Comparative dose-dependence study of FK506 and cyclosporin A on the rate of axonal regeneration in the rat sciatic nerve

The new immunosuppressant drug FK506 (Tacrolimus) increases the rate of nerve regeneration in vivo (Gold et al., 1994; Gold et al., 1995). In the present study, we have examined the dose-dependence of FK506's ability to enhance nerve regeneration. In the first set of experiments, rats received daily s.c. injections of FK506 (2 mg/kg, 5 mg/kg or 10 mg/kg) for 18 days after a sciatic nerve crush injury. Signs of functional recovery in the hind feet appeared earlier than in saline-treated control rats at all three FK506 dosage; recovery was maximally accelerated in the 5-mg/kg group. Light microscopy at 18 days after nerve crush revealed more regenerating myelinated fibers in FK506-treated rats than in controls; this was most apparent in the 5-mg/kg group. Morphometric analysis of axonal areas in the soleus nerve confirmed that axonal calibers were maximally increased in the 5-mg/kg group. In the second set of experiments, the rate of axonal regeneration was determined by radiolabeling the L5 dorsal root ganglion. Regeneration rate for sensory axons was maximally increased (by 34%) in the 5-mg/kg group. In contrast, cyclosporin A (10 or 50 mg/kg; dosages were selected on the basis of the 1/10 lower potency of cyclosporin A) did not significantly alter the rate of axonal regeneration. Cyclosporin A (50 mg/kg) also failed to increase functional recovery or axonal calibers in the soleus nerve. Because the two drugs share a common mechanism for producing immunosuppression (i.e., calcineurin inhibition), these results indicate that FK506's nerve regenerative property involves a distinct, calcineurin-independent mechanism.     

Effects of FK506 on experimental membranous glomerulonephritis induced by cationized bovine serum albumin in rats

BACKGROUND: There have been no reports on the effect of FK5 06, a new immunosuppressive agent, on experimental membranous glomerulonephritis (MN) induced by an exogenous antigen. Therefore we investigated the effects of FK506 on MN induced by cationized bovine serum albumin (C-BSA) in rats. METHODS: Two weeks after the rats were immunized with 1 mg of C-BSA and Freund's complete adjuvant, they received intravenous injections of 3 mg/kg of C-BSA 3 times weekly for 4 weeks. Rats were divided into four groups: group A (n = 5) received intramuscular injections of 3 mg/kg of FK506 daily for 5 days beginning 2 days before the first immunization; group B (n =4) received 1 mg/kg of FK506 daily for 2 weeks beginning 2 weeks after the first immunization; and group C (n =4) received 1 mg/kg of FK506 daily for 2 weeks beginning 4 weeks after the first immunization. Group D (n = 5) received no FK506 and served as the control group. Rats were sacrificed 8 weeks after the first immunization. RESULTS: Administration of FK506 in the preimmunization stage almost completely suppressed the development of MN in group A. Histological findings in groups B and C were similar to those in group D, the control group. However, urinary protein excretion was significantly lower in group B (24+/-46 mg/day) and C (220+/-44 mg/day) than in group D (330+/-61 mg/day). Expression of intracellular adhesion molecule-1 in glomeruli and the number of leukocyte functioning-associated molecules-1 were significantly decreased in groups A, B, and C compared with the control group. Administration of FK506 was not associated with any significant side-effects or histological abnormalities. The whole-blood trough levels of FK506 in groups B and C were 9.1 ng/ml and 9.2 ng/ml respectively. CONCLUSIONS: The efficacy of FK506 was significantly increased when the drug was administered in the early phase of immunization in this model. The present study suggests that FK506 may be useful in patients with intractable nephrotic syndrome such as MN.     

Molecular and biochemical characterization of the protein template controlling biosynthesis of the lipopeptide lichenysin

To assess the duration of immunosuppression in FK506-dosed pigs, an undiluted whole blood assay was established to measure reactivities of T cells in their physiological milieu. PMA and ionomycin were shown to induce IL-2 production in swine blood. The IC50 of FK506 in inhibiting IL-2 production in whole blood and isolated PBMC stimulated with PMA and ionomycin measured 1.2 and 0.04 nM, respectively. These data underscore the influence of red blood cells and plasma proteins on drug potency. IL-2 levels were determined in blood drawn immediately before and 1, 24, 48, and 72 h after iv dosing. For pigs dosed with 0.05 mg/kg, 50% recovery of IL-2 production was observed at 16 h and 100% at 35 h after dosing. For pigs dosed with 0.15 mg/kg, 50% recovery was observed at 38 h and 100% at 72 h. Blood concentrations of FK506 at 50 and 100% recovery of IL-2 production measured 10.8 and 2.2 nM for pigs dosed with 0.05 mg/kg and 6.1 and 1.1 nM for pigs dosed with 0.15 mg/kg, respectively. These concentrations are severalfold higher than predicted from the IC50 of FK506 for inhibiting IL-2 production in the whole blood assay. These data suggest that the true potency of FK506 in blood after dosing is influenced by additional factors, which could include plasma protein binding, the presence of active or interfering metabolites, serum interfering factors, and sequestration of drug in blood cells. Our results demonstrate the utility of an undiluted whole blood assay for assessing the duration of immunosuppression in drug-dosed animals and emphasize the importance of assessing drug potency in the whole blood environment ex vivo.     

Effect of a novel immunosuppressant, FK 506, on autoimmune glomerulonephritis in Brown Norway rats

Mercuric-chloride (HgCl2) induces a lymphoproliferative disorder and autoimmune glomerulonephritis in Brown Norway rats. The effects of a new immunosuppressant FK 506 on this model of glomerulonephritis were studied. Brown Norway rats were treated with HgCl2 according to a standard protocol (HgCl2 1 mg/kg s.c. 3 times/ week). Rats developed proteinuria at day 7, which reached a plateau level at day 14. On day 14, renal histology showed prominent mesangial cellular proliferation and the expansion of mesangial matrix. Electron microscopic study showed the effacement of visceral epithelial foot processes and the microvillous transformation of the visceral epithelium. Immunofluorescence study showed a strong linear staining for IgG and the adhesion molecule ICAM-1 in all glomeruli. Coadministration of FK 506 (1 mg/kg s.c. daily) prevented the appearance of proteinuria at day 14 (621.4 +/- 30.5 vs. 2.2 +/- 2.7 mg/day) and the morphological lesions. These findings suggest that FK 506 could be useful for the therapy of certain types of human glomerulonephritis.     

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.     

The enhanced immunosuppressive efficacy of newly developed liposomal FK506 in canine liver transplantation

Local delivery of immunosuppressants to the graft and lymphatic tissue is a potential appraoch to enhance the immunosuppressive efficacy and to alleviate systemic adverse effects simultaneously. By taking advantage of this method, we developed liposomal FK506. Previous pharmacokinetic study of liposomal FK506 indicated increased FK506 levels in the liver and spleen. Because the liver is the site of the allograft in liver transplantation and the spleen is a major lymphoid tissue, we hypothesized that liposomal FK506 would increase immunosuppressive efficacy in liver transplantation. We evaluated this hypothesis in a canine model. Orthotopic liver transplantation was performed using beagle dogs, and the recipients were divided into the following groups: group I, no immunosuppression (n = 5); group II, 0.05 mg/kg/day of FK506 i.v. in a commercially available i.v. formulation for 14 days (n = 5); and group III, 0.05 mg/kg/day of FK506 i.v. in a liposomal formulation for 14 days (n = 5). All recipients in group I died within 2 weeks. Recipients in group II died within 33 days. In contrast, three recipients in group III survived for more than 200 days (P < 0.05 versus group I or group II). In DNA analysis, splenocyte proliferation activity in group III was significantly suppressed in comparison with group II. These results suggest that liposomal FK506 markedly increase the immunosuppressive efficacy of FK506 in liver transplantation. A local immunosuppressive effect in the grafted liver and significant suppression of splenocyte proliferation might contribute to enhancement of the immunosuppressive efficacy of liposomal FK506.     

Contributions of inflammatory mast cell mediators to alterations in macrophage function after malathion administration

FK506 a new and potent immunosuppressive agent has been shown to be effective in prolonging pancreatic islet allograft survival. The present study was to determine its efficacy in prolonging pig islet xenotransplantation in two different strains of rat recipients. A total of two dosages of FK506 at 1 or 2 mg/kg per day for 2 weeks and then at weekly intervals were tested as monotherapy for their effect on the survival of renal subcapsular xenografts of purified or impure adult pig islets in inbred ACI and outbred Wistar rats. Histological assessment indicated that FK506 at 2 mg/kg per day significantly prolonged purified pig islet xenograft survival and to 7.5 months in two of three ACI recipients. Monotherapy with a lower dosage of FK506 or transplantation with impure pig islets resulted in increased graft survival time over controls, but less than that with the 2 mg/kg per day FK506. The viable pig islet xenografts showed a normal appearance and were readily identified by immunohistochemical staining for insulin and glucagon and further confirmed by immunohistochemical staining with anti-pig islet specific monoclonal antibody clone P44, developed in our laboratory. Mononuclear cell infiltration, mainly of the CD8-positive T-cell subset, increased with the duration of the graft in the recipient. By 7.5 months the majority of the xenografted islet cells were enclosed by the cellular infiltrate. The in vitro perfusion study of pig islets that had survived for 1 or 2 months in vivo showed that they were responsive to glucose stimulation with increase in insulin secretion into the perfusate. The results demonstrated that FK506 significantly prolonged pig islet survival in two rat strains and suggested that it is an effective immunosuppressant for the xenotransplantation model.     

Comparison of the stability of technetium-labeled peptides to challenge with cysteine

-Tacrolimus (FK 506) is a powerful, widely used immunosuppressant. The clinical utility of FK 506 is complicated by substantial hypertension and nephrotoxicity. To clarify the mechanisms of FK 506-induced hypertension, we studied the chronic effects of FK 506 on the synthesis of endothelin-1 (ET-1), the expression of mRNA of ET-1 and endothelin-converting enzyme-1 (ECE-1), the endothelial nitric oxide synthase (eNOS) activity, and the expression of mRNA of eNOS and C-type natriuretic peptide (CNP) in rat blood vessels. In addition, the effect of the specific endothelin type A receptor antagonist FR 139317 on FK 506-induced hypertension in rats was studied. FK 506, 5 mg. kg-1. d-1 given for 4 weeks, elevated blood pressure from 102+/-13 to 152+/-15 mm Hg and increased the synthesis of ET-1 and the levels of ET-1 mRNA in the mesenteric artery (240% and 230%, respectively). Little change was observed in the expression of ECE-1 mRNA and CNP mRNA. FK 506 decreased eNOS activity and the levels of eNOS mRNA in the aorta (48% and 55%, respectively). The administration of FR 139317 (10 mg. kg-1. d-1) prevented FK 506-induced hypertension in rats. These results indicate that FK 506 may increase blood pressure not only by increasing ET-1 production but also by decreasing NO synthesis in the vasculature.     

Immunosuppressants decrease neutrophil chemoattractant and attenuate ischemia/reperfusion injury of the liver in rats

BACKGROUND: Neutrophils may play an important role in the development of liver ischemia/reperfusion injury. We investigated the effects of the immunosuppressants azathioprine (AZA), cyclosporine A (CsA), tacrolimus (FK506), and rapamycin (RPM) on the expression of cytokine-induced neutrophil chemoattractant (CINC) after ischemia/reperfusion of the liver. METHODS: Liver ischemia was induced in male Wistar rats by occluding the portal vein with a microvascular clip for 30 minutes. Rats received two intramuscular injections of AZA (4 mg/kg), CsA (5 mg/kg), FK506 (0.5 mg/kg), or RPM (0.5 mg/kg) 3 and 24 hours before ischemia/reperfusion of the liver. RESULTS: Serum CINC concentrations in untreated animals increased, peaked 6 hours after reperfusion, and thereafter decreased gradually. Pretreatment with AZA, CsA, FK506, and RPM, however, inhibited the increase in serum CINC concentrations after reperfusion. CINC mRNA in liver tissue increased and peaked 3 hours after reperfusion, but was significantly lower in animals treated with AZA, CsA, FK506, and RPM. In vitro CINC production by Kupffer cells harvested from animals treated with AZA, CsA, FK506, or RPM 3 hours after reperfusion was also significantly lower than that observed in untreated animals. Both myeloperoxidase activity and the number of neutrophils accumulating in the liver 24 hours after reperfusion in animals treated with AZA, CsA, FK506, and RPM were significantly lower than in untreated animals. This correlated with lower serum aspartate transaminase, alanine transaminase, and lactate dehydrogenase levels in animals treated with AZA, CsA, FK506, and RPM 24 hours after reperfusion. CONCLUSION: The immunosuppressants AZA, CsA, FK506, and RPM reduce neutrophil accumulation and attenuate ischemia/reperfusion injury of the liver.     

Effect of immunosuppressant FK506 on ischemia-induced degeneration of hippocampal neurons in gerbils

To evaluate the effect of FK506 on delayed neuronal death in gerbils after forebrain ischemia, 84 adult Mongolian gerbils were used in this study. Transient forebrain ischemia was induced by clipping common carotid arteries bilaterally for 5 minutes. One hour after reperfusion we intraperitoneally injected FK506 (1.0 mg/kg), cyclosporin A (CsA) (10.0 mg/kg) or the vehicle solution into each gerbil. In one group, each agent was additionally administered daily 3 more times at 24, 48 and 72 hours after ischemia. The gerbils were killed 4 days or 10 days after transient ischemia, and damage to their hippocampal pyramidal cells was histologically assessed. Additionally, the body temperature was measured following administration of each drug to investigate drug-induced hypothermia. Post-ischemic repeated treatment with FK506 significantly (p < 0.01) reduced degeneration of hippocampal neurons. However, partial treatment did not modify neuronal degeneration. CsA did not show a neuroprotective effect in this study. Drug-induced mild hypothermia (35-37 C) was observed following administration of FK506 or CsA. There was no significant difference in the time course of the body temperature between the FK506 and CsA group. We demonstrated that the repeated FK506 treatment, but not the CsA treatment, reduced ischemia-induced degeneration of hippocampal neurons in gerbils. Although FK506-induced hypothermia might have modified neuronal degeneration, a comparison with CsA indicated that the neuroprotective effect of FK506 was not solely due to hypothermia per se.     

Mechanism of fibrosis in experimental tacrolimus nephrotoxicity

The clinical use of tacrolimus (FK506) is limited by nephrotoxicity. The pathogenesis of fibrosis in chronic FK506 nephrotoxicity remains unknown. Because transforming growth factor (TGF)-beta plays a key role in the fibrogenesis of many diseases, including cyclosporine nephrotoxicity, we studied a salt-depleted rat model of chronic FK506 nephropathy in which clinically relevant FK506 blood levels are obtained and which shows similarities to the lesions described in patients receiving FK506. Pair-fed rats were treated with either FK506 (1 mg/kg/day s.c.) or an equivalent dose of vehicle and were killed at 7 or 28 days. Characteristic histologic changes of tubular injury, interstitial fibrosis, and arteriolopathy developed in FK506-treated rats at 28 days and were accompanied by worsening kidney function, decreased concentrating ability, and enzymuria. FK506-treated kidneys had a progressive increase in the expression of TGF-beta1 and matrix proteins (biglycan, tenascin, fibronectin, and type I collagen). This effect seems to be specific because the expression of type IV collagen, a basement membrane collagen, was not affected. Matrix deposition was present mostly in the tubulointerstitium and vessels in accordance with the FK506 chronic lesion. The expression of plasminogen activator inhibitor-1, a protease inhibitor influenced by TGF-beta, followed TGF-beta1 and matrix proteins, suggesting that the fibrosis of chronic FK506 nephropathy likely involves the dual action of TGF-beta1 on matrix deposition and degradation. Since both peripheral and tissue renin expression were elevated with FK506, the renin-angiotensin system may play a role in the pathogenesis of this condition.     

The significance of eating patterns: an elderly Greek case study

BACKGROUND: Endothelin-1 (ET-1), a novel vasoconstrictor, possibly plays a role in the mediation of ischemia/reperfusion (I/R) injury. Tacrolimus (FK506) and cyclosporin A (CsA) were reported to maintain tissue microcirculation of the liver subjected to I/R. This study investigated the effects of these immunosuppressants on intestinal I/R in terms of intestinal tissue microcirculation associated with ET-1. METHODS AND RESULTS: Male S-D rats were pretreated twice with FK506 (0.2 mg/kg), CsA (10 mg/kg) or only saline solution (0.5 mL). The tissue microcirculation in the control was reduced after I/R (29% +/- 10%) accompanied by hypotension, increased tissue ET-1 expression (25.0% +/- 6.4% to 67.9% +/- 5.0% 60 minutes after reperfusion), and increased ET-1 level in the portal blood (3.4 +/- 0.9 to 23.6 +/- 6.1 pg/mL). FK506 suppressed ET-1 expression (27.3% +/- 5.2%, 4.1 +/- 2.2 pg/mL), maintained microcirculation (96% +/- 16%), and blood pressure, reduced histologic damage, and improved survival. CsA had a similar but weaker effect compared with FK506. An additional experiment was performed with BQ485Na (BQ), an ETA receptor antagonist, to evaluate the genuine role of ET-1. BQ showed almost the same effects as FK506. CONCLUSIONS: FK506 and CsA, particularly the former, maintain microcirculation and protect the tissue from I/R injury by suppressing the production and release of ET-1.     

Limb allograft in rats: studies on optimal maintenance dose of FK506 and development of graft-versus-host-disease (GVHD)

The optimal dose of FK506 on rat limb allograft was investigated across the BN-to-F344 histocompatibility barrier. BN limb allografts were rejected in untreated F344 hosts within 11 +/- 1 days (mean +/- SD) after operation. A single injection of 0.5 mg/kg, 1.0 mg/kg, or 2.0 mg/kg of FK506 on the day of transplantation significantly prolonged graft survival, mean survival times (MST) based on gross sign of skin rejection were 16 +/- 1 days, 19 +/- 1 days, 21 +/- 1 days, respectively. Maintenance doses of 0, 0.25, 0.5, 1.0, or 2.0 mg/kg/week of FK506 after a single administration of 10 mg/kg of FK506 on the day of limb allograft prolonged the graft survival, 63 +/- 10, 68 +/- 20, 87 +/- 23, 98 +/- 30, 136 +/- 20 days, respectively, and showed no evidence of infection or toxic side effects. The regimen of lower maintenance dose of FK506, however, developed chronic GVHD. In the second set of experiments, development of peripheral blood chimeras was studied in PVG-to-ACl limb allograft model. A single injection of 50 mg/kg of the day of transplantation prolonged graft survival and MST was 154 days. The average rates of peripheral blood chimeras were 2 to 6% and there was no relationship between graft survival and peripheral blood chimeras. However, GVHD developed in one of the six recipients at 201 days after operation. In the similar experiments, grafts were irradiated before operation. Peripheral blood chimeras was not observed in there experiments and GVHD was not developed in 300 days after operation. These data suggest that FK506 is quite effective for rat limb allograft survival in dose-dependent manner and GVHD could be prevented by graft irradiation before operation.     

Neuroprotective actions of FK506 in experimental stroke: in vivo evidence against an antiexcitotoxic mechanism

The cellular mechanisms underlying the neuroprotective action of the immunosuppressant FK506 in experimental stroke remain uncertain, although in vitro studies have implicated an antiexcitotoxic action involving nitric oxide and calcineurin. The present in vivo study demonstrates that intraperitoneal pretreatment with 1 and 10 mg/kg FK506, doses that reduced the volume of ischemic cortical damage by 56-58%, did not decrease excitotoxic damage induced by quinolinate, NMDA, and AMPA. Similarly, intravenous FK506 did not reduce the volume of striatal quinolinate lesions at a dose (1 mg/kg) that decreased ischemic cortical damage by 63%. The temporal window for FK506 neuroprotection was defined in studies demonstrating efficacy using intravenous administration at 120 min, but not 180 min, after middle cerebral artery occlusion. The noncompetitive NMDA receptor antagonist MK801 reduced both ischemic and excitotoxic damage. Histopathological data concerning striatal quinolinate lesions were replicated in neurochemical experiments. MK801, but not FK506, attenuated the loss of glutamate decarboxylase and choline acetyltransferase activity induced by intrastriatal injection of quinolinate. The contrasting efficacy of FK506 in ischemic and excitotoxic lesion models cannot be explained by drug pharmacokinetics, because brain FK506 content rose rapidly using both treatment protocols and was sustained at a neuroprotective level for 3 d. Although these data indicate that an antiexcitotoxic mechanism is unlikely to mediate the neuroprotective action of FK506 in focal cerebral ischemia, the finding that intravenous cyclosporin A (20 mg/kg) reduced ischemic cortical damage is consistent with the proposed role of calcineurin.     

FK 506 'rescue' immunosuppression for obliterative bronchiolitis after lung transplantation

PRELIMINARY EXPERIENCE: In a consecutive case series (level V evidence) involving 10 recipients of unilateral lung transplantation (LT) with bronchiolitis obliterans, in conjunction with Fujisawa protocol 93-0-003, the physiologic responses to FK 506 (tacrolimus) "rescue" immunosuppression were assessed. Recipients were 22+/-18 months post-LT and demonstrated progressive allograft dysfunction that was refractory to pulsed-dose methylprednisolone therapy. All recipients received induction immunosuppression with Minnesota antilymphocyte globulin (10 to 15 mg/kg/d) for 5 to 10 days, cyclosporine (CsA) (whole-blood Abbott TDX fluorescence polarization immunoassay (Abbott Inc, Abbott Park, IL)=600 to 800 ng/mL), azathioprine (2 mg/kg/d), and prednisone (tapered to 0.2 mg/kg/d). The "rescue" regimen consisted of oral FK 506 adjusted to maintain a whole-blood Abbott IMX microparticle enzyme immunoassay (Abbott Inc, Abbott Park, IL) of 10 to 15 ng/mL with an initial increase in prednisone (1.0 mg/kg/d) during conversion that was subsequently tapered to 0.2 mg/kg/d. Spirometry was performed monthly in accordance with accepted American Thoracic Society criteria. Recipients were classified in accordance with the International Society for Heart and Lung Transplantation (ISHLT) "Working Formulation for Standardization of Nomenclature and for Clinical Staging of Chronic Dysfunction in Lung Allografts" as stages Ib (n=2), IIb (n=4), and IIIb (n=4) upon entry to the protocol. The deltaFEV1/month relationships during CsA- and FK 506-based regimens were analyzed by linear regression and compared by signed rank test (p<0.05). RESULTS: The deltaFEV1/month slopes were -0.0687+/-0.0221 and +0.0300+/-0.033 L/mo (mean+/-SEM) for CsA and FK 506, respectively (p=0.037). Although no significant spirometric improvement was noted in most recipients, no further decline in FEV1 occurred after conversion to FK 506. Recipients with less severe chronic dysfunction (ie, obliterative bronchiolitis [OB] stages Ib and IIb) stabilized their spirometric indexes at higher levels. Two recipients with OB stage IIIb died of hypercapnic respiratory failure at 6 and 8 months after conversion. CONCLUSIONS: The deltaFEV1/mo slopes stabilized after FK 506 conversion. Earlier conversion may be beneficial in stabilizing FEV1 at a higher plateau. Significant economic impact may be anticipated with FK 506 compared to alternative cytolytic strategies for OB. However, multicenter prospective controlled investigations are necessary to further address the potential role of FK 506 after LT (level I evidence). Furthermore, the ISHLT "Staging of OB Syndrome" may have significant clinical implications vis-à-vis prognosis and potential therapies.     

Quantitative immunohistochemical changes in the endocrine pancreas of nonobese diabetic (NOD) mice

Two-centimeter nerve allografts were transplanted across a major histocompatibility barrier from donor ACI rats into a 0.5-cm gap in the sciatic nerve of recipient Lewis rats and immunosuppressed with FK506, 2 mg/kg per day for 3 months. One group of animals continued to receive intermittent immunosuppression with FK506, 2 mg/kg twice a week for another 2 months, whereas the second group of animals received no further immunosuppression in order to determine whether rejection of nerve allografts can still occur after immunosuppression is withdrawn, even after the axons have regenerated through the nerve graft. The sciatic function index improved from -76.3 at 3 months to -46.6 at 5 months in those animals continuing to receive intermittent immunosuppression, but only improved to -66.8 at 5 months when immunosuppression was discontinued. Similarly, somatosensory evoked potentials demonstrated an improvement in relative latency from 2.3 msec at 3 months to 0.34 msec at 5 months in animals continuing to receive intermittent immunosuppression, but only improved to 1.29 msec at 5 months when immunosuppression was discontinued. Nerve allografts continuing to receive intermittent immunosuppression showed no signs of rejection by light or electron microscopy and no significant difference compared with isografts, whereas nerve allografts whose immunosuppression had been stopped at 3 months showed mild signs of rejection, less regeneration, and a smaller number of nerve fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional and structural characteristics of experimental FK 506 nephrotoxicity

1. FK 506 (Tacrolimus, Prograf) is a novel immunosuppressant which is effective in solid organ transplantation and autoimmune diseases. The lack of a suitable animal model has hindered the study of the nephrotoxicity of the drug which has emerged as a common adverse effect in clinical trials. We report both acute and chronic nephrotoxicity with tacrolimus (FK) in which renal structure and function are worsened by sodium depletion. 2. Pair fed male Sprague-Dawley rats were given FK (3 or 6 mg/kg, p.o.) or vehicle for 7, 21 and 42 days on low salt or normal diet. The FK whole blood trough levels achieved (3-10 ng/mL) were similar to those observed in FK treated transplant patients. 3. In salt depleted animals treated for 7 days, FK (6 mg/kg) decreased renal blood flow and glomerular filtration rate (1.8 +/- 0.1 and 0.2 +/- 0.1 mL/min per 100 g vs 2.9 +/- 0.2 and 1.1 +/- 0.1 mL/min per 100 g in the vehicle group, P < 0.01). 4. After 21 days of treatment of FK on low salt diet but not normal salt, FK induced focal collapse and vacuolization in proximal tubules and discrete or confluent zones of tubulointerstitial oedema and mononuclear cell infiltration. 5. After 42 days in salt depleted rats, there was significant tubulointerstitial scarring that was associated with an increased plasma renin activity (PRA) (64 +/- 10 vs 30 +/- 4 ng AI/mL per h in the vehicle group, P < 0.05). Animals given normal salt diets did not develop significant histological lesions even up to 42 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the developmental toxicity of methacrylonitrile in Sprague-Dawley rats and New Zealand white rabbits

Timed-pregnant Sprague-Dawley (CD) outbred rats and New Zealand White rabbits were dosed by gavage with methacrylonitrile (MACR) in distilled water during major organogenesis. Rats were dosed on Gestational Days (GD) 6 through 15 (0, 5, 25, or 50 mg MACR/kg/day) and rabbits on GD 6 through 19 (0, 1, 3, or 5 mg MACR/kg/day). Maternal clinical status was monitored daily during treatment. At termination (GD 20, rats; GD 30, rabbits), confirmed-pregnant females (25-26 per group, rats; 17-22 per group, rabbits) were evaluated for clinical status and gestational outcome; each live fetus was examined for external, visceral, and skeletal malformations. In rats, no treatment-related maternal clinical signs or mortality were observed, nor was there any adverse effect on maternal body weight or food or water consumption. At necropsy, absolute, relative, and adjusted maternal liver weight was increased at the mid- and high-dose groups, an effect that may be indicative of induction of hepatic enzymes rather than toxicity. In the absence of any indication of maternal toxicity, the no-observed-adverse-effect level (NOAEL) for maternal toxicity in this study was >/=50 mg MACR/kg/day. The NOAEL for developmental toxicity in rats was also >/=50 mg MACR/kg/day. There was no effect of treatment on postimplantation loss, mean fetal body weight per litter, or morphological development. In rabbits, maternal mortality and clinical signs were not dose related. Maternal food consumption, body weight, and liver weight were not adversely affected by treatment. Thus, the maternal NOAEL was >/=5 mg MACR/kg/day. Maternal toxicity, including death, was observed >/=7.5 mg/kg/day in preliminary studies. The developmental NOAEL was also >/=5 mg MACR/kg/day. There was no adverse effect of treatment on postimplantation loss or fetal body weight. A significant decrease in the percentage male fetuses per litter was observed, although there was no effect on total live litter size, suggesting that the reduction in the ratio of live male fetuses in the high-dose group was not biologically significant. MACR had no adverse effect on morphological development. In summary, oral administration of MACR to rats and rabbits during organogenesis, at doses that did not cause persistent maternal toxicity (50 mg MACR/kg/day, rats; 5 mg MACR/kg/day, rabbits), also did not cause any adverse developmental effects.     

Evidence that SMS 201-995 enhances the immunosuppressive effect of FK506

Somatostatin (SS) was originally described as a growth hormone release inhibiting factor synthesised in the hypothalamus. Recently, SS and its receptor (SSTR) have been demonstrated in lymphoid tissues and seem to play a regulatory, largely inhibitory, role in immune responses. The aim of the present study was to check the immunosuppressive effect of a SS derived peptide, the octreotide (SMS 201-995) and to verify whether this molecule acted synergistically with FK506. An immunosuppressive effect of SMS was observed on the proliferation of rat spleen cells induced in vitro, either by polyclonal mitogens such as PHA or by alloantigens. With PHA stimulation, 10(-14) M SMS significantly enhanced the immunosuppressive action of 0.00001 microg/ml FK506. The addition of SMS in MLR (10(-11)-10(-9)M) increased the antiproliferative effect of both 0.0001 microg/ml and 0.00001 microg/ml FK506. In consideration of the extremely low concentration of both drugs that was required to obtain a good immunosuppression in vitro, we verified the association of FK506 and SMS in vivo in an allogeneic skin graft model that used Lewis (Lew) rats as donors and Brown Norway (BN) rats as recipients. BN treated with 0.1 mg/kg FK506 and 0.5-10 microg/kg SMS showed a significant increase in mean skin allograft survival time when compared to either a monotherapy or control group. None of the animals died or showed signs of drug-related toxicity. In conclusion, a combined therapy of SMS and FK506, administered at lower dosages than those that are considered therapeutic, led to an effective immunosuppression without any undesirable side effects.     

Altered proton extrusion in cells adapted to growth at low extracellular pH

Coronary vasodilator and hemodynamic profiles of JTV-506, a newly synthesized 2,2-bis-methoxymethyl benzopyran-derivative potassium channel opener, were evaluated in conscious dogs. JTV-506 (2.5-10 microg/kg, i.v.) elicited dose-dependent increases in coronary blood flow (CBF) and heart rate (HR) but only slight changes in mean blood pressure (MBP). Other vasodilators such as levcromakalim, nicorandil, diltiazem, and nitroglycerin, when administered intravenously, elicited increases in CBF and HR and a decrease in MBP. When dosed orally JTV-506 (0.01-0.1 mg/kg), levcromakalim (0.01-0.1 mg/kg), nicorandil (1-10 mg/kg), and nifedipine (3-30 mg/kg) also elicited increases in CBF and HR and a decrease in MBP. JTV-506 caused a marked increase in CBF with slight changes in HR and MBP. In contrast to JTV-506, however, the changes caused by levcromakalim, nicorandil, and nifedipine were accompanied by a marked increase in HR and a marked decrease in MBP. These results suggest that the action of JTV-506 on hemodynamics is different from that of other vasodilators, including reference potassium channel openers, and that the profile of cardiovascular action of JTV-506 may be useful in the treatment of angina pectoris.