Antigen-specific suppression of inflammatory arthritis by dendritic cells

Purpose Antigen-specific suppression of a previously primed immune response is a major challenge for immunotherapy of autoimmune disease. We have shown that NF-κB inactivation in dendritic cells (modified DC) converts them into cells that tolerize rather than immunize to specific antigen [1]. Antigen-exposed modified DC prevent priming of immunity, and they suppress previously primed immune responses. Regulatory CD4+ T cells, which can transfer antigen-specific tolerance in an IL-10-dependent fashion, mediate the tolerance. We hypothesized that modified DC exposed to arthritogenic antigen would suppress clinical arthritis after disease onset. Methods Antigen-induced arthritis was induced in C57/Bl6 mice by priming to methylated bovine serum albumin (mBSA) antigen followed by challenge injection of mBSA to one knee. Knee swelling was apparent within 2 days, with peak clinical signs apparent at 5 days. Mice were treated with antigen-exposed modified DC between 2 and 6 days after mBSA challenge to the knee joint. Results Clinical arthritis was suppressed in each group receiving mBSA-exposed modified DC within 4 days compared with mice that received either no DC or keyhole limpet hemocyanin-exposed modified DC. Clinical improvement was associated with mBSA-specific tolerance in mice receiving mBSA-exposed modified DC. Tolerance induction was not impaired by concomitant administration of anti-tumor necrosis factor alpha monoclonal antibody. Subsequent rechallenge with intra-articular IL-1 induced flare of arthritis in all groups, which could be effectively suppressed by a second administration of mBSA-exposed modified DC. Conclusions The data indicate that modified DC induce antigen-specific immune suppression in this model of inflammatory arthritis, even after full clinical expression of the disease. These observations have important implications for antigen-specific therapy of autoimmunity.

Methadone maintenance therapy for opioid dependence - A guide to appropriate use

Opioid dependence is a chronic, relapsing condition that is associated with significant morbidity and mortality. Methadone maintenance therapy involves the provision of a controlled supply of an orally administered opioid, thereby stabilising the opioid-dependent patient. Research studies have shown that methadone maintenance reduces illicit opioid use, opioid-related crime, premature mortality and the risk of HIV infection. It is most effective when prescribed at an adequate dosage (usually 60 to 100 mg/day) and when long term maintenance on methadone is the goal of treatment rather than detoxification from all drugs including methadone. Successful long term methadone maintenance is more likely when it takes place within the context of a well established therapeutic relationship and when the medical, social and psychological needs of patients are met either through direct assistance or referral.

An optimized model for rat liver perfusion studies

Conditions which influence the viability, integrity, and extraction efficiency of the isolated perfused rat liver were examined to establish optimal conditions for subsequent work in reperfusion injury studies including the choice of buffer, use of oncotic agents, hematocrit, perfusion flow rate, and pressure. Rat livers were perfused with MOPS-buffered Ringer solution with or without erythrocytes. Perfusates were collected and analyzed for blood gases, electrolytes, enzymes, radioactivity in MID studies, and lignocaine in extraction studies. Liver tissue was sampled for histological examinations, and wet:dry weight of the liver was also determined. MOPS-buffered Ringer solution was found to be superior to Krebs bicarbonate buffer, in terms of pH control and buffering capacity, especially during any prolonged period of liver perfusion. A pH of 7.2 is chosen for perfusion since this is the physiological pH of the portal blood. The presence of albumin was important as an oncotic agent, particularly when erythrocytes were used in the perfusate. Perfusion pressure, resistance, and vascular volume are how-dependent and the inclusion of erythrocytes in the perfusate substantially altered the flow characteristics for perfusion pressure and resistance but not vascular volume. Lignocaine extraction was relatively flow-independent. Perfusion injury as defined by enzyme release and tissue fine structure was closely related to the supply of O-2. The optimal conditions for liver perfusion depend upon an adequate supply of oxygen. This can be achieved by using either erythrocyte-free perfusate at a how rate greater than 6 ml/min/g liver or a 20% erythrocyte-containing perfusate at 2 ml/min/g. (C) 1996 Academic Press, Inc.

Haemonchus contortus: The uptake and metabolism of closantel

Closantel is an anthe lmintic which associates with plasma albumin and is useful for the control of sheep parasites, such as Haemonchus contortus, that ingest blood. However, the utility of closantel for parasite control has been threatened by the emergence of resistance. The mechanisms of resistance are unknown. A closantel-resistant and a closantel-susceptible isolate of H. contortus were compared with respect to the distribution and metabolism of closantel. Neither strain appeared to metabolise closantel in vitro or in vivo. Following treatment of infected sheep with radioactively labelled closantel, isotope levels in closantel-resistant adult H. contortus were significantly lower than in susceptible worms. This reduced accumulation of drug could contribute to closantel resistance by mechanisms such as reduced feeding, failure to dissociate the drug-albumin complex in the gut or increased efflux of closantel from resistant worms. (C) 1997 Australian Society for Parasitology.

Isolated limb perfusion with melphalan for human melanoma xenografts in the hindlimb of nude rats: A surviving animal model

We have established a surviving model of isolated limb perfusion using xenografts of the human melanoma cell line MM 96L injected subcutaneously into the hindlimb of a nude rat, The femoral artery and vein were cannulated via the left renal artery and vein and the hind limb was isolated using tourniquets. The limb was perfused with Krebs Heinseleit buffer at 37 degrees C containing 4.7% bovine serum albumin at a constant flow rate of 4 mi per min for 30-60 min with 100% survival of the animals, Tumour vascularization and blood flow were demonstrated using vascular casts and [Cr-51]-microspheres. Following the addition of melphalan (15 or 100 mu g/ml), drug concentrations in the perfusate, tissues and systemic circulation were determined using high pressure liquid chromatography (HPLC), Systemic leakage, assessed using [I-125]albumin and melphalan and detected by a gamma-counter and HPLC respectively, was <0.5%. The melphalan concentration and tissue flow rate in the tumour deposits were 40 and 30% respectively, when compared with the surrounding subcutaneous tissue, At a dose of 15 mu g/ml, melphalan caused a reduction in tumour growth after 60 min perfusion, and a significant reduction in tumour size was seen when the melphalan dose was 100 mu g/ml. The surviving nude rat model of isolated limb perfusion for recurrent melanoma will allow examination of optimal perfusion conditions, along with the pharmacokinetics, pharmacodynamics and efficacy of melphalan and other drugs.

Melphalan dosing regimens for management of recurrent melanoma by isolated limb perfusion: Application of a physiological pharmacokinetic model based on melphalan distribution in the isolated perfused rat hindlimb

The optimal dosing schedule for melphalan therapy of recurrent malignant melanoma in isolated limb perfusions has been examined using a physiological pharmacokinetic model with data from isolated rat hindlimb perfusions (IRHP), The study included a comparison of melphalan distribution in IRHP under hyperthermia and normothermia conditions. Rat hindlimbs were perfused with Krebs-Henseleit buffer containing 4.7% bovine serum albumin at 37 or 41.5 degrees C at a flow rate of 4 ml/min. Concentrations of melphalan in perfusate and tissues were determined by high performance liquid chromatography with fluorescence detection, The concentration of melphalan in perfusate and tissues was linearly related to the input concentration. The rate and amount of melphalan uptake into the different tissues was higher at 41.5 degrees C than at 37 degrees C. A physiological pharmacokinetic model was validated from the tissue and perfusate time course of melphalan after melphalan perfusion. Application of the model involved the amount of melphalan exposure in the muscle, skin and fat in a recirculation system was related to the method of melphalan administration: single bolus > divided bolus > infusion, The peak concentration of melphalan in the perfusate was also related to the method of administration in the same order, Infusing the total dose of melphalan over 20 min during a 60 min perfusion optimized the exposure of tissues to melphalan whilst minimizing the peak perfusate concentration of melphalan. It is suggested that this method of melphalan administration may be preferable to other methods in terms of optimizing the efficacy of melphalan whilst minimizing the limb toxicity associated with its use in isolated limb perfusion.

The effects of perfusion conditions on melphalan distribution in the isolated perfused rat hindlimb bearing a human melanoma xenograft

An isolated rat hindlimb perfusion model carrying xenografts of the human melanoma cell line MM96 was used to study the effects of perfusion conditions on melphalan distribution. Krebs-Henseleit buffer and Hartmann's solution containing 4.7% bovine serum albumin (BSA) or 2.8% dextran 40 were used as perfusates. Melphalan concentrations in perfusate, tumour nodules and normal tissues were measured using high-performance liquid chromatography (HPLC). Increasing the perfusion flow rates (from 4 to 8 mi min(-1)) resulted in higher tissue blood flow (determined with Cr-51-labelled microspheres) and melphalan uptake by tumour and normal tissues. me distribution of melphalan within tumour nodules and normal tissues was similar for both Krebs-Henseleit buffer and Hartmann's solution; however, tissue concentrations of melphalan were significantly higher for a perfusate containing 2.8% dextran 40 than for one containing 4.7% BSA. The melphalan concentration in the tumour was one-third of that found in the skin if the perfusate contained 4.7% BSA. In conclusion, this study has shown that a high perfusion flow enhances the delivery of melphalan into implanted tumour nodules and normal tissues, and a perfusate with low melphalan binding (no albumin) is preferred for maximum uptake of drug by the tumour.

Percutaneous absorption of sunscreen agents from liquid paraffin: Self-association of octyl salicylate and effects on skin flux

This study provides an investigation of the availability of octyl salicylate (OS), a common sunscreen agent, from liquid paraffin and the effect of OS on skin permeability. A model membrane system to isolate the vehicle effect from membrane permeability has been developed. Partitioning of OS between liquid paraffin and aqueous receptor phases was conducted. Partition coefficients increased with increase in OS concentration. A range of OS concentrations in liquid paraffin was diffused across human epidermis and synthetic membranes into 4% bovine serum albumin in phosphate-buffered saline and 50% ethanol. Absorption profiles of OS obtained from silicone and low-density polyethylene (LDPE) membranes were similar to each other but higher than for the high-density polyethylene [HDPE (3 times)] membrane and human epidermis (15 times). The steady state fluxes and apparent permeability coefficients (K-p') obtained from the diffusion studies showed the same trends with all membranes, except for the HDPE membrane which showed greater increase in flux and K-p' at concentrations above 30%. IR spectra showed that several bands of OS were shifted with concentrations, and the molecular models further suggested that the main contribution to the self-association is from non-1,4 van der Waals interactions.

Synthesis, identification, characterization, stability, solubility, and protein binding of ester derivatives of salicylic acid and diflunisal

O-Acyl esters were prepared from salicylic acid and diflunisal by esterification with the appropriate acyl anhydride (in the presence of sulfuric acid at 80 degrees C) or acyl chloride (in the presence of pyridine at 0 degrees C). Synthesis, identification and characterization of these compounds is described. In vitro hydrolysis, solubility and protein binding studies of these O-acyl esters were performed. For the diflunisal esters, the melting points fell as the side chain was increased from ethyl to pentyl. The melting points showed no significant difference as the length of the side chain was increased from pentyl to heptyl. The aspirin analogues showed a similar trend, The relationship between solubility and carbon chain length agreed closely with that for the melting points with carbon chain length. In vitro non-enzymatic hydrolysis studies concluded that: (1) hydrolysis rate constants generally decreased with carbon chain length; (2) the diflunisal esters have shorter half lives compared with their salicylate counterparts; and (3) the in vitro hydrolysis of these compounds was retarded by the presence of bovine serum albumin. Protein binding experiments showed that the strength of binding of the aspirin and diflunisal analogues to bovine serum albumin increased with carbon chain length. (C) 1997 Elsevier Science B.V.

On the validity of the dispersion model of hepatic drug elimination when intravascular transit time densities are long-tailed

The dispersion model with mixed boundary conditions uses a single parameter, the dispersion number, to describe the hepatic elimination of xenobiotics and endogenous substances. An implicit a priori assumption of the model is that the transit time density of intravascular indicators is approximated by an inverse Gaussian distribution. This approximation is limited in that the model poorly describes the tail part of the hepatic outflow curves of vascular indicators. A sum of two inverse Gaussian functions is proposed as ail alternative, more flexible empirical model for transit time densities of vascular references. This model suggests that a more accurate description of the tail portion of vascular reference curves yields an elimination rate constant (or intrinsic clearance) which is 40% less than predicted by the dispersion model with mixed boundary conditions. The results emphasize the need to accurately describe outflow curves in using them as a basis for determining pharmacokinetic parameters using hepatic elimination models. (C) 1997 Society for Mathematical Biology.

Methimazole and propylthiouracil equally cross the perfused human term placental lobule

Propylthiouracil (PTU) is widely believed to cross the placenta less freely than methimazole (MMI) and is therefore regarded as the preferred drug for treatment of hyperthyroidism in pregnancy. Clinical studies comparing the two drugs show, however, no differences in maternal or fetal thyroid function. We investigated transfer from the maternal to the fetal circuit in the isolated perfused term human placental lobule of low and high doses of PTU (4 mu g/mL and 40 mu g/mL) and MMI(1.5 mu g/mL and 15 mu g/mL) in protein-free perfusate and low doses of both drugs with addition of 40 g/L of bovine albumin. Both drugs readily crossed the placenta, reaching equilibrium in all experiments in about 2 h. Drug concentrations in the two circuits fitted a two compartmental model. Transfer kinetics for the two drugs were similar, nonsaturable, and unaffected by addition of albumin. Clearances (mL.min(-1).g(-1), means +/- SD) of PTU from maternal to fetal circuits were: 0.229 +/- 0.110, 0.216 +/- 0.065, and 0.170 +/- 0.032; and for transfer of MMI: 0.165 +/- 0.025, 0.232 +/- 0.153, and 0.174 +/- 0.009 (for low doses without, low doses with, and high doses without albumin, respectively). Clearances of PTU from fetal to maternal circuits were: 0.147 +/- 0.072, 0.109 +/- 0.014, and 0.116 +/- 0.028; and for transfer of MMI: 0.095 +/- 0.029, 0.122 +/- 0.088, and 0.12 +/- 0.005 (in the same experiments). There was no significant difference between drugs or drug doses and no effect of addition of albumin. We conclude that PTU and MMI have similar placental transfer kinetics.

Interconnected-tubes model of hepatic elimination

The distributed-tubes model of hepatic elimination is extended to include intermixing between sinusoids, resulting in the formulation of a new, interconnected-tubes model. The new model is analysed for the simple case of two interconnected tubes, where an exact solution is obtained. For the case of many strongly-interconnected tubes, it is shown that a zeroth-order approximation leads to the convection-dispersion model. As a consequence the dispersion number is expressed, for the first time, in terms of its main physiological determinants: heterogeneity of flow and density of interconnections between sinusoids. The analysis of multiple indicator dilution data from a perfused liver preparation using the simplest version of the model yields the estimate 10.3 for the average number of interconnections. The problem of boundary conditions for the dispersion model is considered from the viewpoint that the dispersion-convection equation is a zeroth-order approximation to the equations for the interconnected-tubes model. (C) 1997 Academic Press Limited.

The distribution of naloxone to heroin users

Overdose deaths are a manor contributor to excess mortality among heroin users. It has been proposed that opioid overdose morbidity and mortality could be reduced substantially by distributing the opioid antagonist naloxone to heroin users. The ethical issues raised by this proposal are evaluated from a utilitarian perspective. The potential advantages of naloxone distribution include the increased chance of comatose opioid users being quickly resuscitated by others present at the time of an overdose, naloxone's safety and its lack of abuse potential. The main problems raised by the proposal are: the medico-legal complications of medical practitioners prescribing a drug that is most likely to be administered to and by people other than the one for whom it is prescribed; the economic costs of distributing naloxone sufficiently widely to have an impact on overdose morbidity and mortality; and the potentially greater cost-effectiveness of simpler educational interventions. Given the possible benefits of naloxone distribution, it may be worthwhile considering a controlled trial of naloxone distribution to high-risk heroin users.

Tissue and perfusate pharmacokinetics of melphalan in isolated perfused rat hindlimb

Melphalan is commonly used as a cytotoxic agent in isolated limb perfusion for locally recurrent malignant melanoma. The time course of melphalan concentrations in perfusate and tissues during a 60-min melphalan perfusion and 30-min drug-free washout in the single-pass perfused rat hindlimb was examined using a physiologically based pharmacokinetic model. The rat hindlimbs were perfused with Krebs-Heinseleit buffer containing 4.7% bovine serum albumin (BSA) or 2.8% dextran 40 at a constant rate of 3.8 ml/min. The concentration of melphalan in perfusate and tissues was determined by highperformance liquid chromatography. The tissue concentrations of melphalan were significantly higher with the perfusate containing dextran than BSA during the 60-min perfusion. During the washout period, the melphalan concentration in the perfusates decreased rapidly in first few minutes, followed by a slower monoexponential decline. The estimated half life (t(1/2)) for melphalan removal from skin and fat was 59 +/- 2 min for both BSA and dextran perfusates. However, the estimated t(1/2) for melphalan removal from muscle was 79 and 96 min for BSA and dextran washout perfusates, respectively. The predicted concentration-time profiles obtained for melphalan with BSA and dextran perfusates appear to correspond closely to the observed data. This study showed that the uptake of melphalan into perfused tissues is impaired by the use of perfusates in which melphalan is highly bound. Melphalan washout from muscle, but not skin and fat, was facilitated by the use of perfusates in which melphalan is highly protein bound.

Impulse-response studies on tracer doses of [C-14]lignocaine and its multiple metabolites in the perfused rat liver

The outflow-concentration-time profiles for lignocaine (lidocaine) and its metabolites have been measured after bolus impulse administration of [C-14]lignocaine into the perfused rat liver. Livers from female Sprague-Dawley rats were perfused in a once-through fashion with red-blood-cell-free Krebs-Henseleit buffer containing 0 or 2% bovine serum albumin. Perfusate flow rates of 20 and 30 mL min(-1) were used and both normal and retrograde flow directions were employed. Significant amounts of metabolite were detected in the effluent perfusate soon after lignocaine injection. The early appearance of metabolite contributed to bimodal outflow profiles observed for total C-14 radioactivity. The lignocaine outflow profiles were well characterized by the two-compartment dispersion model, with efflux rate << influx rate. The profiles for lignocaine metabolites were also characterized in terms of a simplified two-compartment dispersion model. Lignocaine was found to be extensively metabolized under the experimental conditions with the hepatic availability ranging between 0.09 and 0.18. Generally lignocaine and metabolite availability showed no significant change with alterations in perfusate flow rate from 20 to 30 mt min(-1) or protein content from 0 to 2%. A significant increase in lignocaine availability occurred when 1200 mu M unlabelled lignocaine was added to the perfusate. Solute mean transit times generally decreased with increasing flow rate and with increasing perfusate protein content. The results confirm that lignocaine pharmacokinetics in the liver closely follow the predictions of the well-stirred model. The increase in lignocaine availability when 1200 mu M unlabelled lignocaine was added to the perfusate is consistent with saturation of the hydroxylation metabolic pathways of lignocaine metabolism.