PubMed Health⌕ Search

Biomedical subjects

D K Meijer

Publications and source records attributed to D K Meijer.

At least 91 records · Page 5Linked to original sources
PubMed1997

Drug-targeting to the kidney: renal delivery and degradation of a naproxen-lysozyme conjugate in vivo.

A renal-specific controlled release of an active drug may enable a reduction of the required dose and may provide a reduction of extra-renal toxicity. To achieve renal specific targeting of the NSAID naproxen, the low-molecular-weight protein (LMWP) lysozyme was employed as carrier since it is mainly taken up and catabolized in the proximal tubules of the kidney. A conjugate was synthesized with an average coupling degree of 2 mol naproxen per 1 mol lysozyme in which the drug was directly coupled to the protein via a peptide bond. First, we investigated whether naproxen conjugation affects the renal disposition of lysozyme. As native lysozyme, the conjugate was predominantly and rapidly (within 20 min) taken up by the kidney. The subsequent decrease in renal content reflecting the renal degradation of the conjugated lysozyme molecules appeared also to be similar to that of native lysozyme with a half life of four hours. Second, the effect of lysozyme conjugation on the body distribution of naproxen was studied. An important observation with regard to the aimed reduction in extra-renal side effects was that no detectable amounts of free naproxen were present in the plasma after administration of conjugate. Conjugation of naproxen to lysozyme resulted in a pronounced (70-fold) increase of naproxen accumulation in the kidney. In agreement with the protein disposition study, the conjugate was rapidly taken up by the kidney and subsequently degraded. In conclusion, renal selective targeting of the NSAID naproxen can be obtained by conjugation with the LMWP lysozyme. This concept of drug delivery to the kidney has the potential to improve drug efficacy and safety.

Animals↗
PubMed1997

Targeting naproxen to non-parenchymal liver cells protects against endotoxin induced liver damage.

Non-steroidal anti-inflammatory drugs (NSAID's) could be of value in the treatment of liver disease; however, their use in this situation is limited by renal side effects. Therefore, we explored whether naproxen covalently bound to human serum albumin NAP-HSA) was able to reduce toxicity in an acute model of liver disease induced by endotoxin in rats pretreated with Corynebacterium parvum. In the isolated perfused liver of such animals endotoxin induced cholestasis (0.62 +/- 0.05 vs. 0.24 +/- 0.09 microliter.min-1.g liver-1; p < 0.05), increased vascular resistance (11300 +/- 400 vs. 311000 +/- 2000 dyn.s.cm-5; p < 0.05) and alanine aminotransferase release (22 +/- 9 vs. 149 +/- IU/l; p < 0.05). At the highest dose tested (22 mg/kg, corresponding to 6.0 mumoles naproxen), NAP-HSA normalized ALT release (21 +/- 10 IU/l: p < 0.05) while an equimolar amount of non-targeted naproxen was only partially effective (56 +/- 19 IU/l). A conventional dose of naproxen similarly prevented transaminase release. Cholestasis and increased vascular resistance were also prevented by NAP-HSA. Drug targeting by linking drugs to proteins is a potentially useful approach to maximizing drug effect while minimizing adverse events; this could be particularly useful for compounds with potentially serious adverse effects in patients with chronic liver disease such as the nonsteroidal anti-inflammatory agents used in the present study.

Animals↗
PubMed1997

A physiologic function for alkaline phosphatase: endotoxin detoxification.

Alkaline phosphatase (AP), a common enzyme present in many species including humans, has been studied extensively. Although the enzyme is routinely applied as a marker for liver function, its biologic relevance is poorly understood. The reason for this is obvious: the pH optimum of AP in vitro, as measured with the usual test substrates (+/-10.5), greatly exceeds the physiologic pH range as it occurs in biologic tissues. We now hypothesize that this relatively high pH optimum in vitro is related to dissociation of acidic groups in the protein preparation, which leads to the formation of negatively charged groups in the vicinity of the active site of the enzyme. These negatively charged groups may promote the activity of AP. We examined the possibility that endotoxin is a natural substrate for this enzyme because this phosphorylated substance is able to supply multiple negatively charged residues in the microenvironment of the enzyme at a physiologic pH level. Phosphate groups in the endotoxin molecule are known to be essential for the biologic activities of this bacterial product. The present study demonstrates that in intestinal and renal tissue specimens in vitro, AP is endowed with endotoxin dephosphorylating activity at pH levels closer to the physiologic range. This is also illustrated by our experiments in vivo showing that the toxicity of endotoxin is significantly reduced after exposure to AP preparations, as tested by inducing a local intradermal inflammatory reaction in rats. Collectively, our data suggest that the ubiquitous enzyme AP may accomplish protection against endotoxin, an equally ubiquitous product of Gram-negative bacteria that may cause lethal complications after an infection with these micro organisms.

Alkaline Phosphatase↗
PubMed1997

Relationship between chemical structure and physicochemical properties of series of bulky organic cations and their hepatic uptake and biliary excretion rates.

To obtain more insight in the relationship between physicochemical properties of cationic drugs and their hepatobiliary transport rate, a series of 12 aminosteroidal neuromuscular blocking agents (NMBAs), supplemented with data of four related NMBAs from the literature, were investigated in the isolated perfused rat liver. A significant correlation was found between plasma protein binding and the partition coefficient octanol/Krebs (log P), confirming results from the literature with other organic cations. Evidence was found for a saturable hepatic uptake of several NMBAs, indicating that carrier-mediated uptake processes are involved. Hepatic uptake rate was closely related to the lipophilicity of the compounds; the initial extraction ratio, the apparent clearance and the intrinsic clearance were significantly correlated to log P. We did not find a significant correlation between biliary clearance and lipophilicity in the current series of compounds. Pharmacokinetics analysis of perfusate disappearance and biliary excretion data revealed that a considerable fraction of the dose of these bulky organic cations is stored in the liver and seems to not be directly available for biliary excretion. This finding is in line with earlier observations showing a pronounced accumulation of this type of compounds in mitochondria and lysosomes.

Animals↗
PubMed1997

Dephosphorylation of endotoxin by alkaline phosphatase in vivo.

Natural substrates for alkaline phosphatase (AP) are at present not identified despite extensive investigations. Difficulties in imagining a possible physiological function involve its extremely high pH optimum for the usual exogenous substrates and its localization as an ecto-enzyme. As endotoxin is a substance that contains phosphate groups and is usually present in the extracellular space, we studied whether AP is able to dephosphorylate this bacterial product at physiological pH levels. We tested this in intestinal cryostat sections using histochemical methods with endotoxin from Escherichia coli and Salmonella minnesota R595 as substrate. Results show that dephosphorylation of both preparations occurs at pH 7.5 by AP activity. As phosphate residues in the lipid A moiety determine the toxicity of the molecule, we examined the effect of the AP inhibitor levamisole in vivo using a septicemia model in the rat. The results show that inhibition of endogenous AP by levamisole significantly reduces survival of rats intraperitoneally injected with E. coli bacteria, whereas this drug does not influence survival of rats receiving a sublethal dose of the gram-positive bacteria Staphylococcus aureus. In view of the endotoxin-dephosphorylating properties of AP demonstrated in vitro, we propose a crucial role for this enzyme in host defense. The effects of levamisole during gram-negative bacterial infections and the localization of AP as an ecto-enzyme in most organs as well as the induction of enzyme activity during inflammatory reactions and cholestasis is in accordance with such a protective role.

Alkaline Phosphatase↗
PubMed1997

Differentiation between naproxen, naproxen-protein conjugates, and naproxen-lysine in plasma via micellar electrokinetic capillary chromatography--a new approach in the bioanalysis of drug targeting preparations.

Pharmacotherapy through the targeting of drugs is a promising new approach that requires adequate analytical methods capable of differentiating between the free drug, the drug carrier, and metabolites. Using micellar electrokinetic capillary chromatography (MECC), we report the separation of naproxen (NAP) from NAP covalently coupled to human serum albumin or to mannosylated serum albumin and the metabolite naproxen-lysine. An assay for selective analysis of the different forms of NAP by direct plasma injection was developed with salicylate as internal standard and solute detection by laser-induced fluorescence. Compared with previously applied techniques, including HPLC and total plasma fluorescence, MECC offers the advantage that free and covalently bound NAP can be differentiated in one run and can be accurately monitored in microliter quantities of plasma. Summation of all NAP equivalents determined by MECC revealed data that compare well with those produced by total plasma fluorescence and HPLC.

Animals↗
PubMed1996

Antiviral effects of milk proteins: acylation results in polyanionic compounds with potent activity against human immunodeficiency virus types 1 and 2 in vitro.

A number of native and modified milk proteins from bovine or human sources were analyzed for their inhibitory effects on human immunodeficiency virus type 1 (HIV-1) and HIV-2 in vitro in an MT4 cell test system. The proteins investigated were lactoferrin, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B. By acylation of the amino function of the lysine residues in the proteins, using anhydrides of succinic acid or cis-aconitic acid, protein derivatives were obtained that all showed a strong antiviral activity against human immunodeficiency virus type 1 and/or 2. The in vitro IC50 values of the aconitylated proteins were in the concentration range of 0.3 to 3 nM. Succinylation or aconitylation of alpha-lactalbumin and beta-lactoglobulin A/B also produced strong anti-HIV-2 activity with IC50 values on the order 500 to 3000 nM. All compounds showed virtually no cytotoxicity at the concentration used. Peptide-scanning studies indicated that the native lactoferrin as well as the charged modified proteins strongly bind to the V3 loop of the gp120 envelope protein, with Kd values in the same concentration range as the above-mentioned IC50. Therefore, shielding of this domain, resulting in inhibition of virus-cell fusion and entry of the virus into MT4 cells, may be the likely underlying mechanism of antiviral action.

Aconitic Acid↗
PubMed1996

Mechanism of anti-HIV activity of negatively charged albumins: biomolecular interaction with the HIV-1 envelope protein gp120.

A novel class of polyanionic proteins with potent anti-human immunodeficiency virus type 1 activity, the negatively charged albumins (NCAs), have been reported previously. In vitro antiviral assays established that these compounds preferentially inhibit virus-cell fusion and syncytium formation and that virus-cell binding is less affected. Here the interaction of the NCAs with synthetic peptides composed of 15-36 amino acids and corresponding to different parts of the gp120 envelope protein is described. Among the gp120 peptides tested, binding of the NCAs was observed only with the s0-called V3 loop (amino acids 296-330) and the C-terminal part of gp120. A higher number of negatively charged residues in the albumins resulted in higher binding affinities. NCAs in which, in addition to negative charges, up to 7 or 14 lactose or mannose groups were introduced, respectively did not exhibit increasing binding affinity. In contrast, mannosylated albumin containing about 14 mannose groups showed an increased binding compared with native albumin. Binding of the NCAs to the V3 and C-terminal oligopeptide was competitively inhibited by sulfated polysaccharide heparin and dextran sulfate. This finding indicates that the binding between the gp120 peptides and the NCAs is likely caused by electrostatic interactions. However, the fact that the dissociation constants of dextran sulfate and heparin are orders of magnitude larger compared with the NCAs indicates that the spatial structure of the proteins and/or hydrophobic interactions between the NCAs and the envelope protein may also be involved.

Albumins↗
PubMed1996

Preparation and characterization of conjugates of (modified) human serum albumin and liposomes: drug carriers with an intrinsic anti-HIV activity.

Human serum albumin (HSA) derivatized with cis-aconitic anhydride (Aco-HSA) that was earlier shown to inhibit replication of human immunodeficiency virus type 1 (HIV-1), was covalently coupled to conventional liposomes, consisting of phosphatidylcholine, cholesterol and maleimido-4-(p-phenylbutyryl)phosphatidylethanolamine, using the heterobifunctional reagent N-succinimidyl-S-acetylthioacetate (SATA). The amount of HSA that could be coupled to the liposomes depended on derivatization of the HSA and ranged from 64.2 +/- microgram HSA/micromol total lipid for native HSA to 29.5 +/- 2.7 microgram HSA/micromol total lipid for HSA in which 53 of the epsilon amino groups of lysine were derivatized with cis-aconitic anhydride (Aco53-HSA). Incorporation of 3.8 mol% of total lipid of a poly(ethylene glycol) derivative of phosphatidylethanolamine (PEG-PE) in the liposomes resulted in a lower coupling efficiency of Aco-HSA. The elimination and distribution of the liposomal conjugates in rats in vivo was largely dependent on the modification of the HSA coupled to the liposomes. With native HSA-liposomes, more than 70% of the conjugate was still found in the blood plasma 30 min after i.v. injection in rats, while at this time Aco-HSA-liposomes were completely cleared from the circulation. The rapid clearance of conventional Aco-HSA-liposomes was due to a rapid uptake into the liver and could be considerably decreased by incorporating PEG-PE in the liposomal bilayer. After 3 h 60% of Aco-HSA-PEG-liposome conjugates were found in the blood. In an in vitro anti-HIV-1 assay, the 50% inhibitory concentrations (IC50) for Aco39-HSA-liposomes and Aco53-HSA-liposomes expressed as protein weight, were 2.87 microgram/ml and 0.154 microgram/ml, respectively. When PEG-PE was incorporated, the Aco53-HSA-liposomes retained anti HIV-1 activity (IC50:3.13 microgram/ml). The possibility to modulate the residence time in the bloodstream of Aco-HSA-liposomes and the potent anti-HIV-1 activity of these conjugates, may allow the development of an intrinsically active drug carrier system. By incorporating anti HIV-1 drugs such as AZT into such liposomes a drug delivery system can be designed that might act simultaneously on the virus/cell binding by virtue of the coupled Aco-HSA and on the RNA/DNA transcription of the HIV-1 replication cycle through the nucleoside analogue.

Aconitic Acid↗
PubMed1996

An extended pharmacokinetic/pharmacodynamic model describing quantitatively the influence of plasma protein binding, tissue binding, and receptor binding on the potency and time course of action of drugs.

An extended pharmacokinetic/pharmacodynamic (PK/PD) model is presented, in which the effect of binding of the drug to plasma proteins and to tissue binding sites in a peripheral compartment, and nonspecific and receptor binding in the effect compartment are taken into account. It represents an extension of the classical Sheiner model, and the model proposed by Donati and Meistelman. The present model is characterized by the following parameters: Kue (exit rate constant of unbound drug from the effect compartment), Pue (ratio of the unbound clearances to and from the effect compartment), fue (fraction of drug in effect compartment that is not bound to nonspecific binding sites), Kd (equilibrium dissociation constant of drug-receptor binding), and Rtot (concentration of receptor binding sites in effect compartment). The rate of association and dissociation of the drug-receptor complex can be incorporated in the model. The influence of the pharmacokinetic parameters (V1, V2, fu, fu2, CLu10, CLu20, CLu12, CLu21) and the PK/PD model parameters (kue, Pue, fue, Kd, Rtot) on various dynamic parameters is analyzed. These include potency (single dose needed to produce 90% effect, ED90), constant infusion dosing rate needed to maintain a constant effect of 90%, time to maximum effect (onset time), and duration to 90% recovery. The neuromuscular blocking agent vecuronium is used as an example. It is shown that both potency and time course of action are strongly dependent on the ratio V1/fu, CLu10, kue, Pue (at equipotent doses the time course is not affected by Pue), fue, Kd, and Rtot (only if Rtot is high), whereas they are less affected by the ratio V2/fu2, CLu20, CLu12, and CLu21. In general, the model parameters affect the ED90 and the time course of action in the same direction, e.g., an increase of V1 results in an increase of ED90 and an increase of onset time and duration. However, the unbound clearance CLu10, the intercompartmental unbound clearance CLu12 and the receptor affinity Kd have an opposite effect on ED90 and the time course parameters, e.g., an increase of CLu10 results in an increase of ED90 and a decrease of onset time and duration. This effect may be responsible for the inverse relationship between onset time and potency of neuromuscular blocking drugs observed in animal experiments and clinical studies. We demonstrate that PK/PD analysis using the traditional effect compartment model (Sheiner model) results in an apparent value of keo, which is a function of kue, fue, Kd, Rtot, as well as the unbound drug concentration in the effect compartment Cue. On the other hand, the model proposed by Donati and Meistelman gives correct values of keo (equal to the product fue.kue), but the receptor affinity Kd and the receptor density Rtot obtained by this method are apparent values, which depend on fu, fue, and Pue.

Blood Proteins↗
PubMed1996

Substantial excretion of digoxin via the intestinal mucosa and prevention of long-term digoxin accumulation in the brain by the mdr 1a P-glycoprotein.

1. We have used mice with a disrupted mdr 1a P-glycoprotein gene (mdr 1a (-/-) 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 mdr 1a 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 mdr 1a (-/-) 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 mdr 1a (-/-) mice. Biliary excretion of [3H]-digoxin was not dramatically decreased (24% in wild-type mice versus 16% in mdr 1a (-/-) mice). 5. After a single bolus injection, brain levels of [3H]-digoxin in wild-type mice remained very low, whereas in mdr 1a (-/-) mice these levels continuously increased over a period of 3 days, resulting in a approximately 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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Comparative pharmacokinetic, immunologic and hematologic studies on the anti-HIV-1/2 compounds aconitylated and succinylated HSA.

Charge modification by succinylation or cis-aconitylation of the terminal epsilon NH2 functions of the amino acid lysine in human serum albumin, resulted in polyanionic compounds with an anti-HIV-1 activity in the low nanomolar concentration range. After iv injections in rats of the negatively charged albumins (NCAs), a dose dependent elimination pattern was observed indicating a saturable eliminations pathway. The Michaelis-Menten parameters Vmax and K(m) were 62 +/- 8 micrograms.min-1.kg-1 and 16 +/- 4 micrograms.ml-1 (Clintr 3.9 +/- 1.1 ml.min-1.kg-1) and 74 +/- 6 micrograms.min-1.kg-1 and 11 +/- 2 micrograms.ml-1 (Clintr 6.7 +/- 1.2 ml.min-1.kg-1) for aconitylated-HSA (Aco-HSA) and succinylated-HSA (Suc-HSA) respectively, using 125I-labelled proteins. The volume of distribution (V) of both compounds was approximately 60 ml.kg-1. Coadministration of poly-inosinic acid and formaldehyde treated albumin showed a marked inhibition of blood clearance indicating that the compounds are mainly cleared from the bloodstream by scavenger receptors on liver and spleen endothelial cells and macrophages. The Michaelis-Menten constant K(m) was remarkably higher when the hydrophobic flurophore fluorescein was covalently linked to the protein, indicating that the affinity for the scavenger receptors is largely decreased by FiTC conjugation. The latter observation may have implications for the kinetic behavior of drug-carrier preparations if antiviral drugs like AZT or PMEA are linked to these intrinsic active carriers. In contrast to other polyanionic compounds like heparins and dextran sulfate, these NCAs did not exhibit acute toxicity and had no effect on blood coagulation. They neither had an effect on the lymphocyte proliferation. Studies on immunogenicity of the homologous derivatized albumins in rats did not show a significant response. The present pharmacokinetic and toxicologic data of Suc-HSA and Aco-HSA show that both compounds are interesting preparations for studies in SIV infected monkeys and AIDS patients.

Aconitic Acid↗
PubMed1996

Mechanisms for the hepatic uptake and biliary excretion of tributylmethylammonium: studies with rat liver plasma membrane vesicles.

Hepatic organic cation transport in vitro, using tetraethylammonium (TEA) as a substrate, consists of at least two steps: sinusoidal uptake is stimulated by an inside-negative membrane potential and canalicular membrane transport is mediated by organic cation:H+ exchange (Moseley et al., 1992b). In vivo, however, TEA is poorly excreted into bile. In contrast, larger, more hydrophobic organic cations, such as tributylmethylammonium (TBuMA), undergo significant hepatobiliary excretion. To better characterize hepatic organic cation transport, TBuMA transport was examined in rat canalicular liver plasma membrane (cLPM) and basolateral liver plasma membrane (bILPM) vesicles. In cLPM vesicles, under voltage-clamped conditions, an outwardly directed H+ gradient stimulated [3H]TBuMA uptake consistent with electroneutral TBuMA:H+ exchange; H(+)-dependent [3H]TBuMA uptake was not the result of a H+ diffusion potential. In the absence of a H+ gradient, intravesicular TBuMA trans-stimulated [3H]TBuMA uptake. Substrates for renal and hepatic organic cation:H+ exchange cis-inhibited H(+)-dependent [3H]TBuMA uptake. No ATP-dependent [3H]TBuMA uptake was detected in cLPM vesicles, and the P-glycoprotein substrate, daunomycin, did not cis-inhibit H(+)-dependent [3H]TBuMA uptake. Carrier-mediated [3H]TBuMA uptake exhibited saturability (Km of 0.5 mM and Vmax of 0.5 nmol/mg prot/5 s). In bILPM vesicles, in contrast, a valinomycin-induced intravesicular-negative K+ diffusion potential stimulated [3H]TBuMA uptake. These findings suggest that hepatic transport of TBuMA is similar to TEA but fundamentally different from that of P-glycoprotein substrates, indicating the involvement of at least two separate processes for the hepatobiliary excretion of organic cationic drugs.

Adenosine Triphosphate↗
PubMed1995

Optimization of the reaction conditions for the synthesis of neoglycoprotein-AZT-monophosphate conjugates.

The coupling of the monophosphate derivative of 3-azido-2,3-dideoxythymidine (AZTMP) to glycoproteins by water soluble carbodiimide (1-ethyl-3-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) was greatly improved, relative to a recently reported method, by using also N-hydroxysulfosuccinimide (NHS) in the conjugation reaction. The hydrolysis of the activated AZTMP intermediate, responsible for the low degree of conjugation in the earlier method, could be delayed considerably if the activated phosphate group was converted into an activated ester by addition of NHS. In order to minimize the use of compounds needed for the preparation of AZTMP-protein conjugates, the present study was undertaken to determine if the reaction conditions could be optimized such that a conjugate with 2 AZTMP molecules/mol of neoglycoprotein would result. In addition a low proportion of cross-linked conjugates was desired. Optimization was achieved studying the shape of three-dimensional response surfaces, in which the degree of AZTMP coupling and the percentage of monomeric conjugates were regarded as the relevant responses. It appeared that the optimal conditions for coupling 1-2 mol of AZTMP to 1 mol of glycoprotein were an incubation time of 30 h, an AZTMP amount of 4 mg, an NHS amount between 8 and 15 mg, and a glycoprotein amount of 50 mg.

Antiviral Agents↗
PubMed1995

Deuterium isotope effect on the metabolism of the flame retardant tris(2,3-dibromopropyl) phosphate in the isolated perfused rat liver.

The metabolism of tris(2,3-dibromopropyl) phosphate (Tris-BP) was compared with that of completely deuterated Tris-BP (D15-Tris-BP) in an isolated, recirculating rat liver perfusion system in order to determine the relative quantitative importance of two different biotransformation pathways of Tris-BP: (i) cytochrome P450-mediated metabolism and (ii) GSH S-transferase-mediated metabolism. To accomplish this we quantitated the biliary excretion of S-(3-hydroxypropyl)glutathione (GSOH) as a marker metabolite for cytochrome P450-mediated metabolism and that of S-(2,3-dihydroxypropyl) glutathione (GSOHOH) as a marker metabolite for GSH S-transferase-mediated metabolism. Complete deuterium substitution of Tris-BP significantly decreased the formation of GSOH, whereas there was no effect on the formation of GSOHOH. Because our previous studies showed a large decrease in genotoxicity of D15-Tris-BP compared to Tris-BP, the present results support our hypothesis that cytochrome P450-mediated metabolism is responsible for the genotoxic effects of Tris-BP in the rat liver.

Animals↗