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Biomedical subjects

A Fahr

Publications and source records attributed to A Fahr.

At least 19 recordsLinked to original sources
PubMed2001

The composition-dependent presence of free (micellar) alkylphospholipid in liposomal formulations of octadecyl-1,1-dimethyl-piperidino-4-yl-phosphate affects its cytotoxic activity in vitro.

This study was performed to investigate the effect of cholesterol content, surface charge and sterical stabilization on the physico-chemical properties of liposomes prepared from the cancerostatic alkylphospholipid, octadecyl-1,1-dimethyl-piperidino-4-yl-phosphate (D21266), and their relationship to in vitro cytotoxicity. Stable incorporation of OPP into liposomes was found to be highly dependent on the cholesterol content. 31P-NMR spectroscopy as well as analysis of the lipid composition of OPP-containing liposome formulations revealed an increase in the amount of non-liposome-associated, micellar OPP as the cholesterol content decreased. The fraction of non-liposome-associated OPP constituted about 10% of total OPP when cholesterol was present in equimolar amounts (45.5/45.5 mol %) and increased to approximately 30% at a twofold excess of OPP over cholesterol (58.8/29.4 mol %). In monolayer incorporation studies it was shown that the existence of an increasing micellar pool of lipids leads to increased lipid transfer into the target monolayer. Liposome formulations containing more OPP than cholesterol were also found to display greater cytotoxicity. However, all liposome formulations were less cytotxic than pure (micellar) OPP. Cytotoxicity was not affected by the incorporation of N-methoxy-polyethyleneglycol2000-phosphoethanolamine, a lipid that is known to reduce liposome uptake into phagocytic cells. The results demonstrate that the increase in cell toxicity correlates with the increase in non-liposome-associated, micellar OPP, which can readily exchange into cellular membranes.

Breast Neoplasms↗
PubMed2001

Compartment modeling.

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Administration, Cutaneous↗
PubMed2001

Highly efficient transduction of endothelial cells by targeted artificial virus-like particles.

Targeting the tumor vasculature by gene therapy is a potentially powerful approach, but suitable vectors have not yet been described. We have designed a new type of liposomal vector, based on the composition of anionic retroviral envelopes, that is serum-resistant and nontoxic. These artificial virus-like envelopes (AVEs) were endowed with a cyclic RGD-containing peptide as a targeting device for the a(v)beta3-integrin on tumor endothelial cells (ECs). The packaging of plasmid DNA complexed with low-molecular-weight, nonlinear polyethyleneimine into these AVEs yielded artificial virus-like particles (AVPs) that transduced ECs with efficiencies of up to 99%. In contrast, transduction of a variety of other cell types by these RGD-AVPs was comparably inefficient under the same experimental conditions. This EC selectivity was mediated, in part, but not exclusively, by the RGD ligand, as suggested by the reduced, but still relatively high, transduction efficiency seen with AVPs lacking RGD. The interaction of anionic lipids of the AVPs with ECs may therefore contribute to the observed selective and highly efficient transduction of this cell type. These findings suggest that the targeted AVE technology is a useful approach to create highly efficient nonviral vectors.

Antineoplastic Agents↗
PubMed2001

Liposomal formulations of Cyclosporin A: a biophysical approach to pharmacokinetics and pharmacodynamics.

There are about 20 publications about liposomal formulations of Cyclosporin A (CyA) in the pharmaceutical and preclinical literature. Liposomal formulations were developed in order to reduce the nephrotoxicity of CyA and to increase pharmacological effects. However, conflicting results have been published as to the therapeutic properties of these formulations. This is also true for the change in pharmacokinetics and organ distribution of the liposomally encapsulated CyA as compared to conventionally formulated CyA. Using biophysical methods, it could be shown that CyA is not tightly entrapped in liposomal membranes, despite its high lipophilicity. CyA shows retardation only at high lipid concentrations in blood, following a massive injection of liposomes. This effect may diminish nephrotoxicity, as could be demonstrated by in vitro studies using a model tubule system. The results of these studies can be used to predict the formulation behavior in vivo and to optimize liposomal formulations. When applied in an early phase of the drug formulation process, these types of biophysical experiments can also help minimize animal experiments. However, these basic interaction studies cannot cover all physiological, pharmacological, and toxic effects in animals and humans.

Biophysical Phenomena↗
PubMed2000

Nuclear gene targeting using negatively charged liposomes.

Oligonucleotides are a very useful tool to control gene activity. Oligos work by complementary base-pairing with target sequences either in the nucleus or in the cytosol (Zelphati, O., Szoka, F.C., Jr., 1996. Liposomes as a carrier for intracellular delivery of antisense oligonucleotides: a real or magic bullet? J. Contr. Rel. 41, 99-119). In a new approach using chimeric oligonucleotides (Yoon, K., Cole Strauss, A., Kmiec, E.B., 1996. Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA-DNA oligonucleotide. Proc. Natl. Acad. Sci. USA 93, 2071-2076) conversion of single base mutations with help of intranuclear repair mechanisms maybe an advantageous method to cure genetic diseases which are based on single point mutations. These chimeric oligonucleotides are constructed in a way that they form an intramolecular double strand of DNA and modified RNA-bases. We used a fluorescent labelled pure 68-mer DNA-analogue of a chimeric oligonucleotides to follow the intracellular fate of these kind of genetic material. The oligos were complexed with protamine sulfate and coated with three different liposomal formulations. The AVE-3 formulation shows enhanced properties compared to a classical neutral and negatively charged formulation. Nuclear localisation of oligos could only be observed with the AVE-3 formulation. Furthermore only the negatively charged liposome formulations interact with the protamine-complexed oligonucleotides.

Biological Transport↗
PubMed2000

Improvement of intestinal peptide absorption by a synthetic bile acid derivative, cholylsarcosine.

The potential of the nontoxic bile salt derivative, cholylsarcosine, to enhance the intestinal absorption of peptides was investigated in vitro and in situ. The permeation of the two model peptides octreotide and vasopressin-[arg(8)CT>/=CS, whereas ursodeoxycholic acid exhibited no absorption enhancement. Determination of the cytotoxic potential of the bile salts revealed the same rank order. In rats, octreotide and desmopressin were absorbed from the gastrointestinal-tract with moderate absorption efficiency. Coadministration of bile salts resulted in an increased absorption efficiency. The effect of CS was similar to that of CT. In conclusion, CS shows absorption enhancement properties and a relatively low cytotoxicity. It offers an alternative as absorption enhancer as compared to conventional bile acids which may have a potential cocarcinogenic risk.

Animals↗
PubMed2000

Nuclear transport of oligonucleotides in HepG2-cells mediated by protamine sulfate and negatively charged liposomes.

PURPOSE: The aim of this study was to characterize the intracellular fate and nuclear uptake kinetics of oligonucleotides (ON) that were complexed with protamine sulfate (PS) and negatively charged liposomes at different ratios of ON to PS. METHODS: Double-fluorescence labelling of ON and liposomal lipid was applied to simultaneously monitor the interaction as well as the individual fate of active agent and carrier upon intracellular delivery using confocal laser scanning microscopy (CLSM). A DNA-analogue of a 68-mer intramolecular double-stranded RNA:DNA-hybridoligonucleotide (chimeraplasts) with unmodified phosphate backbone was employed. This construct was condensed with PS and coated with a liposomal formulation (AVE-3 = artificial viral envelope). RESULTS: PS-ON complexes and AVE -3-coated complexes with a defined composition were very effective in nuclear transport of ON for a ON:PS charge ratio of 1:3. Nucleus:cytosol fluorescence ratios peaked at about 10 hrs and started to decrease again at 21 hrs. CONCLUSIONS: AVE associates with PS-condensed ON, and this complex is able to be taken up by cells and to deliver ON to the nucleus. PS-ON complexes are released from the liposomal formulation, mainly as an extranuclear enzymatic degradation of the liposomal phospholipids. The results of the kinetic analysis can be used to optimize transfection protocols with ON in HepG2 cells.

Anions↗
PubMed2000

P-glycoprotein- and mrp2-mediated octreotide transport in renal proximal tubule.

1. Transepithelial transport of a fluorescent derivative of octreotide (NBD-octreotide) was studied in freshly isolated, functionally intact renal proximal tubules from killifish (Fundulus heteroclitus). 2. Drug accumulation in the tubular lumen was visualized by means of confocal microscopy and was measured by image analysis. Secretion of NBD-octreotide into the tubular lumen was demonstrated and exhibited the all characteristics of specific and energy-dependent transport. Steady state luminal fluorescence averaged about five times cellular fluorescence and was reduced to cellular levels when metabolism was inhibited by NaCN. 3. NBD-octreotide secretion was inhibited in a concentration-dependent manner by unlabelled octreotide, verapamil and leukotriene C(4) (LTC(4)). Conversely, unlabelled octreotide reduced in a concentration dependent manner the p-glycoprotein (Pgp)-mediated secretion of a fluorescent cyclosporin A derivative (NBDL-CS) and the mrp2-mediated secretion of fluorescein methotrexate (FL-MTX). 4. This inhibition was not due to impaired metabolism or toxicity since octreotide had no influence on the active transport of fluorescein (FL), a substrate for the classical renal organic anion transport system. 5. The data are consistent with octreotide being transported across the brush border membrane of proximal kidney tubules by both Pgp and mrp2.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2000

Transport of heptafluorostearate across model membranes. Membrane transport of long-chain fatty acid anions I.

Heptafluorostearic acid, an isogeometric derivative of stearic acid, has a pK(a) value of about 0.5. To evaluate the suitability of heptafluorostearate as model compound for anions of long-chain fatty acids in membrane transport, monolayer and liposome studies were performed with lipid mixtures containing phospholipids;-cholesterol-heptafluorostearate or stearate (100:40:20 molar ratios). Transfer of heptafluorostearate and stearate from liposomes to bovine serum albumin (BSA) was followed by measuring the intrinsic fluorescence of BSA. The percentage of heptafluorostearate, equivalent to the amount placed in their outer monolayer, transferred from liposomes (120;-130 nm diameter) to BSA was 55.7 +/- 3.7% within 10 min at 25 degrees C and 55 +/- 2% within 5 min at 37 degrees C. Slow transfer of 22.7 +/- 2.5% of heptafluorostearate at 25 degrees C followed with a half-life of 2.3 +/- 0.4 h and of 20 +/- 4% at 37 degrees C with a half-life of 0.9 +/- 0.1 h until the final equilibrium distributions between BSA and liposomes were reached, 79 +/- 6% to 21 +/- 5% at 25 degrees C and 75 +/- 5% to 25 +/- 4% at 37 degrees C. The pseudounimolecular rate constants for flip-flop of heptafluorostearate equal k(FF,25) = 0.24 +/- 0.05 h(-) and k(FF,37) = 0.6 +/- 0.1 h(-), respectively. By comparison, transfer of stearate required only 3 min to reach equilibrium distribution. The difference between heptafluorostearate and stearate may be explained by a rapid flip-flop movement of the un-ionized fatty acids which exist in different concentrations in accordance with their pK(a) values. Half-life of flip-flop of heptafluorostearate makes it suitable to study mediated membrane transport of long-chain fatty acid anions.

Animals↗
PubMed2000

A closer look at the insertion within helix 54 of the 23S-rRNA of the genus Corynebacterium (and related taxa).

Direct sequencing of the insertion in helix 54 of the 23S-rRNA of different strains of 8 species of the genus Corynebacterium was performed in order to determine the extent of variations of these strains. The results demonstrated that there is considerable variation within different strains of a single species. While this clearly jeopardizes the usefulness of the insertion with regard to species-specific diagnostic probes, the variations were found to concentrate within two clearly defined regions comprising the ascending and descending parts of the distal helix of a putative stemlike secondary structure. The remainder of the insertion sequences seem to be fairly constant within a single species while displaying considerable differences toward sequences of other species of the same genus.

Base Sequence↗
PubMed1999

Pharmacokinetics of sterically stabilized hexadecylphosphocholine liposomes versus conventional liposomes and free hexadecylphosphocholine in tumor-free and human breast carcinoma bearing mice.

The pharmacokinetics of free and different liposomal formulations of hexadecylphosphocholine (HPC) was investigated in tumor-bearing (human mammary tumor MaTu) and tumor-free mice after intravenous and intraperitoneal administration. The levels of HPC were evaluated at different times in serum, normal tissues, and tumor. The purpose was to test the hypothesis that the enhanced therapeutic efficacy of sterically stabilized HPC liposomes in comparison to conventional vesicles and free HPC is due to its pharmacokinetics. Conventional non-compartmental pharmacokinetic analysis and an elaborate three- and four-compartmental model were used for explaining the experimental data. The serum levels of HPC obtained with sterically stabilized liposomes were only consistently higher in comparison to conventional vesicles and free HPC in the first 4 h. In the xenografted MaTu carcinoma, the differences of the HPC content between the different groups are unexpectedly low and do not reflect the high therapeutic activity [5] of sterically stabilized HPC liposomes. Detailed analysis shows that the liposomally encapsulated drug displays a modified pharmacokinetic behavior, which may also involve lymphatic absorption of the liposomal drug.

Animals↗
PubMed1998

Hepatocellular effects of cyclosporine A and its derivative SDZ IMM 125 in vitro.

The novel immunosuppressive drug O-hydroxyethyl-D(Ser)8-cyclosporine (SDZ IMM 125) and cyclosporine A (CyA) were compared in different in vitro models with respect to hepatocellular side effects. SDZ IMM 125 was less lipophilic than CyA and also decreased liposomal membrane anisotropy less. Furthermore, SDZ IMM 125 increased Na+ and Ca++ permeability across the liposomal membranes significantly more than CyA. The uptake of CyA and SDZ IMM 125 into freshly isolated rat hepatocytes was neither saturable, Na+ dependent or temperature sensitive, nor could it be inhibited vice versa, indicating passive diffusion. The diffusion coefficient of CyA was about two times higher than that of SDZ IMM 125, reflecting its higher lipophilicity. In primary hepatocyte monolayers the cellular concentrations of CyA were about two times higher than that of SDZ IMM 125. As an indicator of cholestasis the saturable uptake of cholyltaurine into isolated cells was found to be apparently competitively inhibited to the same extent by both compounds. In isolated perfused rat livers SDZ IMM 125 caused a significantly greater decrease in bile flow than did CyA. Release of lactate dehydrogenase from hepatocyte primary cultures and from isolated perfused livers were determined as parameter of cell damage. In both systems the cytotoxicity of SDZ IMM 125 was significantly higher than that of CyA. The data suggest that SDZ IMM 125 causes greater cholestatic and cytotoxic effects than CyA at equimolar cellular exposure.

Animals↗
PubMed1996

Paclitaxel partitioning into lipid bilayers.

Paclitaxel (taxol) is diterpenoid anticancer drug with a new mechanism of cytostatic action. It is under investigation in clinical trials for treatment of various types of human cancer. A major difficulty in developing paclitaxel as a chemotherapeutic agent in its poor water solubility. In order to improve the bioavailability of paclitaxel, novel vehicle systems such as mixed micelles or liposome-based formulations are being developed. In this study we determined the partition coefficient of paclitaxel partitioning into small unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine using two different methods, namely high-sensitivity titration calorimetry and fluorescence spectrometry. We measured a partition coefficient of Kp approximately equal to 9,500 M-1, a partition enthalpy of Delta H = -25 +/- 3 kcal mol-1 and a free energy of binding of Delta G = -7.9 kcal mol-1. The binding reaction is enthalpy-driven, which can be explained by van der Waals interactions between the hydrophobic drug and the strong temperature dependence of the partition equilibrium. A temperature increase of 10 degrees C reduces the paclitaxel solubility in the lipid phase by a factor of 4.

Lipid Bilayers↗
PubMed1996

Permeation enhancement of octreotide by specific bile salts in rats and human subjects: in vitro, in vivo correlations.

1. The potential of bile salts to improve the enteral absorption of octreotide, an orally active somatostatin analogue, was investigated by a combination of in vitro, in situ and in vivo experiments. 2. Incorporation of octreotide into lipid monolayers (as measured by area increase of the monolayer at constant surface pressure using a Langmuir-Blodgett trough set-up) depended on the type of bile salt used for monolayer pre-treatment. Addition of 20 microM octreotide to the subphase containing 20 microM of the dihydroxylated bile salt ursodeoxycholate (UDCA) causes a 9% increase in area, whereas addition of octreotide to the subphase containing the 7 alpha-enantiomer of UDCA, chenodeoxycholate (CDCA), resulted in an area increase of the lipid monolayer of 20%. Area increase by octreotide alone was not significantly different from the increase of octreotide and UDCA in combination. 3. CDCA and UDCA in combination with octreotide increased the permeability of liposomal membranes for rubidium ions, whereas octreotide alone did not significantly change the permeability. This indicates membrane distortion as a possible cause for the enhanced absorption of octreotide by bile salts. 4. In polarized Caco-2 cell monolayers octreotide exhibited a permeation coefficient of 0.008 +/- 0.004 cm h-1. Addition of 0.2-1% of UDCA to the apical incubation medium had no significant effect upon the permeation coefficient. In contrast, 0.2-1% CDCA in the incubation medium resulted in a significant increase (P < 0.05) of the monolayer permeability of octreotide (0.015-0.037 cm h-1). 5. Octreotide was absorbed as the intact peptide from the gastrointestinal tract in rats with an absorption efficiency of 0.26%. Coadministration of bile salt resulted in a dose-dependent increase in absorption efficiency of the peptide up to 20.2%. The observed effect was more pronounced for CDCA than for UDCA. 6. The effect of CDCA and UDCA on octreotide absorption in vivo was assessed in a pharmacokinetic study with healthy volunteers. After oral administration of 4 mg octreotide in the presence of 100 mg bile salt, an average bioavailability of the peptide of 1.26% was achieved in the presence of CDCA, whereas in the presence of UDCA a bioavailability of only 0.13% was reached. This difference was statistically significant (P < 0.01). 7. In conclusion, the co-administration of CDCA is able to enhance the enteral absorption of octreotide. The in vitro and in situ experiments were predictive for the observed effect in human subjects.

Adult↗
PubMed1996

Utility of PCR in diagnosing pulmonary tuberculosis.

At present, the rapid diagnosis of pulmonary tuberculosis rests with microscopy. However, this technique is insensitive and many cases of pulmonary tuberculosis cannot be initially confirmed. Nucleic acid amplification techniques are extremely sensitive, but when they are applied to tuberculosis diagnosis, they have given variable results. Investigators at six centers in Europe compared a standardized PCR system (Amplicor; Roche) against conventional culture methods. Defined clinical information was collected. Discrepant samples were retested, and inhibition assays and backup amplification with a separate primer pair were performed. Mycobacterium tuberculosis complex organisms were recovered from 654 (9.1%) of 7,194 samples and 293 (7.8%) of 3,738 patients. Four hundred fifty-two of the M. tuberculosis isolates from 204 patients were smear positive and culture positive. Among the culture-positive specimens, PCR had a sensitivity of 91.4% for smear-positive specimens and 60.9% for smear-negative specimens, with a specificity of 96.1%. Analysis of 254 PCR-positive, culture-negative specimens with discrepant results revealed that 130 were from patients with recently diagnosed tuberculosis and 94 represented a presumed laboratory error. Similar analysis of 118 PCR-negative, culture-positive specimens demonstrated that 27 discrepancies were due to presumed uneven aliquot distribution and 11 were due to presumed laboratory error; PCR inhibitors were detected in 8 specimens. Amplicor enables laboratories with little previous experience with nucleic acid amplification to perform PCR. Disease in more than 60% of the patients with tuberculosis with smear-negative, culture-positive specimens can be diagnosed at the time of admission, and potentially all patients with smear-positive specimens can immediately be confirmed as being infected with M. tuberculosis, leading to improved clinical management.

Bacteriological Techniques↗
PubMed1996

Irreversible inhibition of hepatic fatty acid salt uptake by photoaffinity labeling with 11, 11-azistearate.

In order to have a model compound for detection of proteins involved in transport and metabolism of long-chain fatty acid salts by photoaffinity labeling 11,11-azistearate and 11,11-azi[G-3H]stearate (specific radioactivity 2.78 TBq/mmol) were synthesized. The suitability of 11,11-azi[G-3H]stearate for photoaffinity labeling was demonstrated by incorporation into BSA (bovine serum albumin) and H-FABP (hepatic fatty acid salt-binding protein) of rat liver. Repeated photoaffinity labeling resulted in a clear decrease of the binding capacities of both proteins. Labeling of protein mixtures with 11,11-azi[G-3H]stearate showed that binding proteins for long-chain fatty acid salts interact specifically with this probe. Photoaffinity labeling of isolated hepatocytes using 300 microM 11,11-azistearate in the presence of 100 microM BSA resulted in the irreversible inhibition of the uptake of stearate and its analogue 2,2,3,3,18,18,18-heptafluorostearate nearly to the same extent of about 30%. Irreversible inhibition of the uptake of long-chain fatty acid salts by photoaffinity labeling did not alter the mediated transport of cholyltaurine and has no effect on the uptake of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, a compound that crosses the hepatocyte membrane by simple diffusion. The irreversible inhibition of membrane transport by photoaffinity labeling demonstrates the existence of a specific transport system for the uptake of long-chain fatty acid salts into hepatocytes.

Affinity Labels↗