Dirk Meijer

Antiviral activities of lactoferrin

Lactoferrin (LF) is an iron binding glycoprotein that is present in several mucosal secretions. Many biological functions have been ascribed to LF. One of the functions of LF is the transport of metals, but LF is also an important component of the non-specific immune system, since LF has antimicrobial properties against bacteria, fungi and several viruses. This review gives an overview of the present knowledge about the antiviral activities and, when possible, the antiviral modes of action of this protein. Lactoferrin displays antiviral activity against both DNA- and RNA-viruses, including rotavirus, respiratory syncytial virus, herpes viruses and HIV. The antiviral effect of LF lies in the early phase of infection. Lactoferrin prevents entry of virus in the host cell, either by blocking cellular receptors, or by direct binding to the virus particles.

MW PHARM, AN INTEGRATED SOFTWARE PACKAGE FOR DRUG-DOSAGE REGIMEN CALCULATION AND THERAPEUTIC DRUG-MONITORING

The pharmacokinetic software package MW/Pharm offers an interactive, user-friendly program which gives rapid answers in clinical practice. It comprises a database with pharmacokinetic parameters of 180 drugs, a medication history database, and procedures for an individual drug dosage regimen calculation. The included curve-fitting facilities allow estimation of pharmacokinetic parameters on the basis of medication history, taking into account a varying status of the patient with respect to body weight and kidney function, optionally using a Bayesian procedure. The module KinBes performs the evaluation of bioavailability studies, including various methods, and an extensive statistical evaluation of bioequivalence.

Substantial excretion of digoxin via the intestinal mucosa and prevention of long-term digoxin accumulation in the brain by the mdrla P-glycoprotein

1 We have used mice with a disrupted mdrla P‐glycoprotein gene (mdrla (—/—)mice) to study the role of P‐glycoprotein in the pharmacokinetics of digoxin, a model P‐glycoprotein substrate. 2 [3H]‐digoxin at a dose of 0.2 mg kg−1 was administered as a single i.v. or oral bolus injection. We focussed on intestinal mucosa and brain endothelial cells, two major pharmacological barriers, as the mdrla P‐glycoprotein is the only P‐glycoprotein normally present in these tissues. 3 Predominant faecal excretion of [3H]‐digoxin in wild‐type mice shifted towards predominantly urinary excretion in mdrla (—/—) mice. 4 After interruption of the biliary excretion into the intestine, we found a substantial excretion of [3H]‐digoxin via the gut mucosa in wild‐type mice (16% of administered dose over 90 min). This was only 2% in mdrla (—/—) mice. Biliary excretion of [3H]‐digoxin was not dramatically decreased (24% in wild‐type mice versus 16% in mdrla (—/—) mice). 5 After a single bolus injection, brain levels of [3H]‐digoxin in wild‐type mice remained very low, whereas in mdrla (—/—) mice these levels continuously increased over a period of 3 days, resulting in a ∼200 fold higher concentration than in wild‐type mice. 6 These data demonstrate the in vivo contribution of intestinal P‐glycoprotein to direct elimination of [3H]‐digoxin from the systemic circulation and to the pattern of [3H]‐digoxin disposition, and they underline the importance of P‐glycoprotein for the blood‐brain barrier.

Localization of organic anion transporting polypeptide 4 (Oatp4) in rat liver and comparison of its substrate specificity with Oatp1, Oatp2 and Oatp3.

Organic anion transporting polypeptides (rodents: Oatps; human: OATPs) are involved in the absorption and elimination of a wide variety of structurally unrelated amphipathic organic compounds. Several members of this protein family mediate the uptake of substrates across the basolateral membrane of hepatocytes as the first step in hepatic elimination. In contrast to the well-characterized Oatp1 and Oatp2, the localization and substrate specificity of the recently cloned Oatp4 have not been investigated in detail. Therefore, we raised an antibody against the C-terminal end of Oatp4 and localized this 85-kDa protein to the basolateral membrane of rat hepatocytes. Similar to Oatp1 and Oatp2, Oatp4 is a multispecific transporter with high affinities for bromosulfophthalein, dehydroepiandrosterone sulfate, leukotriene C4, and anionic peptides. In addition, we compared the substrate specificity of Oatp4 to that of Oatp3, which so far has mainly been shown to mediate intestinal bile acid transport. Oatp3 had a similar broad substrate specificity, but in general much lower affinities than Oatp4. Thus, while Oatp4 seems to work in concert with Oatp1 and Oatp2 in the basolateral membrane of rat hepatocytes, Oatp3 is a multispecific transport system in the small intestine.

A Novel Strategy to Modify Adenovirus Tropism and Enhance Transgene Delivery to Activated Vascular Endothelial Cells In Vitro and In Vivo

To assess the possibilities of retargeting adenovirus to activated endothelial cells, we conjugated bifunctional polyethylene glycol (PEG) onto the adenoviral capsid to inhibit the interaction between viral knob and coxsackie-adenovirus receptor (CAR). Subsequently, we introduced an alphav integrin-specific RGD peptide or E-selectin-specific antibody to the other functional group of the PEG molecule for the retargeting of the adenovirus to activated endothelial cells. In vitro studies showed that this approach resulted in the elimination of transgene transfer into CAR-positive cells, while at the same time specific transgene transfer to activated endothelial cells was achieved. PEGylated, retargeted adenovirus showed longer persistence in the blood circulation with area under plasma concentration-time curve (AUC) values increasing 12-fold compared to unmodified virus. Anti-E-selectin antibody-PEG-adenovirus selectively homed to inflamed skin in mice with a delayed-type hypersensitivity (DTH) inflammation, resulting in local expression of the reporter transgene luciferase. This is the first study showing the benefits of PEGylation on adenovirus behavior upon systemic administration. The approach described here can form the basis for further development of adenoviral gene therapy vectors with improved pharmacokinetics and increased efficiency and specificity of therapeutic gene transfer into endothelial cells in disease.

The effect of parenterally administered cyclodextrins on cholesterol levels in the rat

The inclusion complex formation of intravenously administered hydroxypropyl-β-cyclodextrin and β-cyclodextrin with endogenous lipids was studied. We tested the hypothesis that complex formation of endogenous cholesterol with cyclodextrins in the bloodstream leads to extraction of cholesterol from the large lipoprotein particles. The relatively small cholesterol–cyclodextrin complexes then leave the bloodstream via capillary pores, and dissociation of the complex in the extravascular compartment finally causes redistribution of cholesterol from blood to tissue. This hypothesis is supported by the following experimental findings. Intravenous administration of cyclodextrins led to a transient decrease in plasma cholesterol levels in a dose-dependent manner, and in vitro cholesterol-cyclodextrin complexes passed dialysis membranes with a molecular weight cutoff of 6000–8000. Further, cyclodextrins increased protein binding of the steroidal drug spironolactone, probably through removal of cholesterol from plasma protein binding sites. Finally, extravascular redistribution was directly demonstrated in histological studies of the kidneys. Glomerular filtration of the cholesterol–cyclodextrin complex is followed by dissociation of the complex in the ultrafiltrate, resulting in cholesterol accumulation in the proximal tubule cells. The cholesterol-β-cyclodextrin complex has a limited aqueous solubility. Crystallization of this complex in renal tissue might explain the nephrotoxicity of parenterally administered β-cyclodextrin. The absence of such crystallization might explain the lower nephrotoxicity of hydroxypropyl-β-cyclodextrin after intravenous administration.

[11] Isolated perfused rat liver technique

Publisher Summary The function of the liver in the metabolism of xenobiotics can be studied with a spectrum of liver preparations ranging from the intact organ in vivo, through perfusion systems, liver slices, isolated hepatocytes, homogenates, and membrane fractions, to purified enzymes.. The use of the isolated perfused liver is also attractive because of the intact architecture of the organ, the possibilities of controlling blood and bile flow, and the ease of the manipulation of the composition of the perfusion medium. Advantages include the large number of perfusate samples that may be collected from and compared with the intact animal and the smaller number of interactions with endogenous compounds. In addition, it is possible to use the concentrations of the substrates and metabolic inhibitors that would not be tolerated in vivo because of their toxicity. Compared with isolated hepatocyte preparations, the perfusion system does not require damaging treatment with Ca 2+ -free solutions and digesting enzymes and the normal functional polarity of cells and their localization in the liver lobule is maintained.

Carrier-mediated transport in the hepatic distribution and elimination of drugs, with special reference to the category of organic cations.

Carrier-mediated transport of drugs occurs in various tissues in the body and may largely affect the rate of distribution and elimination. Saturable translocation mechanisms allowing competitive interactions have been identified in the kidneys (tubular secretion), mucosal cells in the gut (intestinal absorption and secretion), choroid plexus (removal of drug from the cerebrospinal fluid), and liver (hepatobiliary excretion). Drugs with quaternary and tertiary amine groups represent the large category of organic cations that can be transported via such mechanisms. The hepatic and to a lesser extent the intestinal cation carrier systems preferentially recognize relatively large molecular weight amphipathic compounds. In the case of multivalent cationic drugs, efficient transport only occurs if large hydrophobic ring structures provide a sufficient lipophilicity-hydrophilicity balance within the drug molecule. At least two separate carrier systems for hepatic uptake of organic cations have been identified through kinetic and photoaffinity labeling studies. In addition absorptive endocytosis may play a role that along with proton-antiport systems and membrane potential driven transport may lead to intracellular sequestration in lysosomes and mitochondria. Concentration gradients of inorganic ions may represent the driving forces for hepatic uptake and biliary excretion of drugs. Recent studies that aim to the identification of potential membrane carrier proteins indicate multiple carriers for organic anions, cations, and uncharged compounds with molecular weights around 50,000 Da. They may represent a family of closely related proteins exhibiting overlapping substrate specificity or, alternatively, an aspecific transport system that mediates translocation of various forms of drugs coupled with inorganic ions. Consequently, extensive pharmacokinetic interactions can be anticipated at the level of uptake and secretion of drugs regardless of their charge.

Rat liver slices as a tool to study LPS-induced inflammatory response in the liver.

BACKGROUND/AIMS Inflammation in the liver is a complex interaction between parenchymal and non-parenchymal cells, and therefore can not be studied in vitro in pure cultures of these cells. METHODS We investigated whether Kupffer cells in the liver slice are still responsive to an inflammatory stimulus of lipopolysaccharide (LPS), and evoke an inflammatory response in the hepatocytes. RESULTS TNFalpha, IL-1beta and IL-10 were significantly elevated in culture medium of LPS-stimulated rat liver slices. Nitric oxide (NO) production of LPS-treated slices gradually increased from 5 to 24 h (24 h: 81+/-5 microM vs. 14+/-2 microM in control P < 0.05), paralleled by inducible nitric oxide synthase (iNOS) in the hepatocytes, iNOS mRNA was induced after 3 h. NO production but not iNOS induction was significantly inhibited by NOS inhibitors S-methylisothiourea and N(G)-nitro-L-arginine methylester. Both pentoxifylline and dexamethasone inhibited TNFalpha and IL-1beta production, albeit to a different extent, iNOS induction and, as a result thereof, NO production. CONCLUSIONS These results imply that non-parenchymal cells in liver slices are viable and can be activated by LPS. In addition, it is concluded that the upregulation of iNOS in hepatocytes by LPS is caused by cytokines produced by Kupffer cells because inhibition of TNFalpha and IL-1beta production attenuated iNOS induction.

Hepatobiliary and intestinal clearance of amphiphilic cationic drugs in mice in which both mdr1a and mdr1b genes have been disrupted

We have used mice with homozygously disrupted mdr1a and mdr1b genes (mdr1a/1b (−/−) mice) to study the role of the mdr1‐type P‐glycoprotein (P‐gp) in the elimination of cationic amphiphilic compounds from the body. These mice lack drug‐transporting P‐gps, but show no physiological abnormalities under laboratory conditions and have normal bile flow. 3H‐labelled cationic drugs were administered intravenously (i.v.) to mice as a single bolus dose and the disposition of the studied cationic drugs was investigated by focusing on drug secretion into bile, intestinal lumen and urine. Hepatobiliary secretion of the investigated cationic drugs was profoundly reduced in mice devoid of the mdr1‐type P‐gps. In fact, the cumulative biliary output, measured during 1 h, of the small type 1 compounds tri‐butylmethyl ammonium (TBuMA) and azidoprocainamide methoiodide (APM), as well as of the more bulky type 2 cationic drug vecuronium, was reduced by at least 70% in the mdr1a/1b (−/−) mice compared to wild‐type. The intestinal secretion of TBuMA, APM and vecuronium was also profoundly reduced in mdr1a/1b (−/−) mice compared to wild‐type mice. The absence of the mdr1‐type P‐gp resulted in virtual elimination of intestinal secretion of TBuMA and APM (>90% reduced as compared to wild‐type (P=0.0001 and 0.0022, respectively)). The intestinal secretion of the type 2 cation drug vecuronium was reduced by 58% (P=0.0004) compared to the wild‐type mice. Increased renal clearances of both the type 1 compounds TBuMA and APM and also of the type 2 cationic compound vecuronium in the mdr1a/1b (−/−) mice were observed. Furthermore, the balance between hepatic, intestinal and renal clearances of small type 1 organic cations clearly shifted towards a predominant role for renal clearance. Increased renal clearance may be explained by (over)expresion of additional mechanisms for renal organic cation secretion, alternatively they may also point to an as yet undefined role of P‐glycoprotein in kidney physiology and renal secretory function. We conclude that the elimination from the body of a broad spectrum of cationic amphiphilic drugs via liver and intestine, is largely dictated by the activity of mdr1‐type P‐glycoproteins.