Influence of penicillamine and various analogs on matrix-induced bone formation in rats.

D-penicillamine and a variety of analogs have been tested for their ability to interfere with the various stages of bone development using a model for endochondral bone formation. At the highest dose tested (40 mg/rat/day), D-penicillamine inhibited mineralization, D-2-Amino-3-methyl-3-[(2-acetamidoethyl)dithio]butanoic acid (II), at a relatively low dose (10 mg/day), decreased the amount of insoluble collagen in skin, mesenchymal cell proliferation on Day 3, and inhibited bone formation on Day 14. Several other compounds tested, sodium 4-[(D-1, 1-dimethyl-2-amino-2-carboxyethyl)-dithio]butanesulfinate (IV), 2-acetamidoethyl-2-acetamidoethanethiolsulfonate (V), and sodium 4-mercaptobutanesulfinate trihydrate (VI), also inhibited osteogenesis on Day 14.

Lymphocyte surface poisons: disulfides and thiolsulfonates.

Eight disulfides (I-VIII) and a thiolsulfonate (IX) were promising blocking agents of lymphocytes in graft-versus-host reactions (GvHR) without comensurate intracellular effects. The blocking effects were assayed through inhibition of the local GvHR after parental lymphocytes had been incubated with agents at suitable concentrations and then inoculated into F1 hybrid offspring. The intracellular effects were assessed beforehand by measuring the inhibition of [6-3H]thymidine incorporation by lymphocytes in the presence of a wide range of concentrations of agents. Concentration levels which induced no greater than approx. 50% inhibition of the [6-3H] thymidine incorporation were considered to reflect sufficiently small intracellular effects and were used for the subsequent GvHR comparisons. Cellular survival always was 90% or more for the GvHR tests (unless stated otherwise), even when inhibition of thymidine incorporation was as high as 50%; hence the thymidine data are useful not only as guides for dose levels in the GvHR but also as leads to new agents that may show immunosuppressive or anti-leukemic activity through intracellular effects. Structural specificity of the active compounds as cell-surface poisons is evidenced by little or no activity (less than 30% inhibition of GvHR) of 28 other disulfides, 2 trisulfides, 2 Bunte salts, and 8 other thiolsulfonates. Active agents may owe this function to replacement of the H of SH in cell-surface thiol receptors by an SR group. Glutathione did not significantly inactivate agents, probably because the products of reaction also are active disulfides. When two agents (III, IX) were given orally or intraperitoneally to F1 hybrid recipients of untreated parental cells, doses of 10--15 mg/kg produced a GvHR inhibition of 17--53%.

Dose-response studies of penicillamine and related compounds in reducing skin--tensile strength of rats.

Penicillamine (I) and certain related compounds are known to reduce the skin tensile strength (sts) of rat dorsal skin when they are given in the diet. This effect seems significant to studies of the biochemistry of collagen and of diseases of collagen, perhaps including the arthritides. The reduction of sts appears to be caused by diminution of collagen crosslinking. The effects of several such compounds were studied after intraperitoneal (ip) injection, in order to determine structure-activity relationships divorced from possible anorexic or gastrointestinal complications seen after oral dosing, and to examine the relation of ip dose to response. A cyclopentyl analog (II) of I was at least as active as I, showing that structural variants of I can be active when given ip. A dimethylthiazolidine (V) and a zinc chelate (III, rather toxic) of I were nearly as active as I, showing that probable in vivo precursors of I can be obtained that will be active when given ip. The disulfide of I and a zinc chelate of cysteine were inactive. Maximum response for several compounds seemed to occur at intermediate dose levels, with larger or smaller doses affording less sts reduction.

Some immunological effects of penicillamine.

Immunological effects of D- and D,L-penicillamine (PA) were studied in efforts to develop assays for synthetic D or D,L analogs and to contribute to the understanding of the mechanism(s) of action of D-PA in rheumatoid arthritis. At the highest doses tolerated by mice, D,L-PA did not significantly inhibit the development of haemagglutinating antibodies in vivo. In studies in vitro with T lymphocytes, D-PA at 1 mM concentration inhibited both concanavalin A- and phytohaemagglutinin-induced transformation as assayed by [3H]thymidine incorporation, but D-PA concentrations of 5 mM were required to inhibit concanavalin A-induced amino acid uptake. No effect of D-PA was observed either on the induction of cytotoxic T cells or on the attack of specifically sensitized T cells on target cells. It is of interest that D-PA at 1 mM concentration did inhibit lipopolysaccharide-induced transformation, which predominately stimulates B lymphocytes. The effects of PA on the induced transformation of T and B cells deserve further attention for studies with analogs of PA.

Analogs of natural lipids. III. Nonequivalence of methyl groups in methylated phospholipids.

The dimethyl esters of a series of diastereoisomeric cyclopentanoid analogs of phosphatidic acid (A.J. Hancock, M.H. Stokes, and H.Z. Sable. 1977. J. Lipid Res. 18: 81-92.) have been studied by proton NMR spectroscopy at 60, 100, and 300 MHz. The signals of the P-O-CH3 protons near delta 3.80 show the expected doubling due to the 31P-1H coupling. In addition, the spectra of three of the isomers show additional multiplicity the line separation (in Hz) being proportional to the frequency of the spectrometer. This multiplicity is due to the nonequivalence of the two methoxyl groups on phosphorus, predictable from their diastereotopic nature. The same explanation is proposed for similar observations on other compounds made by other authors. The practical utility of symmetry considerations in lipid chemistry is discussed briefly.

Carbon 13 magnetic resonance studies of DL-2-(alpha-hydroxyethyl) thiamin and related compounds. Relation of kinetic acidity to electronic factors in thiamin catalysis.

Carbon 13 NMR spectra have been obtained for aqueous solutions of DL-2-(alpha-hydroxyethyl)thiamin, DL-2-(alpha-hydroxybenzyl)thiamin, DL-2-(alpha-hydroxybenzyl)oxythiamin, and related N-3 methyl and N-3 benzyl analogs. The unusually large downfield shift of the 13C resonance of C-2 of hydroxyethylthiamin suggests that this carbon bears a partial positive charge. This result stands in contrast to results of x-ray crystallographic studies of hydroxyethylthiamin, which place a partial negative charge on C-2 (Pletcher, J., and Sax, M. (1974) J. Am. Chem. Soc. 96, 155-165). A partial positive charge on C-2 helps to explain the facility of carbanion formation at the alpha carbon both enzymatically and in model systems. The rates of proton-deuteron exchange of (C-alpha)-H with solvent deuterium, and of release of aldehyde to regenerate thiamin have been measured for hydroxyethylthiamin and analogs. The differences in kinetic acidity of (C-alpha)-H and of rates of aldehyde release are rationalized in terms of differing electron-withdrawing abilities of the substituents attached to N-3, and appear not to be related to intramolecular basic catalysis of these processes by the C-4' amino group.

Conformation of complexes of thiamin pyrophosphate with divalent cations as studied by nuclear magnetic resonance spectroscopy.

The binding of Ni-2+ and Mn-2+ to thiamin phosphate and thiamin pyrophosphate (thiamin-PP) has been compared with the binding of these ions to oxythiamin phosphate and oxythiamin pyrophosphate, analogues of thiamin in which the C-4 amino group has been replaced by an -OH group. The replacement of the NH2 group results in reduced basicity of N-1 of the pyrimidine ring of oxythiamine derivatives. The effects of pD, ligand concentration, and temperature on the binding of metal ions to N-1 have been studied by observing the metal ion-induced shifting and broadening of the C-6-H signal of these compounds. The results indicate the following: (a) the metal ion is held near N-1, resulting in a "folded" conformation, because of a favorable bonding interaction between N-1 and the metal ion rather than for general conformational reasons alone; and (b) the amount of "folded" conformation present in the different pyrophosphate complexes at neutral pH follows the order: Ni-2+-thiamin-PP greater than Mn-2+-thiamin-PP greater than Mn-2+-oxythiamin-PP and Ni-2+-oxythiamin-PP It is concluded that the strength of the metal ion-pyrimidine interaction in the "folded" conformation depends strongly both on the coordination affinity of the metal ion and on the basicity of N-1. Since the interaction of the phosphate-bound metal ion with the pyrimidine ring in the Mg-2+-thiamin-PP complex is probably weaker than the corresponding interaction in the Mn-2+-thiamin-PP complex, these results predict that the Mg-2+-thiamin-PP complex in solution, at neutral pH, exists predominantly in an "unfolded" conformation.