Lars Ståhle

AAPS-FDA workshop white paper: Microdialysis principles, application, and regulatory perspectives

Many decisions in drug development and medical practice are based on measuring blood concentrations of endogenous and exogenous molecules. Yet most biochemical and pharmacological events take place in the tissues. Also, most drugs with few notable exceptions exert their effects not within the bloodstream, but in defined target tissues into which drugs have to distribute from the central compartment. Assessing tissue drug chemistry has, thus, for long been viewed as a more rational way to provide clinically meaningful data rather than gaining information from blood samples. More specifically, it is often the extracellular (interstitial) tissue space that is most closely related to the site of action (biophase) of the drug. Currently microdialysis (μD) is the only tool available that explicitly provides data on the extracellular space. Although μD as a preclinical and clinical tool has been available for two decades, there is still uncertainty about the use of μD in drug research and development, both from a methodological and a regulatory point of view. In an attempt to reduce this uncertainty and to provide an overview of the principles and applications of μD in preclinical and clinical settings, an AAPS-FDA workshop took place in November 2005 in Nashville, TN, USA. Stakeholders from academia, industry and regulatory agencies presented their views on μD as a tool in drug research and development.

High‐dose ribavirin in combination with standard dose peginterferon for treatment of patients with chronic hepatitis C

Improved treatment regimens for patients with chronic hepatitis C, genotype 1 and high viral load are needed. Increasing the dose of ribavirin has increased the response rate, but experience with doses of more than 1,200 mg/day is limited. The aim of this study was to investigate the safety and tolerance to treatment with a high and individualized dose of ribavirin in combination with peginterferon. Ten patients with chronic hepatitis C, genotype 1 and high viral load were treated with peginterferon alfa‐2a and ribavirin for 48 weeks in a prospective trial. The initial ribavirin dose was individualized and calculated from a pharmacokinetic formula based mainly on renal function. Ribavirin plasma concentrations were monitored, and the dose was adjusted to reach the target concentration. Hemoglobin was monitored, and patients were treated with erythropoietin and blood transfusions when indicated. After dose adjustments, the mean dose of ribavirin was 2,540 mg/day (range, 1,600‐3,600) at week 24. The main side effect was anemia, which was controlled with erythropoietin. Two patients required blood transfusions. One patient was withdrawn at week 24 because of a lack of viral response, and one patient at week 39 because of side effects, primarily interferon associated. At follow‐up (≥24 weeks posttreatment), nine of ten patients had undetectable HCV RNA and thus were cured by standard definitions. In conclusion, a high dose of ribavirin according to an individualized schedule is feasible but associated with more frequent and serious side effects such as anemia. The viral response merits further evaluation. (HEPATOLOGY 2005;41:275–279.)

A comparison between three methods for estimation of extracellular concentrations of exogenous and endogenous compounds by microdialysis

In vitro models simulating tissues were used to test the validity of three methods for determining the free concentration of drugs and endogenous compounds in the extracellular space by means of microdialysis. Theophylline was used as a model substance. Recovery is defined as the proportion of compounds extracted from the medium surrounding the probe. The water recovery method is the use of recovery, determined in a water solution, to estimate concentrations in other media. This method was shown to underestimate the surrounding concentration when the effective rate of diffusion is smaller in the other medium than in water. The difference method measures the net transport over the dialysis membrane at varying concentrations in the perfusion medium. The point of equilibrium, where no net transport takes place, is used to estimate the surrounding concentration. The perfusion rate method involves two phases. In the first phase (calibration), surrounding media with different diffusion characteristics were used as tissue models. The amount recovered at different perfusion rates was measured and a multivariate regression method was used to calculate a mathematical model. In the second phase, the mathematical model was used to predict the concentration in the surrounding medium in new experiments. The two latter methods gave satisfactory predictions of the surrounding concentrations. Protein binding did not affect the methods. It is concluded that the difference method and the perfusion rate method may be used to estimate the in vivo concentration of drugs and endogenous compounds.

Ribavirin treatment in dialysis patients with chronic hepatitis C virus infection – a pilot study

Standard treatment for chronic hepatitis C is with interferon (IFN)‐α and ribavirin for 6–12 months. In dialysis patients only interferon therapy is currently used due to the lack of knowledge concerning ribavirin dosage and side‐effects. The aim of this study was to investigate if ribavirin can be added to interferon when treating dialysis patients with hepatitis C.

Dosage of ribavirin in patients with hepatitis C should be based on renal function: a population pharmacokinetic analysis.

A combination of interferon alfa and ribavirin is standard therapy for chronic hepatitis C virus (HCV). Ribavirin dosage is currently based on body weight. The aim of this study was to critically evaluate current dosage recommendations on the basis of a population pharmacokinetic analysis. The data consisted of 383 ribavirin plasma concentration samples collected from 63 patients undergoing treatment of HCV. Forty-four patients had normal range serum creatinine with an estimated glomerular filtration rate (GFR = estimated creatinine clearance) of 57–144 mL/min. Another 19 patients had renal impairment with a GFR of 5–57 mL/min. Population factors were age, gender, body weight, serum creatinine, and GFR. A population pharmacokinetic analysis with a two-compartment model was carried out using nonlinear mixed effect modeling. Ribavirin clearance was found to be linearly dependent on renal function with a small nonrenal clearance dependent on body weight and age. Estimated GFR was a significantly better predictor of ribavirin clearance than body weight alone. There remained a significant 40% interindividual variability in ribavirin total clearance not explained by estimated GFR and body weight. The volume of distribution was large and proportional to body weight (V = 44.3 × body weight), which resulted in a long half-life (100–500 hours, depending on GFR) and a long time to steady state (3–12 weeks). Ribavirin dosage should mainly be based on renal function and not, as currently recommended, on body weight alone. A ribavirin-dosing schedule based on GFR and body weight to reach an intended target concentration is proposed. Ribavirin monitoring may be useful for optimizing HCV treatment not only in patients with renal insufficiency but also in other patients considering the time to steady state and the interindividual variability in ribavirin clearance.

Efavirenz plasma concentrations in HIV-infected patients: inter- and intraindividual variability and clinical effects.

Efavirenz is a drug subject to extensive metabolism, mainly by the cytochrome P-450 isoenzyme CYP2B6, known to exhibit extensive interindividual variability. The aim of the present study was 2-fold: to investigate the relationship between plasma concentration and clinical effects of efavirenz and to investigate the extent of the inter- and intraindividual variability of the plasma concentration measurements. From an open clinic, 68 HIV-positive patients on efavirenz-containing treatment were recruited. From each patient 1 to 5 samples were collected; 43 had more than 1 sample taken. Most samples were taken 10–24 hours after the latest dose. Efavirenz was analyzed by high-performance liquid chromatography with UV detection. The data were analyzed by the variance component model analysis of variance. Efavirenz concentrations were reproducible, and intraindividual variability constituted only 16% of the total variance. Thus, 84% of the variance was attributed to interindividual variability. The incidence of primary treatment failure was related to low plasma concentrations with a geometric mean concentration of 6.1 μmol/L compared with 8.7 μmol/L in those responding to therapy (P < 0.05). If a cutoff of 7 μmol/L is used, 10 of 13 failing to respond were below this level compared with 15 of 45 in those responding. It is concluded that efavirenz plasma concentration measurement gives reproducible results predictive of primary treatment failure. A lower bound for the therapeutic level of 7 μmol/L is proposed, and data from other authors suggests that an upper level of 13 μmol/L may be applied.

Multivariate data analysis and experimental design in biomedical research.

Publisher Summary This chapter presents multivariate statistical methods for the design and analysis of experiments that can substantially facilitate the research process. Biomedical research at the beginning of the century usually involves relatively few measurements on many subjects. While measurement is expensive, the cost for experimental subjects is often low. The methods for data analysis developed during the same period––tests, analysis of variance, and multiple regression––are optimized for this situation with many subjects and few measurements. In medicinal chemistry, it is customary to take a large number of measurements to characterize drugs pharmacologically and chemically. A physicochemical description of a drug molecule in quantitative terms is best made by a number of measurements to include information about properties relevant to the drug effect. Likewise, the pharmacological effects of the drug have to be assessed in an array of pharmacological model systems to make possible reasonable predictions about therapeutic value and side-effects.

Drug distribution studies with microdialysis. III: Extracellular concentration of caffeine in adipose tissue in man.

Microdialysis was applied to estimate the extracellular concentration of caffeine in subcutaneous abdominal adipose tissue of five healthy volunteers after oral administration of approximately 5 mg/kg (300 or 400 mg) of caffeine. The peak extracellular levels were in the range of 20 - 80 microM. The time-course in blood and in extracellular fluid was similar but the plateau concentrations were not closely correlated. The estimated mean concentration of five individuals was similar in blood and extracellular fluid. The intraindividual variation between probes was found to be small compared to the interindividual variation (8% versus 43%). It is concluded that microdialysis yield useful data on drug distribution in man and that distribution to adipose tissue may not strictly follow the concentrations in blood. A comparison with available information of the in vitro properties of caffeine shows that the levels attained in the extracellular fluid were too small to significantly affect phosphodiesterase but sufficiently high to block adenosine receptors.

Evidence that plasma concentration rather than dose per kilogram body weight predicts ribavirin-induced anaemia.

Summary.  Ribavirin in combination with interferon alpha‐2 or pegylated interferon is the standard treatment for chronic hepatitis C. The current dosage recommendations for ribavirin are based on body weight (bw). Ribavirin is mainly eliminated by the kidneys and we have recently shown that ribavirin plasma concentrations are determined primarily by renal function. It is therefore reasonable to hypothesize that side‐effects of ribavirin, i.e. anaemia, should be more closely related to plasma concentrations of ribavirin than to the dose per kg bw. A total of 108 consecutive patients eligible for treatment of chronic hepatitis C were studied. Ribavirin concentrations in plasma were measured by high‐performance liquid chromatography (HPLC)‐UV after solid‐phase extraction in trough samples taken 4, 8 and 12 weeks after the treatment commenced. A total of 213 samples were obtained and the change in the haemoglobin level and the creatinine concentration was measured in addition to ribavirin. The dose of ribavirin per kg bw did not correlate with the drop in haemoglobin level induced by ribavirin. The concentration of ribavirin was non‐linearly related to the drop in the haemoglobin level as revealed by fitting a standard Hill equation type dose–response curve. The half maximal drop in haemoglobin was obtained at 4.4 μm. The results from this study suggest that the anaemia induced by ribavirin depends primarily on the concentration of ribavirin, and not on the dose per kg bw. This lends further support to the idea that ribavirin should be dosed according to renal function.

Circulating Proprotein Convertase Subtilisin Kexin Type 9 Has a Diurnal Rhythm Synchronous With Cholesterol Synthesis and Is Reduced by Fasting in Humans

Objective—To gain insight into the function of proprotein convertase subtilisin kexin type 9 (PCSK9) in humans by establishing whether circulating levels are influenced by diurnal, dietary, and hormonal changes. Methods and Results—We monitored circulating PCSK9 in a set of dynamic human experiments and could show that serum PCSK9 levels display a diurnal rhythm that closely parallels that of cholesterol synthesis, measured as serum lathosterol. In contrast to these marked diurnal changes in cholesterol metabolism, serum low-density lipoprotein (LDL) cholesterol levels remained stable during the diurnal cycle. Depletion of liver cholesterol by treatment with the bile acid–binding resin, cholestyramine, abolished the diurnal rhythms of both PCSK9 and lathosterol. Fasting (>18 hours) strongly reduced circulating PCSK9 and lathosterol levels, whereas serum LDL levels remained unchanged. Growth hormone, known to be increased during fasting in humans, reduced circulating PCSK9 in parallel to LDL cholesterol levels. Conclusion—Throughout the day, and in response to fasting and cholesterol depletion, circulating PCSK9 displays marked variation, presumably related to oscillations in hepatic cholesterol that modify its activity in parallel with cholesterol synthesis. In addition to this sterol-mediated regulation, additional effects on LDL receptors may be mediated by hormones directly influencing PCSK9.