Binding of substance P to monolayers and vesicles made of phosphatidylcholine and/or phosphatidylserine.

Analyses of interactions between substance P (SP) and phospholipids were performed by combined surface pressure and surface potential measurements in monolayers and by 13C-NMR experiments on liposomes. This study was carried out using synthetic SP molecules: [1-13C-Gly9]SP and [1-13C-Gly2]SP. Injection of SP into the aqueous subphase led to an expansion of phosphatidylcholine (PtdCho) or phosphatidylserine (PtdSer) monolayer surface area. An apparent association constant of SP for PtdSer was estimated to be around 10(6)-10(-7) M-1. The surface potential delta V/n varied linearly with the molecular area whereas the variation of surface pressure was biphasic, suggesting that at least two binding states contributed to the monolayer expansion. These two states Si (SP is inserted into the bilayer) and Ss (SP is stuck on the surface) were observed on vesicular membranes by 13C-NMR. The kinetic of interconversion between these two states can be estimated by NMR, the Ss state being the stablest one. No perpendicular insertion of SP into these vesicular preparations seemed to occur, as previously postulated. However, SP might form aggregates in contact with these model systems, leading to a loss of permeability of the lipid vesicles.

Influence of the replacement of amino acid by its D-enantiomer in the sequence of substance P. 2. Conformational analysis by NMR and energy calculations.

The D-enantiomer of residues 2, 4, 5, 6, 7, 8, 10 and 11 was introduced in the sequence of Substance P: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH1. The achiral glycine was replaced by a D-Ala residue. The conformations of the D-substituted analogues were analysed by NMR and molecular energy calculations. Introduction of a D-amino acid in the address sequence of SP (1 to 5) distorted the helical structure of [D-Pro2]SP and [D-Pro4]SP. A D-glutamine in position 5 hampered the formation of an helix, the core of [D-Gln5]SP was stabilized by the presence of two beta-turns. The exact fitting of both Phe7 and Phe8 in the binding pocket can be achieved by either an alpha- or a 3(10) helix or two beta-turns types I and II'. Replacement of an amino acid in the message sequence, 6 to 11, drastically decreased the potencies of the corresponding analogues, different conformational modifications were observed. [D-Gln6]SP presented two beta-turns, however, the types of beta-turns should orientate the side-chains in such a way that [D-Gln6]SP did not fit in the binding site. The conformations of [D-Phe7]SP and [D-Phe8]SP were completely changed, a more or less extended structure being observed. Modifications in the Gly-Leu-Met-NH2 sequence did not affect the helical structure of the core of [D-Ala9]SP, [D-Leu10]SP and [D-Met11]SP, but decreased the percentage of extended structure of the C-terminal tripeptide.

Influence of the replacement of amino acid by its D-enantiomer in the sequence of substance P. 1. Binding and pharmacological data.

The D-enantiomer of residues 2, 4, 5, 6, 7, 8, 10 and 11 was introduced in the sequence of Substance P: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. The achiral glycine residue was replaced by a D-Ala residue. Regarding NK-1 binding potencies or activities, changing to the D-enantiomer in positions 2, 4 or 5 did not modify the pharmacological patterns of the resulting peptides. Introduction of a D-residue in the 6 to 11 sequence drastically decreased the potency of the D-analogues with the exception of [D-Leu10]SP which was found only three times less potent than SP in contracting the guinea-pig ileum. No clear cut evidence between the binding potencies and activities on NK-1, NK-2 and NK-3 assays, was observed which allows a more rational design of tachykinins antagonists.

Dielectric study of the molecular mobility and the isothermal crystallization kinetics of an amorphous pharmaceutical drug substance.

During the development of new pharmaceutical products based on drug substances in their amorphous form, the molecular mobility of an amorphous active ingredient was characterized in detail within a very broad time-temperature range. The relation between the isothermal crystallization kinetics and the dynamics of this amorphous substance was investigated. First, dynamic dielectric spectroscopy (DDS) and the thermostimulated current (TSC) techniques were used to analyze the molecular mobility of the amorphous drug substance over a wide frequency and temperature range (the drug substance is referred to as SSR in this text and was chosen as a model glass-forming system). Two relaxation processes, corresponding to different molecular motions, were identified. The beta(a)-relaxation process, associated with intramolecular oscillation of small dipolar groups, followed Arrhenius temperature behavior over the entire time-temperature domain that was studied. However, the main alpha(a)-relaxation process, assigned to the dielectric manifestation of the dynamic glass transition of the amorphous phase, was described by Vogel-Fulcher-Tammann (VFT) and Arrhenius behavior above and below the glass transition temperature (T(g)) respectively. The physical meaning of these complex dynamics is explained in the context of the Adam and Gibbs (AG) model, by the temperature dependence of the size of cooperatively rearranging regions (CRR) that govern the time scale of delocalized molecular motions. The distinction between the molecular mobility and the structural relaxation of amorphous systems below T(g) is discussed. This work shows that the complementary nature of both DDS and TSC techniques is essential to directly analyze the intramolecular and molecular motions of disordered phases over a wide time-temperature range above and below the T(g). Second, real-time dielectric measurements were carried out to determine the isothermal crystallization kinetics of the SSR amorphous drug. Whatever the crystalline form obtained over time in the crystallization process, the decrease of the dielectric response of amorphous phase, which is characteristic of the isothermal crystallization, was studied to monitor the time dependence of the degree of crystallinity. The characteristic crystallization time, derived from Kohlrausch-Williams-Watt (KWW)-Avrami analyses performed at different temperatures, followed an Arrhenius temperature dependence. Behaviors specific to the molecular mobility of the amorphous drug substance were compared with the characteristic crystallization time. It was concluded that the crystal growth process of the SSR drug seems to be controlled by the intramolecular motions involving the beta(a)-relaxation mode and not by the molecular motions responsible for the alpha(a)-relaxation mode in the range of temperatures >T(g). Subsequent studies will focus on the crystallization process of the SSR drug in the glassy state (T < T(g)).