UCL- UniversitŽ catholique de Louvain
Faculty of Medicine
THE SCHOOL OF PHARMACY
SCIENTIFIC ACTIVITIES
1996
School of PharmacyUniversitŽ catholique de Louvain
SFAR 7360
Avenue Mounier 73
B-1200 Brussels
Belgium / / Secretariat office:
Annie Celis, Josiane Coremans
& VŽronique Picart
Phone : #-32-2-764 73 60
Fax : #32-2-764 73 63
E-mail :
President : R.Verbeeck
Academic secretary : V. PrŽat
Biopharmacy
lBiological dosimetry on people accidentally or professionally exposed to ionizing radiations
lClinical evaluation of new therapeutic approaches
lMetabolism of immunosuppressive drugs and immunosuppressive activity of the metabolites
lDrug Glucuronidation
lPlasma protein binding and pharmacokinetics microdialysis sampling
lBiodegradable polymers as drug deliver systems
lElectrically enhanced transdermal drug delivery
l Nanoparticles and microspheres as drug and antigen delivery systems
Other sections of this document (Introduction, Pharmacochemistry, Pharmacotoxicology and Staff Profiles) are available for download at For a full publication list, see < THEMESp 39
Clinical evaluation of new therapeutic approaches
S. Ibrahim (FACM), C. Bruno-Dusart (FACM) and P.M. Tulkens (FACM)
Supported by the industry (Schering Plough Belgium and Bristol-Myers Squibb)
Clinical trials are the ultimate and most critical final step in the development of new drugs and are also essential to rationally improve their use. In this context, our efforts are directed towards the development of optimized therapeutic schemes for antibiotics, based on the knowledge of the pharmacodynamic and pharmacokinetic parameters governing their efficacy and/or toxicity.
The final and the most important aspect in drugs development is their clinical use. Pharmacology and toxicology have therefore an obvious application which is the definition of the proper use of drugs, and their permanent improvement. In this context, we use the knowledge gained by our studies on the pharmacology of antiinfective drugs to design and perform clinical trials directed to these goals. These studies attempt to apply to the clinical environment the concepts of pharmacodynamics and toxicology which were proven useful in our experimental studies. We also set up the necessary monitoring procedures when these require non-routine investigations or develo-pments. In this context, the following clinical evaluations are being performed.
Administration of aminoglycosides in a Òonce-a-dayÓ schedule
This new model of administration was proposed on the basis of experimental studies demonstrating its potential for being more, or at least as effective than the conventional schedules (3 times a day), while causing less toxic reactions. The lower toxicity stems from the saturable character of aminoglycoside uptake by target tissues (kidney, inner ear). Moreover, the toxicity induced by aminoglycosides proceeds by successive thresholds from recoverable alterations towards irreversible damage. Keeping the level of alterations below a critical threshold results therefore in an effective protection of the patient. The 'once-a-day' schedule new scheme was successfully tested in several patient populations and is now introduced in a large number of applications of these drugs (suitable modifications of the official package inserts have been made in several countries including Belgium for two aminoglycosides so far).
Evaluation of phospholipiduria as a means to monitor aminoglycoside-induced renal alterations
We developed this approach in parallel to the above studies on aminoglycosides, because it provides a direct way to monitor non invasively the early alterations which aminoglycosides cause in kidney cortex. A series of patient populations have been examined so far, ranging from premature children, neonates, young females, patients suffering from cancer with or without granulocytopenia, to intensive care patients and HIV positive patients. Drugs examined so far have included netilmicin and amikacin. A trial with tobramycin is ongoing. Results show that phospholipiduria is a useful marker of impending toxicity in correlation with the duration and extent of treatment. It is also a useful monitoring criteria for assessing the safety of new schedules. It can also be used experimentally to assess the effectiveness of nephroprotectants. The same approach has been used to assess the safety of azithromycin in patients, in connection with the potential of this drug to cause phospholipidosis (see above).
Evaluation of §-lactam antibiotics in special applications in relation with their pharmacodynamic properties
In contrast with aminoglycosides, §-lactam antibiotics must be administered several times a day, because their serum concentration must ideally remain above the MIC of the offending organisms for the whole period of treatment. While this can be easily achieved in simple applications of these drugs, little is known concerning this aspect in special situations where pharmacokinetic parameters can vary largely from what is observed in 'normal' patients. In this context, we have explored the validity of the registered scheme of administration of aztreonam (a §-lactam antibiotic with specific activity against Gram (-) bacteria) in two populations at high risk of infection, namely, patients undergoing liver transplantation and patients suffering from cystic fibrosis. We could demonstrate that the officially registered schedules and dosages were markedly inadequate for cystic fibrosis patients. We are currently designing clinical trial aimed at testing rationally designed new schemes, based on the specific pharmaco-kineticsof aztreonam in this patient population and the pharmacodynamic criteria explained above.
RESEARCH THEMESp 40
Collaborations
lCentre hospitalier universitaire, Li ge (Belgium)
lCliniques universitaires St-Luc, Bruxelles (Belgium)
lAkademische Zietenhuis, Gent (Belgium)
lH™pital Erasme, Bruxelles (Belgium)
lH™pital St. Pierre, Bruxelles (Belgium)
Expertises
lClinical trials of new drugs and new schemes of administrations.
lMonitoring of renal alterations by non-invasive approaches.
References
lS. IBRAHIM,, M.P. DERDE, L. KAUFMAN, F. CLERCKX-BRAUN , Ph. JACQMIN, J. DONNEZ and P.M. TULKENS (1990) Analysis of safety, pharmacokinetics and efficacy of once-a-day administration of netilmicin and amikacin vs their conventional schedules in pelvic inflammatory disease patients. Renal Failure 12:199-203.
lP.M. TULKENS (1991) Pharmacokinetic and toxicological evaluation of the once-a-day regimen versus conventional schedules of netilmicin and amikacin. J. Antimicrob. Chemother. 27(C):49-61.
lJ.P. LANGHENDRIES, O. BATTISTI, J.M. BERTRAND, A. FRAN‚OIS, J. DARIMONT, S. IBRAHIM and E. SCALAIS (1993) Once-a-day administration of amikacin in neonates: assessment of nephrotoxicity and ototoxicity. Dev. Pharamacol. Ther., 120:220-230
lS. IBRAHIM, J.P. LANGHENDRIES, A. BERNARD and P.M. TULKENS (1994) Urinary phospholipids excretion in neonates treated with amikacin. Int. J. Clin. Pharm. Res. 14:149-156.
lS. IBRAHIM, B.K. KISHORE, P. LAMBRICHT, G. LAURENT and P.M. TULKENS (1991) Effect of aminoglycosides and of coadministration of poly-L-aspartic acid on urinary phsopholipids excretion: A comparative Study. In: P.H. Bach, N.J. Gregg, M.F. Wilks and L. Delacruzz, (eds). Nephrotoxicity:Mechanisms, Early Diagnosis and Therapeutic Management.Marcel Dekker, New York, 105-109.
Phospholipiduria as an early indicator of aminoglycoside-induced nephro-toxicity. The figures compares the level of urinary phospho-lipids in cancer patients receiving 6 mg/kg/day of netilmicin as a single injection or divided in 3 injections during 7 days. Despite the fact that the interindividual variations are important; it clearly appears that the thiol administration causes a more rapid and elevated phospho-lipidurva than the qd administration. The rise caused by tid treatment shows also a slower reversibility (From Van der Auwera et al, 1991)
RESEARCH THEMESp 41
Metabolism of immunosuppressive drugs and immunosuppressive activity of the metabolites
G. Lho‘st (FATC), M. Nickmilder (FATC), R. Verbeeck (FATC)
Metabolites of the recent immunosuppressive agents possessing a macrolide structure or a cylosporine like structure such as FK-506, rapamycin and SDZ-IMM-125 respectively, may retain or not the immunusuppressive activity were investigated and were found in the case of FK-506 to be related to some intra-molecular interactions of polar groups with the FK-506 binding region and are still under investigation for the other drugs. Also the influence of Phase II reaction on the immunosuppressive activity of the metabolites will be studied.
Several disciplines, including chemical ecology, seek to understand the molecular basis of information transfer in biological systems, and general molecular strategies are beginning to emerge. Often these strategies are discovered by a careful analysis of naturel products and their biological effects. Cyclosporin A, FK-506, and rapamycin are produced by soil microorganisms, SDZ lMM 125 being the hydroxyethyl derivative of D-serine cyclosporin A. They are being used or considered as clinical immunosuppressive agents. They interrupt the cytoplasmic portion of T-cell signaling by forming a complex with a binding protein FKBP-12 in the case of FK-506 and rapamycin and cyclophilin A (CyPA) in the case of cyclosporin A (CsA). This complex in turn inhibits a protein target and the best understood target is calcineurin which is inhibited by FK-506-FKBP-12 and CyPA-CsA.
The T-cell Acfivation Process
The chemical structure of FK-506 is illustrated in the figure. Despite having different structures, the mechanism of action of FK-506 and CyA are very simiar. On the other hand, FK-506 and rapamycin share certain structural similarities. Their identical binding domain led to the belief that these two agents would also have similar mechanism of action. However, despite this similarity, the effector elements are structurally different, and the two agents exert their immunosuppressive activity by different mechanisms. CyA and FK-506 act at an early stage in the T-cell activation process, blocking the T-cell receptor-mediated signal transduction pathway prior to late signalling events, rapamycin blocking the signal at a later stage.
Intracellutar Binding
In order to mediate their effects, CyA, FK-506 and rapamycin must each bind to a cytosolic target protein. Each agent binds to these binding proteins (known as immunophilins) with high affinity. The first immunophilin to be identified was cyclophilin, which complexes with CyA. The FK-506 binding protein (FKBP) was identified later. Both FK-506 and rapamycin bind to FKBP as a result of their identical binding domain, The target of the immunophilin-drug complexes is the calcium and calmodulin-dependent protein phosphatase, calcineurin, which is composed of two subunits Aand B. Calcineurin isan essential element in the signal transduction pathway, carrying information from the cell membrane to the nucleus, in order to stimulate the synthesis of an important cytokine IL-2. FK-506 and CyA both act as a molecular "glue" binding the calcineurin-calmodulin complex to immunophilin molecules, which do not interact under normal conditions.
RESEARCH THEMESp 42
FK-506 in vitro metabolism and site of intra-molecular interactions of FK-506 metabolites
As reported in the figure FK-506 (Tacrolimus) is a potent immunosuppres-sive macrolide produced by Streptomyces tsukubaensis possessing a lactone P-450 3A dependent mixed fonction oxygenase enzymic system to several metabolites including a FK-506 O-demethylated, hydroxylated, O-demethylated hydroxylated metabolites as well as an isomerized epoxide and a C36-C37 FK-506 dihydrodiol. Metabolism of FK-506 to demethyla-ted metabolites has been demonstrated in incubations with liver microsomes from animal treated with dexamethasone or erythromycin, known inducers of the cytochrome P-450 3A isozyme. Some of the FK-506 demethylated metabolites and the C36-C37 FK-506 dihydrodiol metabolite are also subniitted, as demonstrated for FK-506, to ring-and open chain tautomerism effects, involving the C9 FK-506 carbonyl group as demonstrated by FAB mass spectrometry. FK-506 metabolites, where intra-molecular interactions with certain polar groups at the FK-506 C9 position are not observed, are retaining their in vitro immunosuppressive activity since interaction with the human immunophilin at position C9 remains possible.
Collaborations
lD. Latinne (UCL-IMEX)
References
lG. LHOèST, N. MATON and R.K. VERBEECK (1993) Isolation and Identification of a novel isomerized epoxide metabolite of FK-506 from erythromycin induced rabbit liver niicrosomes. Drug Metab. Disp., 21,850-854
lG. LHOèST, N. MATON, A. LAURENT and R.K. VERBEECK (1994) Isolation and identification of a FK-506 C36-C37 dihydrodiol from erythromycin induced rabbit liver microsomes. J. Pharm. Biomed. Analysis, 12,235-241
lG. LHOèST, N. MATON, D. LATINNE, A. LAURENT, and R.K. VERBEECK (1994) 15-Desmethyl FK-506 and 15-31 -Desmethyl FK-506 from human liver niicrosomes: Isolation, Identification (by Fast Atom Bombardment Mass Spectrometry and NMR), and Evaluation of in Vitro Immunosuppressive Activity. Clini. Chem. 40,740-744
lG. LHOèST, R.K. VERBEECK, N. MATON, P. MUTHELET and D. LATINNE (1995) The in Vitro Immunosuppressive Activity of the C,, -demethylated Metabolite of FK-506 is Govemed by Ringand Open-Chain Tautomerism Effects. J. Pharm. Exp. Ther., 274, 622-626
RESEARCH THEMESp 43
Drug glucuronidation
R. Verbeeck (FATC), F. Brunelle (FATC), C. Meunier (FATC), D. Lambert (CMFA)
Many drugs and/or their phase I metabolites undergo glucuronide conjugation leading in most cases to inactive conjugates. Some of these glucuronides may undergo §-glucuronidase catalyzed hydrolysis. Such glucuronidation-deglucuronidation futile cycling will modulate the duration and intensity of the activity/toxicity of the aglycone. This phenomenon is investigated in rats in vitro and in vivo using diflunisal and ketoprofen as model compounds. In addition, the role of extrahepatic tissues to the overall glucuronidation of propofol is studied in rat and man.
Glucuronidation is an important metabolic pathway leading to polar conjugates of unchanged drug and/or its phase I metabolites. UDP-glucuronosyltransferases (UDPGT's) are located in the smooth endoplasmic reticulum in cells of different organs. Their function is to transfer glucuronic acid from UDP-glucuronic acid (UDPGA) to a suitable endogenous or exogenous substrate containing a hydroxyl, carboxyl or amino group. In most cases these conjugates are inactive and many pharmacologists therefore firmly believe that glucuronidation stands for detoxification. However, in recent years it has become evident that this is not always true and that the pharmacological activity of a glucuronide conjugate may be higher than that of the parent compound. The most notorious example is morphine-6-glucuronide which has analgesic activities much higher than those of morphine itself.
Whether glucuronidation leads to an inactive conjugate of a pharmacologically active molecule or results in the formation of an active glucuronide, the rate at which a compound undergoes this phase II reaction can significantly influence the intensity and duration of its activity and/or toxicity. It is therefore very important to study the factors which may affect drug glucuronidation. We are currently investigating 2 different apects related to the glucuronidation of drugs:
Regulation of drug glucuronide production by futile cycling in vitro and in vivo
Often overlooked in the discussion of the regulation of drug glucuronide formation in vitro and in vivo is the involvement of futile cycling via §-glucuronidase and possibly esterase. The term futile cycling is used because conjugated metabolites of drugs, endogenous substrates and toxic chemicals (e.g. carcinogens) may undergo successive cycles of synthesis to the glucuronide and hydrolysis back to the aglycone. The enzyme which deconjugates glucuronides, §-glucuronidase, is widely distributed in mammalian tissues with a particularly high activity in the liver. It is localized intracellularly in lysosomes and endoplasmic reticulum. In addition, hydrolysis of acyl glucuronides may be catalyzed by esterases. Using diflunisal and ketoprofen as a model compounds, we are studying the glucuronidation and hydrolysis of drug glucuronides in rats in vitro (microsomes, hepatocytes, isolated perfused rat liver) and in vivo. Results of studies using liver microsomes have clearly shown the contribution of §-glucuronidase-catalyzed hydrolysis to the overall formation formation of the acyl glucuronide of diflunisal (DAG) in the rat. The microsomal formation rate of the phenolic glucuronide of diflunisal (DPG) is not influenced by the §-glucuronidase activity because DPG has a very low affinity towards this enzyme in comaparison to DAG. Using saccharo-1,4-lactone, an inhibitor of §-glucuronidase, the role of futile cycling of DAG in the overall elimination of diflunisal is being investigated in the rat both in vitro (isolated perfused liver) and in vivo.
Extrahepatic glucuronidation in man and rat
Although the liver plays a major role in drug metabolism, major drug metabolizing enzymes such as cytochromes P450 and UDPGT's are also present at other sites. Biotransformation in the gastrointestinal tract is of particular interest after oral administration because bioavailability may be diminished by intestinal presystemic metabolism. In addition, in disease states such as severe cirrhosis of the liver, these extrahepatic pathways may compensate, in part, for impaired hepatic elimina-tion. We have been studying the extrahepatic metabolism of propofol, an intravenous anesthetic agent, in rats and man, both in vitro and in vivo. The results of these studies indicate that the small intestine and possibly the kidney contribute to the overall elimination of propofol in both species.
Collaborations
lL. Van Obbergh (UCL, ANES)
RESEARCH THEMESp 44
References
lF.M. BRUNELLE and R.K. VERBEECK (1993) Glucuronidation of diflunisal by rat liver microsomes. Effect of microsomal §-glucuronidase activity. Biochem. Pharmacol. 46:1953-1958
lR.J. HERMAN, G.R. LOEWEN, D.M. ANTOSH, M.R. TAILLON, S. HUSSEIN and R.K. VERBEECK (1994) Analysis of polymorphic variation in drug metabolism. III. Glucuronidation and sulfation of diflunisal in man. Clin. Invest. Med .17:297-307
lF.M. BRUNELLE, A.A. RAOOF, J. DE VILLE DE GOYET and R.K. VERBEECK (1996) Glucuronidation of diflunisal, (-)-morphine, 4-nitrophenol and propofol in liver microsomes of two patients with Criggler-Najjar syndrome type II. Biopharm. Drug Disp. 17, 311-317
lF.M. BRUNELLE and R.K. VERBEECK (1996) Glucuronidation of diflunisal in liver and kidney microsomes of rat and man. Xenobiotica, 26, 123-131
lA.A. RAOOF, L.J. VAN OBBERGH, J. DE VILLE DE GOYET and R.K. VERBEECK (1996) Extrahepatic glucuronidation of propofol in man: possible contribution of gut wall and kidney. Eur. J. Clin. Pharmacol.,50, 91-96
Theoretical effect of modifications of § glucuronidase activity on the net formations and excretion of glucoronide and the elimination of the parent compound in the case of a conjugation-deconjugation cycle
RESEARCH THEMESp 45
Plasma protein binding and pharmacokinetics microdialysis sampling
R. Verbeeck (FATC), P. Evrard (FATC), N. Van Brandt (FATC),
Plasma protein binding of drugs is an important phenomenon affecting the distribution and elimination of many drugs. We are studying the interindividual variability in the plasma protein binding of midazolam and sufentanil in ICU patients and its effect on the pharmacokinetics and activity of these compounds. In addition, microdialysis techniques have been developed in our laboratory which permit the in vivo measurement of unbound concentrations of drugs in blood and other tissues (e.g. CNS) of small laboratory animals. Using this technique several projects are underway not only to study the in vivo plasma protein binding and pharmacokinetics of drugs, but also to investigate the transport of compounds across the blood brain barrier.