Improved methods for transplanting split-heart neonatal cardiac grafts into the ear pinna of mice and rats.

The rodent heterotopic ear-heart transplant method is a useful alternative to the more technically demanding vascularized graft technique. We modified the procedure to improve efficiency and used it in mice and rats to determine the survival times of both isologous and allogeneic grafts and compare reference immunosuppressants. Bisected rat and mouse cardiac (split-heart) isografts were uniformly viable up to 4 weeks postimplant; however, by 24 weeks only 90% of Lewis rat or C3H mouse split-heart isografts retained electrocardiographic activity, regressing to 81% by 60 weeks for the Lewis rat and to less than 50% for the C3H mouse by 43 weeks post-implant. The potency of tacrolimus, sirolimus, and cyclosporine for prevention of allograft rejection was comparable whether using split-hearts or whole hearts in the Balb/C to C3H mouse model. The maximally effective doses at 2 weeks postimplant for intraperitoneally administered tacrolimus, sirolimus, cyclosporine, and oral leflunomide with Brown-Norway (BN) to Lewis rat ear-split-heart allografts (0.3, 0.1, 3.0, 10, mg/kg/day, respectively) agreed extremely well with published data for the rat primary vascularized heterotopic heart model. This reproducible and efficient transplantation model was improved by using split-hearts to double available donor tissue, a gonadotropin-enhanced breeding strategy that enables routine use of low-fecundity inbred rats as donors, implantation devices that speed and simplify the procedure, and defined electrocardiographic evaluation criteria to maximize sensitivity and provide an objective endpoint for defining rejection.

Nephrotoxicity studies of the immunosuppressants tacrolimus (FK506) and ascomycin in rat models.

The nephrotoxic potential of ascomycin, the C21-ethyl analogue of FK506, was defined and ways explored to enhance its detection. After 14-day dosing in the Fischer-344 rat, FK506 and ascomycin reduced creatinine clearance by >50% at doses of 1 and 3 mg/kg, i.p., respectively. Ascomycin also had a 3-fold lower immunosuppressive potency in a popliteal lymph node hyperplasia assay, resulting in an equivalent therapeutic index consistent with a common mechanistic dependence on calcineurin inhibition. Renal impairment with different routes of administration was correlated with pharmacokinetics. Sensitivity of detection was not adequate with shorter dosing durations in rats with unilateral nephrectomy or in mice using a cytochrome P-450 inhibitor, SKF-525A. In 14-day studies, nephrotoxicity was not induced by continuous i.p. infusion of ascomycin at 10 mg/kg/day or daily oral administration (up to 50 mg/kg/day) in rats on a normal diet, nor by continuous i.v. infusion (up to 6 mg/kg/day) in rats on a low salt diet to enhance susceptibility. The lack of toxicity at high oral doses of FK506 or ascomycin, and the finding of non-linear oral pharmacokinetics of ascomycin show that this drug class has an oral absorption ceiling. The negative results with continuous infusion suggest that ascomycin nephrotoxicity is governed by peak drug levels. In addition to defining ways to meaningfully compare the nephrotoxic potential of FK506 derivatives, these results have implications for overall safety assessment and improved clinical use.

Discovery of less nephrotoxic FK506 analogs and determining immunophilin dependence of immunosuppressant nephrotoxicity with a novel single-dose rat cisplatin potentiation assay.

Comparing nephrotoxicity of numerous drug analogs is impractical with chronic in vivo models. We devised a new cisplatin potentiation assay (CISPA) that sensitively detects renal injury as a serum creatinine increase when only one dose of test compound is followed by cisplatin. Reference nephrotoxins known to act on various sites in kidney tubules, glomeruli or renal papilla were all detected by the CISPA at single doses that without cisplatin gave little change, which showed that this simple, sensitive assay has broad potential utility for mechanistic studies of nephrotoxicity. We used the CISPA both to probe the nephrotoxic mode of action of immunosuppressants and to search for safer compounds. Although several non-nephrotoxic immunosuppressants were inactive, cyclosporine, FK506, ascomycin (C21-ethyl-FK506) and rapamycin were nephrotoxic in the CISPA at single doses equal to the daily amounts required to reduce creatinine clearance with 14 days of treatment. Similar therapeutic indices were derived comparing toxicity by either method to prevention of rat ear-heart allograft rejection. C18-OH-ascomycin, an FK506-binding protein (FKBP) antagonist, reversed in vivo immunosuppression by FK506 and ascomycin in the rat, and pretreatment in the CISPA blocked FK506 and ascomycin nephrotoxicity, which showed a common immunophilin dependence. Rapamycin nephrotoxicity was unaffected (as with cyclosporine), which indicated that binding to FKBP was not required. Rapamycin nephrotoxicity thus appears mechanistically unrelated to its immunosuppressive mode of action. Screening with the CISPA enabled discovery of A-119435, a less nephrotoxic ascomycin analog having a 10-fold higher therapeutic index.

Dunaimycins, a new complex of spiroketal 24-membered macrolides with immunosuppressive activity. III. Immunosuppressive activities of dunaimycins.

The immunosuppressive effects of the dunaimycins, a new complex of spiroketal 24-membered macrolides, were compared to cyclosporin A, ascomycin, and rapamycin. Each dunaimycin was a potent inhibitor of the mitogenic response observed in mixed murine splenocyte or human leukocyte cultures, and like immunosuppressive drugs these compounds were relatively less potent inhibitors of the constitutive proliferation of murine EL4 thymoma cells. Dunaimycin D4S showed no selectivity in inhibiting the mitogenic response of spleen cells to concanavalin A, pokeweed mitogen, lipopolysaccharide, or phytohemagglutinin. Cyclosporin A and ascomycin did not inhibit interleukin 2 dependent proliferation, whereas the dunaimycins and rapamycin blocked the uptake of [3H]thymidine in mixed cultures supplemented with exogenous interleukin 2. In addition, dunaimycin D4S had no apparent affinity for cyclosporin A or FK-506 immunophilins. Although the dunaimycins inhibited the activity of Na+, K(+)-ATPase, inhibition of this enzyme appeared insufficient to explain the biological activity of these new macrolides. Over a narrow concentration range, dunaimycin D4S showed in vivo immunosuppressive activity in the murine popliteal lymph node hyperplasia model.

C5a structural requirements for neutrophil receptor interaction.

A number of C5a modifications were tested to determine effects on receptor binding to polymorphonuclear leukocyte (PMNL) membrane receptors and triggering of PMNL chemokinesis and myeloperoxidase (MPO) release. Site-directed mutagenesis was used to probe relationships of key C-terminal residues, and suggested a role for additional sites, particularly Lys19-20. A synthetic peptide based on C5a 19-30, weakly inhibited C5a binding. Potency of the C-terminal octapeptide, a full agonist, was markedly improved by a single Phe substitution for His67, and a Phe point mutation at this site was shown to enhance activity of the full recombinant protein.

Identification of receptor-binding residues in the inflammatory complement protein C5a by site-directed mutagenesis.

C5a is an inflammatory mediator potentially involved in a number of diseases. To help define which of its 74 residues are important for receptor binding and response triggering, changes in the amino acid sequence of C5a were introduced by site-directed mutagenesis. Synthetic C5a-encoding genes incorporating point mutations were expressed in Escherichia coli, and the mutant proteins were purified to homogeneity. Modifications of the C5a molecule causing parallel reductions in binding to polymorphonuclear leukocyte membranes and in stimulation of polymorphonuclear leukocyte locomotion (chemokinesis) suggest that carboxyl-terminal residues Lys-68, Leu-72, and Arg-74 interact with the receptor. Substitutions in the disulfide-linked core of C5a revealed involvement of Arg-40 or nearby residues, because potency losses were associated with only localized conformational changes as detected by NMR. Surprisingly, a substitution at core residue Ala-26, which did not alter C5a core structure, appeared from NMR results to reduce potency by causing a long-distance conformational change centered on residue His-15. Thus, at least three discontinuous regions of the C5a molecule appear to act in concert to achieve full potency.

High-level C5a gene expression and recovery of recombinant human C5a from Escherichia coli.

Poor expression of a synthetic gene for the inflammatory mediator, C5a, was observed in E. coli grown in rich media. Varying the media composition markedly improved expression, although C5a levels still declined rapidly at the end of log phase. Using a protease-deficient strain, C5a was recovered at stationary phase in high yield (13 mg/liter of culture). Recovery was dependent on guanidinium hydrochloride extraction to solubilize the protein and glutathione treatment to promote correct folding. Two-thirds of the C5a retained an amino-terminal methionine. Both forms of recombinant C5a had activity similar to serum-derived C5a in binding to human neutrophil receptors and inducing chemotaxis. The 700-fold improvement in yield made it feasible to obtain gram amounts of C5a and provides an efficient system for site-directed mutagenesis.

Rhodium(III) complexes as genotoxic agents: photochemical effects and their implications.

The genetic toxicology of coordination compounds of transition metals has been of considerable interest since the application of cis-platinum(II) to the therapy of solid tumors. The nature of reactions of such compounds with DNA is still unclear, despite intensive investigation. In this study, several coordination compounds of rhodium(III) were tested for DNA-damaging activity and mutagenicity in bacterial assays in an attempt to understand both the chemical species involved in interactions with DNA and any structural requirements for such interactions. For several complexes it appears that dissociation of a ligand from the complex precedes reactions with DNA. This conclusion stems from the finding that photosensitive complexes of rhodium(III) are often many times more toxic to repair-deficient bacterial stains of E. coli K12 when incubated in the light than when incubated in the dark. Similar responses were seen for mutagenicity in S. typhimurium strain TA100. However, reversion of strain TA102 was largely independent of light exposure. Comparisons between mutagenicity and DNA-damaging activity revealed that the 3 activities measured sorted with some independence among the different compounds tested. Thus, the profiles for crosslink formation and/or generation of oxidative mutagens (mutagenicity in S. typhimurium strain TA102), mutagenicity in TA100 and DNA-damaging activity for the various groups of complexes showed many of the theoretically possible combinations of response in the assays. It is possible, then, that there are different structural requirements for DNA-damaging activity and mutagenicity respectively. This may indicate that synthesis of coordination compounds with specific genotoxic properties is possible. Such syntheses may provide complexes for study of DNA-metal interactions and could, later, direct an approach to the design of new antitumor agents.

The effects of anti-inflammatory and other agents on the rat dermal arthus reaction.

The inhibitory effects of selected drugs on the Arthus reaction, a model of immune-complex-induced tissue injury, were studied. The reverse passive Arthus reaction (RPAR) was elicited in the dorsal skin of rats, using bovine serum albumin and the gamma-globulin fraction of rabbit anti-BSA. The optimal amounts of antigen and antibody required to elicit the reaction, as well as the reaction kinetics, were examined. Chlorpheniramine, cyproheptadine, d-penicillamine, chloroquine, indomethacin, phenylbutazone, naproxen, and mefenamic acid were found to be inactive despite high doses; aspirin and ibuprofen were only weakly active. Hydrocortisone and colchicine were strong inhibitors of the RPAR: the calculated ED50 values were 13 mg/kg p.o. and 0.3 mg/kg i.v., respectively. The RPAR exhibits a different sensitivity to drug inhibition than conventional models of inflammation (e.g., carrageenin paw edema) end may be useful to detect new types of anti-inflammatory agents.

[Dose requirement and definition of the image with new types of mammographic systems (author's transl)]. / Dosisbelastung und Abbildungseigenschaften neuer Mammographiesysteme.

Several film-screen-systems have been tested under clinical conditions in order to check their utility for mammography (Dupont, Kodak, 3M, Agfa, Auer). The aim was to find the balance between the resolution of the details and the minimal radiation dose.