A review of membrane sampling from biological tissues with applications in pharmacokinetics, metabolism and pharmacodynamics

European Journal of Pharmaceutical Sciences 17 (2002) 1–12 www.elsevier.nl / locate / ejps

Mini review

A review of membrane sampling from biological tissues with applications in pharmacokinetics, metabolism and pharmacodynamics a b b c, Kenneth E. Garrison , Stephanie A. Pasas , Joshua D. Cooper , Malonne I. Davies * a Department of Chemistry, College of the Ozarks, Point Lookout, MO 65726, USA Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS, USA c Bioanalytical Systems, Inc., BAS Kansas Research Laboratory, 2095 Constant Ave., Lawrence, KS 66047, USA b

Received 22 June 2001; received in revised form 28 January 2002; accepted 29 July 2002

Abstract This review provides an overview of membrane sampling techniques, microdialysis and ultrafiltration, and cites illustrations of their applications in pharmacokinetics, metabolism and / or pharmacodynamics. The review organizes applications by target tissue and general type of information gleaned. It focuses on recently published microdialysis studies (1999 to this writing) and offers the first review of ultrafiltration sampling studies. The advantages and limitations of using microdialysis and ultrafiltration sampling as tools for obtaining pharmacokinetic and metabolism data are discussed. Numerous examples are described including studies in which several types of data are collected simultaneously. Reports that study local metabolism of drug delivered through the probe are also presented.  2002 Elsevier Science B.V. All rights reserved. Keywords: Membrane sampling; Microdialysis; Ultrafiltration; In vivo samples; Pharmacokinetics; Pharmacodynamics; Drug metabolism

1. Introduction The rapid growth seen in the pharmaceutical world is rate limited by a number of steps from synthesis to market. Of 10 000 compounds synthesized, only one may make it to market (DiMasi, 1996). Although many of the compounds entering the pipeline prove unsuccessful, the number of compounds reaching the in vivo testing stage has increased dramatically over the past decade. This critical step in the approval of a potential drug involves various animal studies including pre-clinical trials. Many traditional in vivo techniques are labor and animal intensive in terms of sample collection, cleanup and preparation. Membrane sampling techniques such as ultrafiltration (UF) and microdialysis (MD) offer unique advantages for in vivo drug testing that compliment traditional techniques. During recent years, membrane sampling techniques such as MD and UF have gained popularity. These techniques have several inherent characteristics and advantages that complement traditional methodologies and can substantially reduce the number of animals used for pre*Corresponding author. Tel.: 11-785-864-3927; fax: 11-785-8644466. E-mail address: [email protected] (M.I. Davies).

clinical studies. To obtain an n56 tissue concentration– time profile of an analyte using traditional methodology involved the sacrifice of six animals at each of eight time points over 3 h, a total of 48 animals (numbers from Sabol and Freed, 1988). In contrast, microdialysis sampling generated the n56 profile with 19 time points in the 3 h using only six animals. Fig. 1 illustrates this reduction in animals required for a study. The quality of data, quantity of useful information, and reduction in number of animals and labor requirements all point towards the utilization of these methods (Janle and Kissinger, 1996). Development of MD sampling took place in the neurosciences where today it is an established technique. Success in the neurosciences paved the way for its utilization in other tissues and biological fluids. MD and UF are parallel techniques that obtain samples by collecting small molecules across a semi-permeable membrane. Each type of probe acts as a generic sampling system that provides samples that reflect the extracellular fluid (ECF) concentrations of compounds. Table 1 lists these and other characteristics of membrane sampling, summarizes the advantages and indicates methods to which they apply. MD and UF can be used in conjunction with a wide range of analytical techniques since both generate proteinfree samples. MD is especially amenable to direct on-line

0928-0987 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0928-0987( 02 )00149-5

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Fig. 1. Microdialysis sampling compared to direct tissue assay method. The microdialysis experiment (upper illustration) used six animals to produced an n56 concentration–time profile with 19 time points at uniform time intervals over 3 h (vertical bars represent sampling intervals). Direct tissue assay (lower panel) required 48 animals to produce the n56 profile with only eight time points. (Based on Sabol and Freed, 1988).

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Table 1 Characteristics and resulting advantages of membrane sampling Characteristic

Advantage

Applies to MD

UF

Minimal perturbation of surrounding tissue

Represents normal physiology Long-term sampling in awake animals Simultaneous multiple site sampling

* * *

* * *

Pre-experiment (pre-dose) samples

Animal serves as its own control Fewer animals required overall

* *

* *

Protein-free samples

No further enzymatic action Samples the unbound fraction No loss due to sample clean-up / preparation Direct on-line coupling to analytical system

* * * *

* * *

Samples reflect the ECF concentration

Multiple analytes can be profiled High relative recovery of analytes

*

* *

No net change in fluid volume

Long-term, continuous sampling Good temporal resolution

* *

Diffusion is bi-directional

Deliver drug via probe to observe local metabolism without systemic involvement

*

Can be used in conscious animals

Minimal restraint and handling of the animal Allows simultaneous activity monitoring Concurrent collection of biological fluids and wastes is possible

* * *

analysis. Depending on the experimental goals, the target analyte(s) may be endogenous (i.e., neurotransmitters monitored for changes in concentration in response to drug administration), exogenous (such as a drug and its metabolites) or a combination of both. Analytical challenges result from the small sample volume, typical low concentration of analyte(s), high ionic strength, and possible interference from similar compounds. Non-separation-based methods generally can be used to monitor one analyte at a time, whereas separation-based methods allow the detection of multiple analytes in each sample. Table 2 summarizes the analytical methods used for MD and UF. The following references provide additional information about analytical methods used with membrane sampling (Davies et al., 2000; Davies and Lunte, 1997; Kissinger, 1991; Wages et al., 1986). Today, approximately 700 papers are published each year that involve MD.1 Reviews of MD sampling applications in various disciplines appear regularly and MD has been the exclusive focus of recent issues in the literature (Lunte, 1999; Sawchuk and Elmquist, 2000b). Among the microdialysis topics recently reviewed: pharmacokinetic applications (Davies, 1999; de la Pena et al., 2000; de Lange et al., 2000a; Verbeeck, 2000); drug delivery and disposition studies (Boschi and Scherrmann, 2000; Chu and Gallo, 2000; Elmquist and Sawchuk, 2000); clinical use (Joukhadar et al., 2001; Muller, 2000); studies of central nervous system drugs and transporters (Hammar1

Based on results using the PubMed search engine for the term ‘microdialysis’ each year from 1997 through 2000.

* * *

lund-Udenaes, 2000; Sawchuk and Elmquist, 2000a); and sampling neurotransmitters (Westerink and Timmerman, 1999). This review presents an overview of the advantages and limitations of MD and of UF. Research reports illustrating applications of MD in pharmacokinetics, metabolism and / or pharmacodynamics have been drawn from articles published between 1999 and this writing. The application examples are organized by target tissue and by types of information collected. Special emphasis has been given to research that involves simultaneous multiple-site sampling and / or concurrent collection of several types of data. Although UF is a less utilized technique with fewer publications, it merits inclusion in this review because of its potential for use in drug development.

2. Microdialysis

2.1. Microdialysis background Microdialysis is a diffusion controlled process. The sampling is accomplished by implanting a probe consisting of a short length of hollow-fiber dialysis membrane affixed to narrow bore inlet and outlet conduits. Several typical probe designs are shown in Fig. 2. A solution (perfusate), isotonically matched to the ECF, is pumped slowly through the probe, usually at flow rates in the range of 0.5–5.0 ml / min, and the outflow (dialysate) is collected for analysis. In a variation on the usual MD procedure, several recent reports have used MD or similar probes with

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Table 2 Summary of advantages and limitations of analytical and detection methods commonly used for MD and UF Method

Advantage

Limitation

Non-separation based

Continuous monitoring Near real time detection

One analyte at a time

Mass spectrometry

Sensitivity and selectivity

High salt sample causes plugging, interference, noise

Biosensors

Selective Membrane sampling reduces protein fouling

May have slow response

Immunoassay

Sensitivity

Time consuming

Radioimmunoassay (RIA)

Separation based

Cross-reactivity RIA requires radioactive materials Aqueous-based samples

Small sample volume

Liquid chromatography

Direct injection of dialysate

Temporal resolution limited by sample volume required

Capillary electrophoresis

Utilizes small sample volume Increased speed and efficiency

High ionic samples can cause band broadening

UV–Vis

Near universal Rugged

Sensitivity and selectivity

Fluorescence

High sensitivity

Not all compounds are native fluorophores

Laser-induced fluorescence

Very sensitive Improved selectivity

Derivatization required for other compounds

Electrochemical

High sensitivity Simplicity Widespread applicability to biomolecules

Ruggedness Not all compounds are electroactive

Immunoassay

Sensitivity

Time consuming

Detection for separation methods

Radioimmunoassay (RIA)

Mass spectrometric

Cross reactivity RIA requires radioactive materials Sensitivity and selectivity

High salt sample causes plugging, noise, interference

Compiled from Davies et al. (2000), Davies and Lunte (1997), Kissinger (1991) and Wages et al. (1986).

simultaneous push and pull pumping to avoid fluid leakage through the membrane associated with the push only configuration (Asai et al., 1996; Patterson et al., 2001). The driving force for the movement of molecules across the membrane is the concentration gradient established between the ECF and the fluid within the probe lumen. Small molecules diffuse into (recovery) or out of (delivery) the probe while large molecules such as proteins and molecules bound to proteins are excluded. MD is not typically carried out at equilibrium conditions so the concentration of an analyte in the sample will be some

fraction of the actual concentration in the surrounding ECF. The relationship between the analyte concentration in the dialysate and that in the ECF may be thought of as an extraction efficiency and is often called relative recovery. Detailed discussions of probe calibration issues have been ` published (Song and Lunte, 1999; Stahle, 2000; Stenken, 1999). When MD sampling is conducted in an awake animal, its movement must be constrained with specialized containment systems which prevent tangling of the fluid lines. Although local tissue disturbance is minimal, the surgery

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Fig. 2. Typical microdialysis probe designs (probes not drawn to scale). (A) Concentric cannula probe and intracerebral guide for sampling from brain tissue. (B) Flexible concentric cannula probe for intravenous use. (C) Linear probe for peripheral tissues such as muscle, liver, or dermis. (D) Flow-through or shunt probe for sampling from rat bile duct. (Used with permission of Bioanalytical Systems, West Lafayette, IN.)

to implant the probe may be invasive depending on the target tissue. For example, placing a probe in the liver, bile duct or kidney requires opening the abdominal cavity and thus is more complicated than placing a probe in skin or skeletal muscle. Given the hydrophobic nature of the membranes commonly used and their functional molecular weight cut off, MD sampling may not be suitable for all potential analytes.

2.2. Microdialysis applications Microdialysis sampling from a single tissue can provide information about the therapeutically relevant tissue ECF concentration of a drug and its metabolites or concentrations of physiologically important endogenous compounds in their tissue of origin. Applications that collect several types of data, sample from multiple sites and / or

increase the duration of the study provide benefits for in vivo drug testing while concurrently reducing the number of animals used and the labor requirements of these studies.

2.2.1. Pharmacokinetics by microdialysis in a single tissue or fluid 2.2.1.1. Central nervous system Microdialysis sampling for the evaluation of blood– brain barrier (BBB) penetration is an example of the complementary pairing of membrane sampling with serial blood sampling (Sawchuk and Elmquist, 2000a). While blood samples reflect the plasma concentration, the MD probe provides the profile of the drug in brain tissue. A comparison of two compounds using brain MD with serial blood sampling in rats was carried out by Scott and Heath

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to assess the BBB permeability of MDL 100 907 and its metabolite MDL 105 725 (Scott and Heath, 1998). Hinton and Hudson (1999) combined MD with blood sampling in a study of brain concentration and kinetics of enadoline. Comparative pharmacokinetic data by MD from two brain regions (hippocampus and frontal cortex) with serial blood sampling was obtained by Walker et al. (2000) for the antiepileptic drug, lamotrigine. Mather et al. (2000) combined brain MD, blood sampling, and regional brain homogenates to study the equilibration of thiopental enantiomers across the BBB. A study of ziconotide by Newcomb et al. (2000) used in vivo MD to assess the drug’s bioavailability and influx from blood to brain. In addition to BBB permeation, MD can also help elucidate the mechanisms involved in drug transport (Hammarlund-Udenaes, 2000; Sawchuk and Elmquist, 2000a). MD probes transversely implanted in the cortex of genetically altered mice were used by Xie et al. (1999) to investigate the role of P-glycoprotein in the BBB transport of morphine. Similarly, P-glycoprotein involvement in brain distribution of several fluoroquinolones was studied by de Lange et al. (2000b). Numerous MD studies have been conducted in spinal cord tissue or fluid. Hoizey et al. (2000) implanted MD probes into the spinal cord of rats to monitor the pharmacokinetics of gacyclidine enantiomers. A combination loop probe-injection catheter was used by Kroin et al. (2000) to monitor the conversion of the prodrug, dexamethosone sodium phosphate, to free dexamethosone in the lumbar subarachnoid space in rats. A recent study by ´ Clement et al. (2000) determined the intrathecal bioavailability of a mixture of bupivacaine and lidocaine using MD in either the epidural or intrathecal space in rabbits. Ummenhofer et al. (2000) simultaneously implanted multiple probes in the cerebrospinal fluid at various locations along the spine of pigs enabling the researchers to compare the spinal distribution and clearance of four intrathecally administered opioids.

2.2.1.2. Blood stream Compounds in blood samples are subject to enzyme action prior to preparation and clean-up. Since MD sampling excludes proteins, enzymatic activity ceases as the sample crosses the membrane and they can be analyzed without preparation or clean-up steps (Verbeeck, 2000). The preparation or clean-up steps themselves may change the metabolite to parent drug ratio. Valuable information is lost when this alteration or masking of different forms of the drug or its metabolites occurs. Ye et al. (2000) demonstrated this in a study of an isothiazolone, PD161374, and its metabolites. The parent isothiazolone exists in equilibrium in vivo with its thiol and disulfide forms. Preparation steps for blood samples reduced the compound solely to the thiol, thus removing any information about equilibrium distribution among the forms. By using MD sampling from the jugular vein of awake rats, the authors were able to monitor the concentration–

time profiles of all three forms without changing the equilibrium distribution. The classic design for studying enterohepatically cycled compounds is by hepato-duodenal cannulation between donor and recipient rats with serial blood samples collected from each animal. Tsai et al. (2000) varied the classic design by using a MD probe in the jugular vein of each rat to monitor chloramphenicol and its glucuronide.

2.2.1.3. Eye Another example of MD sampling used to investigate the drug concentration at the site of action is the application of MD in eyes to assess ocular drug delivery (Rittenhouse and Pollack, 2000a). Nakashima et al. (1997) implanted MD probes in the anterior chamber of rabbit eyes to determine the time course for delivery of ocularly applied cyclosporin. Rittenhouse et al. (1998) conducted MD in the aqueous humor of both dogs and rabbits to study the absorption and disposition of propranolol administered either topically or by intrathecal injection. More recently, the same group followed the pharmacokinetics of propranolol using ocular MD in conscious rabbits (Rittenhouse et al., 1999).

2.2.1.4. Dermis /subcutaneous tissue In vitro evaluations using excised skin do not always provide an accurate estimate of a drug’s in vivo transdermal penetration. Dermal or subcutaneous MD can provide this information. The transdermal penetration of ondansetron hydrochloride in the presence and absence of oleic acid was monitored by MD in anesthetized rats by Ding et al. (2000). Zhou et al. (1999) coupled dermal MD directly on-line with capillary electrophoresis with electrochemical detection to monitor delivery of nicotine from a dermal patch to the skin of an awake rat. Brunner et al. (1998) compared skin blister fluid, dermal MD, and saliva sampling in humans to assess peripheral pharmacokinetics of paracetamol (acetaminophen) concluding that MD results corresponded well with the serum profile. Valproic acid pharmacokinetics, monitored by MD samples from human subcutaneous fluid, was found by Lindberger et al. (1998) to correlate with pharmacokinetics of unbound drug in plasma. A series of MD probes implanted in the forearm of volunteers were used by Benfeldt et al. (1999) to evaluate the impact of skin barrier perturbation on the dermal penetration of salicylic acid. A novel combination of in vivo pharmacokinetics in peripheral tissues with in vitro pharmacodynamics has been reported for the evaluation of antibiotics. Delacher et al. (2000) in a study of ciprofloxacin and Frossard et al. (2000) studying fosfomycin, determined the in vivo pharmacokinetic profiles in human subcutaneous adipose using MD sampling. The profiles were then recreated in vitro in bacterial cultures to obtain pharmacodynamic data as bacterial counts versus time.

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2.2.1.5. Bile Originally introduced by Scott and Lunte (1993) the flow-through or shunt probe enables monitoring of compounds in the bile of rats without interruption of flow. Heppert and Davies (1999b) used the shunt probe coupled on-line with LC to monitor the concentration–time profile of phenolphthalein glucuronide, after a dose of phenolphthalein (known to enterohepatically cycle), in rats with intact or diverted bile flow. The concentration of phenolphthalein glucuronide in the bile of intact and diverted rats declined in parallel for 90 min, then declined less rapidly in the intact rats. Tsai et al. (1999) have used the shunt design coupled on-line with microbore LC to monitor biliary excretion of chloramphenicol and its glucuronide in rats. More recently, the same group reported a study of the pharmacokinetics of cefepime in rat bile using the shunt probe on-line with LC (Chang et al., 2001). 2.2.2. Simultaneous multiple tissues microdialysis The small size of the MD probe and no net change in fluid volume permits simultaneous sampling from different tissues / fluids or from multiple sites within an organ. For example, concurrent MD sampling from brain and blood provides improved temporal resolution and reduces sample preparation time for BBB studies. Chang et al. (2000) determined the BBB penetration of cefoperazone with the jugular dialysates analyzed on-line and brain dialysates directed to a fraction collector for later analysis. Sveigaard and Dalgaard (2000) sampled by MD from blood and brain for investigation of BBB penetration of a series of analogous tetrahydropyridines. Alternatively, sampling from different tissues in the same animal can provide comparative distribution and kinetic data. Tokunaga et al. (2000) inoculated one caudate nucleus of rats with L9 glioma cells. MD probes were later implanted in each caudate nucleus. This provided comparative data about the distribution and pharmacokinetics of a novel cisplatin derivative in tumor and healthy brain tissue. Wu et al. (2000) monitored melphalan concentration by MD in rat skin and muscle during isolated limb perfusion. An on-line LC system was used by McLaughlin et al. (2000) to determine tirapazamine and its reduced metabolites in blood and muscle dialysates collected simultaneously from awake rats. Cefpirome concentrations in human subcutaneous adipose and muscle tissues following infusion or bolus dose were monitored by MD in a study by Hollenstein et al. (2000). 2.2.3. Pharmacodynamic data by microdialysis sampling A common experimental design using MD sampling for pharmacodynamics involves monitoring changes in the concentration of one or more endogenous compounds in tissues. These changes can be monitored in a single animal over long periods with good temporal resolution. In a recent study of this design, Seppa¨ et al. (2000) monitored striatal concentrations of dopamine and its metabolites following an acute nicotine dose in rats maintained at

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different temperatures. Bourne et al. (2001) determined dopamine and metabolites, acetylcholine and choline, in brain dialysates from guinea pigs. The study investigated a D 1 receptor subtype antagonist, SCH 23390, for protection against soman-evoked seizures. Yamazaki and Akiyama (1996) conducted MD sampling in the heart of anesthetized cats to monitor the change in norepinephrine concentration as the result of desipramine delivered via the MD probe. Gilinsky et al. (2001) determined myocardial norepinephrine in conscious rats obtaining similar pharmacodynamic results from the local delivery of desipramine. Kawada et al. (2000) have also used cardiac MD to monitor epinephrine and norepinephrine as pharmacodynamic markers in a study of chronic adriamycin treatment in rabbits. Rittenhouse and Pollack (2000b) applied ocular MD to monitor ascorbate as a marker of aqueous humor production in rabbit eyes following the administration of propranolol.

2.2.4. Simultaneous pharmacokinetic and pharmacodynamic data by microdialysis Studies that simultaneously collect different sets of data, such as pharmacokinetic and pharmacodynamic, maximize the potential of MD sampling for reducing animal numbers and the labor intensity of pre-clinical studies. Heppert and Davies (1999a) applied MD sampling to establish concentration–time profiles of caffeine in blood, brain and muscle while simultaneously monitoring a pharmacodynamic effect (horizontal motion activity) in awake rats. Huff and Davies (submitted) studied the pharmacokinetics of methylphenidate in blood and brain by MD simultaneously with two pharmacodynamic effects: changes in dopamine concentration in brain and the horizontal motion activity in awake rats. Maximum increase in dopamine and motion occurred concurrently with peak methylphenidate concentrations in brain and blood. Dual site MD, in venous blood and striatum, and arterial blood samples combined with pain threshold determination were used by Bouw et al. (2000) to study the processes involved in the delay of antinociceptive effect of morphine in rats. The authors concluded that morphine was actively effluxed at the BBB accounting for 85% of the observed effect delay. 2.2.5. Microdialysis for in vivo study of local metabolism The bi-directional nature of diffusion through the probe membrane makes possible the investigation of local metabolism without systemic involvement. MD was employed by Darbin et al. (2000) to compare metabolism of endogenous compounds in glioma and healthy brain tissue in a single animal. Rats, previously inoculated in one striatum with C6 glioma cells, were implanted with a probe in each striatum. Glucose, lactate and pyruvate were monitored from each probe as markers of local metabolism. Freed et al. monitored the formation of metabolites as substance P was delivered from the perfusate to rat brain (Freed et al., 2000).

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F-DOPA (6-[ 18 F]-fluoro-L-DOPA), an imaging agent, is widely used with positron emission tomography (PET) studies to study neuropsychiatric disorders. The methods determine a single time course for all 18 F labeled compounds. By implanting MD probes in the striatum of rats, DeJesus et al. (2000) determined separate time courses for F-DOPA and five resulting tagged metabolites. Microdialysis probes constructed with large pore size dialysis membranes (molecular weight cutoff of 100 kDa compared to the usual 12–30 kDa membranes) were used to introduce macromolecules locally into human muscle tissue in a study reported by Rosdahl et al. (2000). Glucose, lactate and urea concentrations were monitored before and during the local delivery of insulin via the probe. The authors determined that large pore membranes can be useful for studies involving large peptides and that such studies allowed assessment of local events while avoiding potentially adverse systemic reactions. Stenken et al. (2001) have published a mathematical model for the prediction of metabolite concentrations in the MD probe during local metabolism experiments. Both in vitro and in vivo experiments were conducted to test the model. The in vivo experiments used probes implanted in the liver of rats and perfused with a solution containing acetaminophen. Dialysates were analyzed to determine the concentration of acetaminophen and its locally formed metabolites.

2.2.6. Microdialysis probe as a drug delivery device In MD, the direction of net flux of a compound is determined by its concentration gradient making the probe a potential drug delivery device. Using intraocular MD, Waga (2000) was able to deliver therapeutic concentrations of ganciclovir in the vitreous of rabbit eyes via the probe. The standard delivery method for the drug is a sustained release intravitreal implant, a method that does not easily accommodate altering the dosage. Waga concludes that MD, as a drug delivery method, would greatly facilitate dose adjustments. Waga and Ehinger (2000) used MD probes with large pore membranes (molecular weight cutoff of 100 kDa) to deliver nerve growth factor to the vitreous of rabbit eyes. The authors note that intraocular MD probes have been shown to function for up to 3 weeks and that a removable ocular MD probe system was reported for long-term use (up to 6 months). Thus, ocular MD probes may be suitable for repeated and / or long-term administration of drugs to the eye without the need for repeated intravitreal injections.

Fig. 3. Ultrafiltration probe with needle hub assembly and vacutainer used to supply negative pressure. The needed pressure gradient can also be supplied by a peristaltic pump. The number and length of membrane loops are varied to optimize sampling from different sizes of animals or from different tissues. (Used with permission of Bioanalytical Systems, West Lafayette, IN.)

MD that are advantageous in drug testing. The UF probe consists of one or more hollow dialysis fiber loops connected to a microbore impermeable tube (Janle and Kissinger, 1993). A diagram of a UF probe is shown in Fig. 3. The loops are placed in the tissue of interest and a negative pressure gradient is used to create a flux of ECF, including low molecular mass solutes, across the porous hydrophobic membrane. This forced transport results in analyte recoveries of greater than 90% for small molecules. In contrast to MD, the relative recovery of analyte(s) is independent of the flow rate of ECF through the probe (Linhares and Kissinger, 1992). The UF probes are larger than MD probes, thus limiting their use in some tissues. UF probes have been used in animals ranging from mice to horses (Janle and Sojka, 2000). Since UF involves the removal of ECF from the site of implantation, the probe must be implanted in tissues with high fluid turnover that can tolerate a loss of fluid (Janle and Kissinger, 1996). The technique is more feasible in larger animals because the relative fluid loss is less and the sample vial can be attached to a vest or harness so the animal need not be tethered (Linhares and Kissinger, 1992). In addition, the rate of fluid flux across the probe can change over time and the time required to collect samples can alter throughout a study. Therefore, UF sampling is best suited for studies that do not require high temporal resolution.

3.2. Ultrafiltration applications 3. Ultrafiltration

3.1. Ultrafiltration background Ultrafiltration has been used to investigate the ECF of a variety of analytes. UF possesses some benefits parallel to

UF sampling has been applied to pharmacokinetics and to monitor endogenous compounds. Subcutaneous space is the most common target site, however, saliva, blood and bone have also been sampled using UF. The space available for the probe and the high fluid turn over make these ideal sites for this technique.

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3.2.1. Pharmacokinetic studies Linhares and Kissinger (1992) applied UF sampling to monitor theophylline in human saliva and acetaminophen in subcutaneous tissue in rats. In another study, they developed a micellular electrokinetic chromatography method to track the concentration of theophylline in ultrafiltrates from the subcutaneous space of rats (Linhares and Kissinger, 1993b). Mice have not been widely used in pharmacokinetic studies because the repeated removal of blood samples from an animal with such a small blood volume can significantly alter the action of the drug being studied. However, Janle and Kissinger (1993) employed subcutaneous UF sampling in mice to study the in vivo pharmacokinetics of cefazolin following intraperitoneal dosing. Drug profiles from UF sampling were comparable to those obtained by MD.

3.2.2. Endogenous compounds and pharmacodynamics Ultrafiltration probes have been used to monitor glucose, lactate, other small molecules, and various ions of physiological importance. As indicators of disease states, these molecules also serve as pharmacodynamic markers for therapeutic compounds. In the first published reports of UF, Ash et al. (1992, 1993) implanted UF probes in laboratory animals and in diabetic patients. They demonstrated that glucose concentrations in subcutaneous ultrafiltrate matched blood glucose values determined at the same time. Endogenous concentrations of glucose in streptozotocin-induced diabetic mice were monitored by Janle et al. (1992). Daily subcutaneous UF samples provided average glucose concentrations and eliminated the intraday variation seen with blood sampling. The glucose concentrations in a single mouse were monitored for over 40 days evidencing the long lifetime of the probe. In a similar investigation, Janle and Kissinger (1996) used UF sampling to track subcutaneous glucose and creatinine concentrations in dogs with some studies lasting up to 6 months. Moscone et al. (1996) and others combined UF sampling with a biosensor for continuous monitoring of subcutaneous glucose. Subcutaneous and intravenous glucose was monitored in rats by Kaptein et al. (1997). The UF samples were analyzed with a flow injection system using a biosensor for detection. The same group compared ultraslow MD with UF for subcutaneous monitoring of glucose and lactate in rats (Kaptein et al., 1998). They have also monitored glucose and lactate in humans (Tiessen et al., 1999). Most recently, this group incorporated an UF probe in a cardiac catheter to monitor glucose and lactate in swine heart during myocardial ischemia (Tiessen et al., 2001). Ultrafiltration sampling has also been demonstrated for large biomolecules such as proteins. Schneiderheinze and Hogan (1996) investigated UF in vitro and in vivo for sampling model proteins in the 6–68 kDa molecular mass range. UF probes implanted subcutaneously in rats were

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held under continuous vacuum for about 5 h. The collected ultrafiltrate contained two proteins with molecular weights of about 10 and 27 kDa. Linhares and Kissinger (1993a) used UF probes, implanted subcutaneously in rats, to monitor endogenous concentrations of sodium, potassium, calcium and phosphate ions. They also followed changes in potassium concentration under conditions of hyperinsulinemia. In another study, Spehar et al. (1998) conducted UF sampling from multiple sites in horses to monitor sodium, potassium and calcium ion concentrations in subcutaneous space and muscle for comparison with plasma concentrations of the ions. Calcium and magnesium are important indicators of bone physiology. Janle et al. (1992) recently monitored concentrations of both metal ions in ovine bone using UF sampling. The probes were implanted in muscle, bone, and subcutaneous tissue of sheep and remained functional for 35–40 days. The UF probe in bone enabled study of mineral distribution in a tissue not previously accessible in awake animals. Both short- and long-term chemical changes in bone were investigated and compared to the data collected concurrently from the other tissues. Fluctuations in the concentrations of these two minerals can indicate a loss in bone integrity. Data obtained in these experiments may provide valuable clues about many bone degenerating diseases. The UF probe in bone offers the potential for investigating, at the site of action, the pharmacokinetic and pharmacodynamics of drugs to treat degenerative bone disease.

4. Conclusion The use of membrane sampling methods have increased in recent years. Blood sampling still remains the gold standard in terms of the number of research applications. However, the serum concentration of a drug does not always reflect the more therapeutically relevant concentration, namely the ECF concentration in the target tissues. In contrast to blood sampling, membrane sampling can target specific tissues thus providing access to pharmacokinetics and / or pharmacodynamics at the site of action for many compounds. Both MD and UF can sample physiologically significant compounds, such as pharmacodynamic markers, for the appraisal of drug action. As a drug delivery device, the MD probe permits evaluation of local metabolism without systemic involvement. The discussion of single tissue pharmacokinetics illustrates the scope of tissues investigated by these techniques. Multiplesite sampling and reports obtaining several types of data from a single study demonstrates the value of membrane sampling to minimize the time invested, labor intensity, and number of animals used for pre-clinical in vivo testing during drug development.

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Acknowledgements Financial support for this work was provided by National Cancer Institute Training Grant [ T32 CA09242 (K.E.G. and J.D.C.), National Science Foundation grant [ 443700 (J.D.C.) and by grant [ IND22220 from Bioanalytical Systems, Inc. (S.A.P.). We also acknowledge R. Scott Martin, Kathleen Heppert and Brian Huynh for helpful discussions during the preparation of this review.

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