Fasted and fed state human duodenal fluids: Characterization, drug solubility, and comparison to simulated fluids and with human bioavailability.

Accurate in vivo predictions of intestinal absorption of low solubility drugs require knowing their solubility in physiologically relevant dissolution media. Aspirated human intestinal fluids (HIF) are the gold standard, followed by simulated intestinal HIF in the fasted and fed state (FaSSIF/FeSSIF). However, current HIF characterization data vary, and there is also some controversy regarding the accuracy of FaSSIF and FeSSIF for predicting drug solubility in HIF. This study aimed at characterizing fasted and fed state duodenal HIF from 16 human volunteers with respect to pH, buffer capacity, osmolarity, surface tension, as well as protein, phospholipid, and bile salt content. The fasted and fed state HIF samples were further used to investigate the equilibrium solubility of 17 representative low-solubility small-molecule drugs, six of which were confidential industry compounds and 11 were known and characterized regarding chemical diversity. These solubility values were then compared to reported solubility values in fasted and fed state HIF, FaSSIF and FeSSIF, as well as with their human bioavailability for both states. The HIF compositions corresponded well to previously reported values and current FaSSIF and FeSSIF compositions. The drug solubility values in HIF (both fasted and fed states) were also well in line with reported solubility data for HIF, as well as simulated FaSSIF and FeSSIF. This indicates that the in vivo conditions in the proximal small intestine are well represented by simulated intestinal fluids in both composition and drug equilibrium solubility. However, increased drug solubility in the fed vs. fasted states in HIF did not correlate with the human bioavailability changes of the same drugs following oral administration in either state.

Solution structure of RP 71955, a new 21 amino acid tricyclic peptide active against HIV-1 virus.

The structure of RP 71955, a new tricyclic 21 amino acid peptide active against human immunodeficiency virus 1, was determined. Its amino acid composition was inferred from the results of fast atom bombardment mass spectrometry, nuclear magnetic resonance, Raman spectroscopy, and amino acid analysis. Its sequence could not be determined classically, using Edman degradation, given the lack of a free terminal NH2. It was deduced from the interpretation of interresidue nuclear Overhauser effects and confirmed by the sequencing of peptides obtained by limited chemical hydrolysis. It was found to be CLGIGSCNDFAGCGYAVVCFW. An internal amide bond between the NH2 of C1 and the gamma-COOH of D9 was observed, as well as two disulfide bridges, one between C1 and C13 and one between C7 and C19. The three-dimensional structure of RP 71955 was determined from nuclear magnetic resonance derived constraints using distance geometry, restrained molecular dynamics, nuclear Overhauser effect back calculation, and an iterative refinement using a full relaxation matrix approach. Analogies between the structure of RP 71955 and some functional domains of gp41, the transmembrane protein of human immunodeficiency virus 1, suggest hypotheses concerning the mode of action of RP 71955.

Conformations in crystals and solutions of d(CACGTG), d(CCGCGG) and d(GGCGCC) studied by vibrational spectroscopy.

Crystals of self complementary DNA hexamers d(CACGTG), d(CCGCGG) and d(GGCGCC) were grown by vapour diffusion technique and studied by microRaman and microIR spectroscopies. The oligonucleotides were studied in parallel in solution by vibrational spectroscopy. A B- greater than Z transition was detected by Raman spectroscopy during the crystallization process for d(CACGTG). Vibrational spectroscopy shows that the d(GGCGCC) crystals adopt a B geometry. On the contrary the d(CCGCGG) sequence which is shown to be able to undergo in solution or in films quite easily the B- greater than Z transition, remains trapped in crystals in a geometry which may correspond to an intermediate conformation often proposed in models of the B- greater than Z transition. The crystals used in this study were characterized by X-ray diffraction. The unit cell and space group have been determined.

IR and UV studies on stability and conformations of short DNA duplexes containing a no-base residue: coexistence of B and Z conformations.

Tridecamers containing a central no-base residue (X) have been synthesized and hybridized to their complementary strands, so as to constitute duplexes consisting of two hexamers separated by central mismatched X-A or X-T pairs. The effect of the introduction of this deoxyribose derivative on duplex stability was investigated by measuring UV absorbance as a function of salt concentration and temperature. As expected, the duplexes containing the abnormal base pairs (X-T and X-A) are less stable when compared to the totally complementary duplexes (A-T and T-A). The X-T mismatched duplex shows the most unstable thermodynamical behaviour. The conformational changes of these duplexes were studied by IR spectroscopy in condensed phase as a function of water content. At high relative humidity, the IR spectra show that these tridecamers form B-type double stranded duplex structures. If the water content is decreased, only the duplexes m5CGm5CGCTXAGCTTC GCGCGAATCGAAG and, to a lesser degree, m5CGm5CGCTXAGCTTC GCGGCATTCGAAG undergo a partial B---Z transition involving the methylated hexamer, the conformation of the second segment remaining of the B type. These results show that only one apurinic residue leads to a flexible junction between B and Z forms in a short duplex containing 5-methyl-2'-deoxycytidines.

Conformational flexibility in the right-handed and in the left-handed double helices of Na+-d(m5C-G-T-A-m5C-G) studied by IR spectroscopy.

The X ray diffraction study of a d(m5C-G-A-T-m5C-G) crystals has shown the existence of a 2 conformation while the Raman spectroscopy study of the same fragment in solution showed that then the oligonucleotide adopted a B geometry. Infrared spectroscopy has allowed us to study this sequence in films in a wide range of hydrations and to vary the water content of the sample at different rates. We have thus obtained four I.R. spectra, of the B and 2 conformations respectively at 100% and 98% relative humidities, of another 2 form with a different geometry of the phosphate groups at relative humidities below 98% and in addition a fourth spectrum recorded after a rapid dehydration of the sample which is then blocked in a right-handed form at low R.H. In this case the structure of the phosphodiester chain may be not uniform. The results are discussed by comparison with previously obtained I.R. spectra of poly d(G-C), poly d(A-C). d(G-T), d'm5C-G-A-m5C-G-T-G-C-G) d(m5C-G-C-G-m5C-G) and d(C-G-m5C-G).

Raman spectroscopy of Z-form poly[d(A-T)].poly[d(A-T)].

Helical structures of double-stranded poly[d(A-T)] in solution have been studied by Raman spectroscopy. While the classical right-handed conformation B-type spectra are obtained in the case of sodium chloride solutions, a Z-form Raman spectrum is observed by addition of nickel ions at high sodium concentration, conditions in which the inversion of the circular dichroic spectrum of poly[d(A-T)] is detected, similar to that observed for high-salt poly[d(G-C)] solutions [Bourtayre, P., Liquier, J., Pizzorni, L., & Taillandier, E. (1987) J. Biomol. Struct. Dyn. 5, 97-104]. The characterization of the Z-form spectrum of poly[d(A-T)] is proposed by comparison with previously obtained characteristic Raman lines of Z-form poly[d(G-C)] and poly[d(A-C)].poly[d(G-T)] solutions and of d(CG)3 and d(CGCATGCG) crystals [Thamann, T. J., Lord, R. C., Wang, A. H.-J., & Rich, A. (1981) Nucleic Acids Res. 9, 5443-5457; Benevides, J. M., Wang, A. H.-J., van der Marel, G. A., van Boom, J. H., Rich, A., & Thomas, G. J., Jr. (1984) Nucleic Acids Res. 14, 5913-5925]. Detailed spectroscopic data are presented reflecting the reorientation of the purine-deoxyribose entities (C2'-endo/anti----C3'-endo/syn), the modification of the phosphodiester chain, and the adenosine lines in the 1300-cm-1 region. The role played by the hydrated nickel ions in the B----Z transition is discussed.

Z form of poly d(A-C).poly d(G-T) in solution studied by Raman spectroscopy.

Poly d(A-C).poly d(G-T) structures have been studied in solution by Raman spectroscopy, in presence of Na+, Mn2+ and Ni2+ counterions. Increase of the Na+ concentration or addition of Mn2+ ions up to 1M MnCl2 does not modify the B geometry of the polynucleotide. On the contrary, in conditions of low water activity (4M NaCl), the presence of small amounts of nickel ions (65 mM) induces a left-handed geometry of the DNA. The shift of the guanine line located at 682 cm-1 in B form to 622 cm-1 reflects unambiguously the C2'-endo/anti-greater than C3'-endo/syn reorientation of the deoxyribose-purine entities. Moreover modifications in the phosphate backbone lines indicate that the polymer is in a Z conformation. New or displaced lines corresponding to adenosine vibrations are correlated with the left-handed structure. An interaction of the Ni2+ ions specifically with the N7 site of purines, combined with a low water activity is necessary to promote the B-greater than Z transition.

Infrared and Raman studies show that poly(dA).poly(dT) and d(AAAAATTTTT)2 exhibit a heteronomous conformation in films at 75% relative humidity and a B-type conformation at high humidities and in solution.

The decadeoxynucleotide d(AAAAATTTTT)2 in duplex form and the double-helical polynucleotide poly(dA).poly(dT) have been studied by Raman and infrared (IR) spectroscopy under a variety of environmental conditions. The IR spectra have been taken of cast films and compared to the IR spectra of the alternating poly(dA-dT), which shows clear B-genus and A-genus vibrational spectra under conditions of high (greater than 92%) and low (75%) relative humidity (RH). From the IR data, it is shown that d-(AAAAATTTTT)2 and poly(dA).poly(dT) adopt a B-genus conformation in films with high water content. When the relative humidity of the film is decreased, the IR spectra reflect a gradual evolution of the geometry of both d(AAAAATTTTT)2 and poly(dA).poly(dT) into a form intermediate between the B genus and A genus, but the IR spectrum of a pure A genus has not been obtained. In these DNAs at 75% RH, the IR bands of adenosine have the same frequencies as those found in poly(dA-dT) at 75% RH where the local furanose conformation is C3' endo/anti, but the thymidine frequencies do not resemble those of poly(dA-dT) at 75% RH but rather those of poly(dA-dT) at high humidities. It is concluded that both poly(dA).poly(dT) and d(AAAAATTTTT)2 adopt a fully heteronomous duplex geometry in cast films at low humidity. For studies in aqueous solution the Raman effect was employed. As a model for the heteronomous conformation in solution, the duplex poly(rA).poly(dT) was used.(ABSTRACT TRUNCATED AT 250 WORDS)