)- and 5-phenyl substituted 3-cyano-6-methyl-2-oxaalkylaminopyridines]. / Potentielle Kardiotonika. 2. Mitteilung: Synthese und pharmakologische Eigenschaften von 5-(pyrid-4-yl)- und 5-phenylsubstituierten 3-Cyan-6-methyl-2-oxaalkylamino-pyridinen.

The authors describe the preparation, the physicochemical properties and the results of evaluation of positive inotropic and vasodilator activities in a series of 5-(4-pyridinyl)- and 5-phenyl-substituted 3-cyano-6-methyl-2-oxaalkylamino-pyridines. Some of the compounds are comparable in their positive inotropic potency to that of amrinone and cause, additionally, a decrease in blood pressure.

[Crystal modifications of 3-cyan-6-methyl-5-(pyrid-4-yl)-1,2-dihydropyrid-2-one (milrinone)]. / Kristallmodifikationen von 3-Cyan-6-methyl-5-(pyrid-4-yl)-1,2-dihydropyrid-2-on (Milrinon).

The preparation and characterisation of crystalline alpha-, beta- and gamma modifications of 3-cyan-6-methyl-5-(4-pyrid-4-yl)-1,2-dihydropyrid-2-on e are described. The thermic transformation of alpha- and beta-modification into gamma-modification was proved by thermogravimetric analysis (t.g.a.), differential scanning calorimetry (d.s.c.), IR and powder diffraction pattern. In dissolution behaviour no significant differences were found between the modifications.

[X-ray structural analysis of 3 crystalline modifications of 3-cyan-5-methyl-5-(pyridinyl)-1,2-dihydro-pyrid-2-one (milrinone)]. / Röntgenstrukturanalytische Untersuchungen an drei kristallinen Modifikationen des 3-Cyan-6-methyl-5-(pyrid-4-yl)-1,2-dihydropyrid-2-on (Milrinon).

The crystal and molecular structures of the alpha-, beta-, and gamma-modification of the cardiotonic compound milrinone have been determined by X-ray structure analyses. In all modifications the molecules exist in the lactam form. Because of steric hindrances the pyridine rings deviate considerably from coplanarity. The dihedral angle amounts to 45 degrees in milrinone-alpha, 43.7 degrees in milrinone-beta, and 57.5 degrees in milrinone-gamma. In the three crystal structures molecules are connected via hydrogen bonds forming dimers. These are centrosymmetric in milrinone-alpha and -beta. In milrinone-gamma the molecules within a dimer are symmetry related by a twofold axis. The dimers are linked by charge transfer interactions. This leads to infinite chains in the alpha- and beta-modification and to infinite layers in milrinone-gamma.

On the blood-brain barrier to peptides: [3H]gonadotropin-releasing hormone accumulation by eighteen regions of the rat brain and by anterior pituitary.

After intracarotid injection of [3H]gonadotropin-releasing hormone ([3H]GnRH) the mean accumulation of radioactivity per unit wet weight of 18 investigated brain samples and the anterior pituitary was 0.38 +/- 0.11% g-1 of the injected tracer dose. This indicates a low but measurable brain uptake of the peptide. The brain uptake of [3H]GnRH in blood-brain barrier (BBB)-protected regions is 5% of that of separately investigated [3H]OH. In BBB-free regions the accumulation of radioactivity was more than 25-fold higher than in BBB-protected regions. The accumulation of [3H]GnRH among regions with BBB varies less than among regions with leaky endothelia. The data presented for [3H]GnRH are similar to those for other peptides so far investigated.

Direct tritium-labelling into histidine of luteinizing hormone-releasing hormone agonists and use of the tracers in studies on proteolytic breakdown.

Analogs of luteinizing hormone-releasing hormone (LHRH) having higher biological activity than LHRH itself are being mainly used to study the biological effects and the mechanism of action of LHRH. In the present study, conditions for the direct 3H-labelling at the histidine residue of analogs of LHRH were worked out, circumventing the synthesis of precursor peptides for labelling. [D-Phe6,desGly10]-LHRH ethylamide and [D-Ser(But)6,desGly10]-LHRH ethylamide were tritiated by tritium gas and a 10% Pd/Al2O3 catalyst to high specific radioactivities. The labelled peptides are sufficiently stable to be used in biochemical studies. The degradability of the analogs by homogenates of various tissues of rats was compared with that of the native LHRH. The analogs were shown to be distinctly degradable, but to a lower extent. The kidney homogenate degrades the analogs [D-Phe6,desGly10]- and [D-Ser(But)6,desGly10]-LHRH ethylamide with 35 and 50%, respectively, of the velocity observed with LHRH, whereas the degradation velocity of the analogs by a homogenate of the hypothalamus and pituitary is only 10% of that of LHRH. It is suggested that the lower degradability of the analogs at peripheral sites and target sites (pituitary, ovary) explains partly their higher biological activity.

Some observations indicating a low brain uptake of [3H]Nle11-Substance P.

The studies concerning the problem of whether an exogenous neuropeptide is able to enter the brain tissue were extended to the undecapeptide Substance P (SP). The amount of radioactivity 15 s after intracarotid injection of [3H] Nle11-SP or [14C] inulin was determined in 18 brain regions and the anterior pituitary of male rats. As compared to the reference [14C] inulin (mean +/- SD: 0.143 +/- 0.009% of the injected radioactivity per g tissue wet weight), the amount of radioactivity was higher after [3H]Nle11-SP injection (0.233 +/- 0.039%, p less than 0.001). Statistically significant differences could be found particularly in cortical and caudal areas as well as in the circumventricular organs studied. These observations do not refute the assumption that a low brain uptake of the labelled neuropeptide occurred due to an accumulation within structures of the blood-brain barrier and/or a penetration of the barrier system.

Protection of methionine in peptides during iodination by sulfonium salt formation.

A method for the prevention of methionine oxidation during iodination of tyrosine containing peptides is reported. The methionine containing peptide is converted into the corresponding S-tert.-butylsulfonium derivative, which is iodinated using iodine monochloride. After removal of the S-tert.-butyl group and purification, sulfoxide-free 3,5 diiodotyrosine (Dit) peptides were obtained. Dit8-substance P, Dit8-physalaemin 6-11 and Dit1, Met5-enkephalin were synthesized by this route. Tritium labeling of Dit1, Met5-enkephalin yielded 3H-enkephalin with a specific radioactivity of 38 Ci/mmol.

Tritium labeling of gonadotropin releasing hormone in its proline and histidine residues.

3,4-dehydroproline9-GnRH prepared by solid phase peptide synthesis was tritiated catalytically under various conditions yielding 3H-GnRH with specific radioactivities in the range from 35-60 Ci/mmol and full LH releasing activity in vitro. Using palladium/alumina catalyst, the tritiation of the double bond occurs within ten minutes. Investigation of the tritium distribution between the amino acid residues showed a remarkably high incorporation of tritium into the histidine residue (11 to 37%). On the basis of this observation, the tritium labeling of GnRH and angiotensin I by direct catalytic hydrogen-tritium exchange was found to be useful for the labeling of these peptides at remarkably high specific radioactivity.