Factors influencing magnitude and duration of target inhibition following antibody therapy: implications in drug discovery and development.

Antibodies or antibody-related fusion proteins binding to soluble antigens in plasma form an important subclass of approved therapeutics. Pharmaceutical companies are constantly trying to accelerate the pace of drug discovery and development of these antibodies and identify superior candidates in face of significant attrition rates. Understanding the interplay between drug- and target-related factors on magnitude and duration of target inhibition is imperative for successful advancement of these therapeutics. Simulations using a target-mediated drug disposition model were performed to evaluate the influence of antibody-target binding affinity, baseline target concentration, and target turnover on magnitude and duration of soluble target inhibition. These simulations assumed intravenous dosing of the antibody and evaluated multiple parameters over a wide range. These simulations reveal that improvement in affinity reaches a point of diminishing returns following which further improvement in affinity does not alter the magnitude and more importantly the duration of target inhibition. Evaluation of unbound antibody and target kinetics indicated that point of diminishing returns in duration of inhibition was due to target-mediated binding and subsequent elimination of antibody at later time points. Similarly, influence of baseline target concentration and target turnover on magnitude and duration of target inhibition in plasma is shown. Additionally, the fraction of dose eliminated via target mediated elimination (Fel(™)) can be a useful tool to enable selection of strategies to increase duration of target inhibition. The implications of these simulations in drug discovery and development with regard to target identification, antibody optimization, and backup candidate selection are discussed.

Effects of methylprednisolone and its liver-targeted dextran prodrug on ischemia-reperfusion injury in a rat liver transplantation model.

PURPOSE: To evaluate the effectiveness of a liver-targeted dextran prodrug (DMP) of methylprednisolone (MP) in cold preservation-warm reperfusion injury associated with liver transplantation. METHODS: The effects of donor pretreatment with single 5 mg/kg doses of MP or DMP on ischemia-reperfusion damage to the liver were studied after 8 or 24 h of cold preservation in both isolated perfused rat liver (IPRL) and syngeneic orthotopic rat liver transplantation (OLT) models. RESULTS: In IPRL studies, donor pretreatment with DMP, and to a lesser degree MP, significantly improved the uptake of hyaluronic acid (HA), a marker of endothelial cell function, following 8 h of cold preservation. However, neither pretreatment was protective after 24 h of preservation. In the OLT model using 24-h-preserved livers, the seven-day survival of untreated grafts was 50%. DMP pretreatment of donors significantly improved graft survival to 100%, whereas MP pretreatment was ineffective. Additionally, only DMP significantly increased the blood glucose concentrations and decreased the plasma concentrations of tumor necrosis factor-alpha after OLT. Other measured markers of liver injury were not affected by either pretreatment. CONCLUSIONS: Selective delivery of methylprednisolone to the liver as a donor pretreatment strategy improves 24-h preserved graft survival in the OLT model.

Attenuation of acute rejection in a rat liver transplantation model by a liver-targeted dextran prodrug of methylprednisolone.

BACKGROUND: The use of methylprednisolone (MP) and other corticosteroids for the treatment of acute liver allograft rejection is associated with severe toxicities in nontarget tissues. Therefore, selective delivery of MP to the liver may improve its efficacy and alleviate its side effects. We investigated the effects of a novel liver-targeted dextran prodrug of MP (DMP) in an orthotopic rat liver transplantation (OLT) model. METHODS: The model consisted of a high responder rejection strain combination (Dark Agouti donors and Lewis recipients). Liver recipients were intravenously administered saline or a single subtherapeutic dose of MP (5 mg/kg) as the parent drug (MP) or its prodrug (DMP). Different groups were then monitored for graft survival or euthanized 5 or 9 days posttransplantation. Plasma chemistry, including alkaline phosphatase and bilirubin, allograft histology, and survival duration were determined. RESULTS: Untreated recipients exhibited elevated plasma levels of liver injury markers, progressive portal and venous inflammation and cellular infiltration in liver allografts, and a mean graft survival time (MST) of 10.5 days. MP treatment did not alter any of these parameters. In contrast, a single dose of DMP resulted in a decrease in plasma levels of liver injury markers, a decrease in histological grade of rejection on day 5, and a substantial increase in MST (27.5 days). CONCLUSIONS: These results demonstrate attenuation of acute rejection following local (allograft) immunosuppression with a single subtherapeutic dose of MP delivered as a liver-targeted prodrug. Dextran prodrugs may be useful for selective delivery of immunosuppressants to the liver following liver transplantation.

Effects of duration of ischemia and donor pretreatment with methylprednisolone or its macromolecular prodrug on the disposition of indocyanine green in cold-preserved rat livers.

PURPOSE: Cold preservation of the liver before transplantation may change uptake and excretory functions of hepatocytes. We hypothesized that an increase in the duration of preservation would result in a progressive decrease in the hepatic uptake and/or biliary excretion of indocyanine green (ICG), which would be attenuated by pharmacologic interventions. METHODS: Donor rats (n = 40) were administered saline (control) or single 5 mg/kg doses of methylprednisolone (MP) or its liver-targeted prodrug (DMP) 2 h prior to liver harvest. Following preservation in cold University of Wisconsin solution for 0, 24, 48, or 72 h, livers were reperfused in a single-pass manner for 30 min in the presence of ICG (approximately 4 microg/ml), followed by 60 min of ICG-free perfusion. The inlet, outlet, and bile concentrations of ICG were measured periodically by high performance liquid chromatography (HPLC), and kinetic parameters were estimated. RESULTS: Effects of duration of preservation: In unpreserved livers, a significant portion of ICG dose (16%) was effluxed from the liver during the washout period. Cold preservation for 24-72 h progressively increased (p < 0.05) the efflux of ICG (>2-fold at 72 h). Similarly, average extraction ratio showed a modest (30-40%) decrease with increasing preservation time (p < 0.05). However, biliary excretion of ICG showed the most sensitivity to the preservation time (14 to >800-fold decline). Effects of pretreatment: DMP caused significant (p < 0.05) increases in biliary ICG levels (>12-fold) and bile flow rates (6-15-fold) of preserved livers. Although MP pretreatment significantly (p < 0.05) increased (6-fold) bile flow rates in 48-h preserved livers, its effects on biliary ICG levels were not significant (p > 0.05). CONCLUSIONS: Biliary excretion of ICG is the most sensitive kinetic parameter to prolonged cold ischemia-reperfusion injury in a rat liver perfusion model. The injury may be significantly attenuated by pharmacologic pretreatment of the liver donors.

Hepatic disposition of cyclosporine A in isolated perfused rat livers.

PURPOSE: To develop an isolated perfused rat liver model to study the hepatic disposition of cyclosporine A (CyA) in both sexes. METHODS: Livers were isolated from male (n = 6) and female (n = 7) rats and perfused with a physiological buffer in a single-pass manner. A bolus 1-mg dose of CyA was injected into the inlet catheter and periodical samples (0-15 min) were collected from the outlet perfusate. The concentrations of CyA in the outlet perfusate, collected bile (0-15 min), and liver tissue (at the end of perfusion) were quantitated by HPLC and subjected to statistical moment analysis. RESULTS: The dilution curves of CyA in the outlet perfusate exhibited unusually long terminal phases due to large volume of distribution of the drug (approximately 100 mL/g) and its slow release from binding sites in the liver (net release rate constant of approximately 0.020 min(-1)). This was in contrast to the rapid uptake of the drug, indicated by significant amounts of the intact drug (>40%) taken up during one single pass through the liver. Consequently, the liver tissue:perfusate distribution ratio of CyA was very high (approximately 220). No significant differences were found between the male and female livers in any of the estimated parameters. CONCLUSIONS: The tissue binding of cyclosporine A is substantial, slowly reversible, and gender-independent in isolated perfused rat livers.

Attenuation of Kupffer cell activation in cold-preserved livers after pretreatment of rats with methylprednisolone or its macromolecular prodrug.

PURPOSE: Activation of hepatic Kupffer cells (KCs) during organ preservation and subsequent reperfusion causes release of proinflammatory mediators and is responsible, at least in part, for rejection of transplanted livers. Our hypothesis was that donor pretreatment, before liver harvest, with methylprednisolone (MP) or its dextran prodrug (DMP) would reduce KC activation. METHODS: Adult donor rats were administered a single 5-mg/kg (MP equivalent) IV dose of MP or DMP or saline 2 h before liver harvest. The livers were then stored in University of Wisconsin solution for 24, 48, or 96 h (n = 4/treatment/time). A recirculating perfusion model was used to study, for 180 min, the release of KC activation markers, tumor necrosis factor (TNF)-alpha and acid phosphatase, and other biochemical indices from the cold-preserved livers. RESULTS: Cold ischemia-reperfusion resulted in release of substantial levels of TNF-alpha in untreated groups. Pretreatment of rats with MP or DMP caused a significant (p < 0.0001) reduction in TNF-alpha AUC in the perfusate, with no significant differences between MP and DMP. The maximum inhibitory effect of MP (77.5 +/- 10.2%) was observed after 48 h of preservation, whereas DMP showed maximal inhibition of TNF-alpha AUC at both 24 (74.5 +/- 15.8%) and 48 (74.8 +/- 12.6%) h of preservation. Similarly, both MP and DMP resulted in a significant (p < 0.0004) decrease in acid phosphatase levels of cold-preserved livers. However, neither pretreatment had any substantial effect on the levels of other biochemical markers. CONCLUSIONS: Both MP and DMP pretreatments decreased the release of TNF-alpha and acid phosphatase from livers subjected to cold ischemia preservation. Therefore, pretreatment of liver donors with MP or its prodrug decreases KC activation by cold ischemia-reperfusion.

Dextran-methylprednisolone succinate as a prodrug of methylprednisolone: local immunosuppressive effects in liver after systemic administration to rats.

PURPOSE: The purpose of this work was to study the local immunosuppressive effects of systemically administered methylprednisolone (MP) and its prodrug, dextran-methylprednisolone (DMP), in rat livers. METHODS: Single 5 mg/kg (MP equivalent) doses of MP or DMP were injected intravenously to rats, and livers were isolated at different time points (0-72 h; n = 4/time point). Isolated livers were stimulated ex vivo with bacterial lipopolysaccharide, and outlet perfusate and bile samples were analyzed for their concentrations of tumor necrosis factor (TNF)-alpha by enzyme-linked immunosorbent assay. The area under the perfusate TNF-alpha concentration-time curve (AUC) was used as a measure of immune response. Hepatic concentrations of MP and DMP were also measured by high-performance liquid chromatography. RESULTS: Both MP and DMP resulted in a decrease in lipopolysaccharide-induced increase in TNF-alpha AUC. MP injection 8 h before liver isolation resulted in a maximum of 50% decrease in TNF-alpha AUC. Compared with MP, the maximum effect of the prodrug (DMP) was both more intense (approximately 80% reduction in TNF-alpha AUC) and delayed (maximum inhibition at 24 h). Overall, the area under the effect (% inhibition of TNF-alpha)-time (%inhibition-h) for DMP (3,680 +/- 406) was approximately four times more than that for the parent drug (846 +/- 114). Whereas the MP concentrations in the liver were not quantifiable after the injection of the parent drug, relatively large concentrations of DMP and regenerated MP were found in the liver of DMP-injected rats. CONCLUSIONS: After systemic administration to rats, both MP and DMP exhibit local immunosuppressive effects in the liver. The local effects of the prodrug (DMP), however, appear to be more intense and sustained than those of the parent drug (MP).

Microsomal cytochrome P450 levels and activities of isolated rat livers perfused with albumin.

PURPOSE: We recently showed that the perfusion of isolated rat livers with perfusates containing bovine serum albumin (BSA) would significantly stimulate the release of tumor necrosis factor (TNF)-alpha. Here, we hypothesize that BSA-induced increase in the release of TNF-alpha, and possibly other cytokines, would affect cytochrome P450 (CYP)-mediated drug metabolism. METHODS: Rat livers were perfused ex vivo for 1, 2, or 3 h with a physiologic buffer containing or lacking 1% BSA (n = 4-5/group). At the end of perfusion, liver microsomes were prepared and analyzed for their total CYP, CYP2E1, CYP3A2, and CYP2C11 protein contents and the activities of cytochrome c reductase, CYP2E1, CYP3A2, CYP2C11, CYP2E1, CYP2D1, CYP1A1, and CYP2B1/2. In addition, the concentrations of various cytokines and nitric oxide were quantified in the outlet perfusate. RESULTS: In the absence of BSA, the perfusate levels of all measured cytokines and nitric oxide were low. However, when the perfusate contained BSA, the levels of TNF-alpha, interleukin-6, and nitric oxide increased significantly (p < 0.005). Perfusion of the livers for 3 h with the BSA-containing perfusate resulted in significant (p < 0.05) decreases in the total CYP (41%), CYP2E1 (59%), CYP3A2 (68%), and CYP2C11 (50%) protein contents and activities of cytochrome c reductase (31%), CYP2E1 (66%), CYP3A2 (54%), and CYP2G11 (51%). In contrast, perfusion of livers for 1 or 2 h with the BSA perfusate did not have any significant effect on CYP-mediated metabolism. The CYP1A2, CYP2D1, and CYP2B1/2 activities were not affected by BSA, regardless of perfusion time. CONCLUSION: Addition of BSA to perfusates, which is a routine practice in isolated rat liver studies, can reduce CYP-mediated drug metabolism by a mechanism independent of protein-binding effect.

High-performance liquid chromatographic analysis of cyclosporin A in rat blood and liver using a commercially available internal standard.

All the available HPLC assays of cyclosporin A (CyA) use internal standards that are not commercially available. Our purpose was to develop an HPLC assay for measurements of CyA in rat blood and liver using a commercially available internal standard (I.S.). After the addition of tamoxifen (I.S.), blood (0.25 ml) or the liver homogenate (1 ml) samples were extracted into a mixture of ether:methanol (95:5). The residue after evaporation of the organic layer was dissolved in 200 microl of an injection solution and washed with 1 ml of hexane before analysis. The separation was achieved using an LC-1 column (70 degrees C) with a mobile phase of methanol-acetonitrile-0.01 M KH(2)PO(4) (50:25:25, v/v) and a flow-rate of 1 ml/min. Detection was at 205 nm. Cyclosporin A and I.S. eluted at 5 and 7 min, respectively, free from endogenous peaks. Linear relationships (r>0.98) were observed between the CyA:I.S. peak area ratios and the CyA concentrations within the range of 0.2-10 microg/ml for blood and 0.1-4 microg/ml for the liver homogenates. The intra- and inter-run C.V.s and errors for both the blood and liver samples were <15%. The extraction efficiency (n=5) was close to 100% for both CyA and I.S. in both blood and liver homogenates. The lower limit of quantitation of the assay was 0.2 or 0.1 microg/ml based on 250 microl of blood or 1 ml of liver homogenate, respectively. The assay was capable of measuring blood and liver concentrations of CyA in a rat injected intravenously with a single 5-mg/kg dose of the drug.