Hans-Hubert Borchert

Tetracycline-HCl-loaded poly(dl-lactide-co-glycolide) microspheres prepared by a spray drying technique: influence of γ-irradiation on radical formation and polymer degradation

Tetracycline-HCl (TCH)-loaded microspheres were prepared from poly(lactide-co-glycolide) (PLGA) by spray drying. The drug was incorporated in the polymer matrix either in solid state or as w/o emulsion. The spin probe 4-hydroxy-2,2,6, 6-tetramethyl-piperidine-1-oxyl (TEMPOL) and the spin trap tert-butyl-phenyl-nitrone (PBN) were co-encapsulated into the TCH-loaded and placebo particles. We investigated the effects of gamma-irradiation on the formation of free radicals in polymer and drug and the mechanism of chain scission after sterilization. Gamma-Irradiation was performed at 26.9 and 54.9 kGy using a 60Co source. The microspheres were characterized especially with respect to the formation of radicals and in vitro polymer degradation. Electron paramagnetic resonance (EPR) spectroscopy, gel permeation chromatography (GPC), differential scanning calorimetry (DSC), high-performance liquid chromatography (HPLC), gas chromatography-mass spectroscopy (GC-MS), and scanning electron microscopy (SEM) were used for characterization of the microspheres. Using EPR spectroscopy, we successfully detected gamma-irradiation induced free radicals within the TCH-loaded microspheres, while unloaded PLGA did not contain radicals under the same conditions. The relatively low glass transition temperature of the poly(dl-lactide-co-glycolide) (37-39 degrees C) seems to favor subsequent reactions of free radicals due to the high mobility of the polymeric chains. Because of the high melting point of TCH (214 degrees C), the radicals can only be stabilized in drug loaded microspheres. In order to determine the mechanism of polymer degradation after exposure to gamma-rays, the spin trap PBN and the spin probe TEMPOL were encapsulated in the microspheres. gamma-Irradiation of microspheres containing PBN resulted in the formation of a lipophilic spin adduct, indicating that a polymeric radical was generated by random chain scission. Polymer degradation by an unzipping mechanism would have produced hydrophilic spin adducts of PBN and monomeric radicals of lactic or glycolic acid. These degradation products were not detected by EPR. This result is confirmed by the observation that possible diamagnetic reaction products of low molecular weight, consisting of TEMPOL and lactide or glycolide monomers, could not be detected by GC-MS. While an irradiation dose-dependent decrease in molecular weight of PLGA could be verified in agreement with the literature, TCH content of the microspheres was not affected by the exposure to gamma-rays. It can be concluded that EPR spectroscopy in combination with GPC, DSC, and HPLC allows a detailed characterization of the impact of gamma-sterilization on biodegradable parenteral drug delivery systems.

Correlation between genotype and phenotype of the human arylamine N-acetyltransferase type 1 (NAT1)

Arylamine N-acetyltransferase 1 (NAT1) conjugates several aromatic amines and their N-hydroxylated metabolites by N- or O-acetylation. NAT1 genotype and phenotype is known to be variable in human populations. In this study, we set out to measure the functional relevance of the frequent NAT1 gene variants for the activity in human red blood cells. Healthy German volunteers (N = 314) were genotyped for NAT1 alleles *3, *4, *10, *11, *14, and *15 using polymerase chain reactions and restriction fragment length pattern analysis, and NAT1 enzyme kinetic parameters were measured in a subset of 105 individuals using p-aminobenzoic acid as specific substrate. There was no functional difference between NAT1 alleles *4 and *10. In particular, there was no trend of increasing activity from NAT1*4/*4 to *4/*10 and *10/*10. Carriers of the NAT1 *11 and *14 alleles had a statistically significant lower enzyme activity compared with carriers of the *3, *4, or *10 alleles. Compared with the wild-type genotype NAT1*4/*4, activity of the NAT1*11/*11, NAT1*11/*10, and NAT1*11/*4 genotypes was reduced by 20.7%, 35.7%, and 31.5%, respectively. Activity of the NAT1*10/*14 and NAT1*4/*14 genotypes was reduced by 49.8% and 55.6%, respectively. The difference in NAT1 activity between the *4/*11 and *4/*14 genotypes was also significant (P < 0.01). The carrier of the NAT1*15/*15 genotype had no detectable enzyme activity. In conclusion, functional consequences of NAT1 mutations were tested in a large population. Activity in carriers of NAT1 alleles *3, *4, and *10 did not differ, alleles NAT1*11 and *14 appeared to be low activity alleles, and allele NAT1*15 had no activity.

Concordance between enzyme activity and genotype of glutathione S-transferase theta (GSTT1).

Blood samples from 140 healthy German volunteers were used to further characterize the genetic polymorphism of the human theta class glutathione S-transferase 1 (GSTT1). For measurements of GSTT1 activity, hemolysates were incubated in vitro with different concentrations of dichloromethane. The resulting enzymatically mediated production of formaldehyde was determined colorimetrically by the Nash reaction. GSTT1 genotyping was performed by polymerase chain reaction (PCR) methods using genomic DNA from total white blood cells. The prevalence of homozygous deletion of the GSTT1 gene was 19.3% (95% confidence limits: 12.2-27.7%). There was a high agreement between genotyping and phenotyping data. The individuals with the null genotype had a rate of formaldehyde production below the limit of quantification. In addition, in the group of GSTT1-positive individuals, we could differentiate highly active people (35.7%) from individuals with an intermediate enzyme activity (45.0%). It can be concluded that the PCR method is suitable to quickly genotype large populations, whereas the phenotyping assay at present offers the advantage of differentiating heterozygously from homozygously active subjects. Our results confirm the ethnic differences in the prevalence of the homozygous deleted genotype which were previously observed and seem to exist even between closely related ethnic groups such as German and Swedish populations.

Non-destructive and localized assessment of acidic microenvironments inside biodegradable polyanhydrides by spectral spatial electron paramagnetic resonance imaging

Spectral spatial electron paramagnetic resonance imaging (e.p.r.i.) was applied to characterize the microenvironment inside 2 mm thick poly(1,3-bis-p-carboxyphenoxypropane-co-sebacic anhydride) (14) polymer discs. Incorporated nitroxides are highly immobilized in the dry polymer. Exposure to 0.1 M phosphate buffer (pH 7.4) resulted in the formation of a front of degraded polymer from outside to inside. The localized measurement of pH values inside the polymer matrix was performed, using a pH sensitive nitroxide (pKa 6.1). A pH gradient was found between the degrading polymer and the buffer, but also inside the polymer. The pH inside the delivery matrix rises with time for 4.7 to 7.4. The results demonstrate that difficulties in the characterization of the pH, one of the most important parameters for drug delivery and polymer degradation, can be overcome by spectral spatial e.p.r.i.

Influence of Drug Treatment on the Microacidity in Rat and Human Skin—An In Vitro Electron Spin Resonance Imaging Study

AbstractPurpose. The possibilities of the noninvasive examination of microacidity5 in different depths of the skin in vitro was explored, and the impact of drug treatment on the pH inside the skin was studied. Methods. Spectral-spatial electron spin resonance imaging (ss-ESRI) and pH-sensitive nitroxides were used to obtain a pH map of rat and human skin in vitro. Results. The dermal application of therapeutically used acids, such as salicylic acid and azelaic acid, caused a plain change of microacidity (pH) inside the skin. Species-linked differences between rat and human skin samples with respect to penetration and microacidity were found. Conclusions. ESRI has been shown to be a new and completely noninvasive method to monitor microacidity in different skin layers and on the skin surface. This nondestructive method allows serial measurements on skin samples to be performed without any preparatory steps.

Nitroxide metabolism in the human keratinocyte cell line HaCaT.

Metabolism of different nitroxides with piperidine structure used as spin labels in electron spin resonance (ESR) studies in vitro and in vivo was investigated in human keratinocytes of the cell line HaCaT by GC and GC-MS technique combined with S-band ESR. Besides the well known reduction of the nitroxyl radicals to the ESR silent hydroxylamines as primary products our results indicate the formation of the corresponding secondary amines. These reductions are inhibited by the thiol blocking agent N-ethylmaleimide and by the strong inhibitors of the thioredoxin reductase (TR) 2-chloro-2,4-nitrobenzene and 2,6-dichloroindophenol. The competitive inhibitor TR inhibitor azelaic acid and the cytochrome P-450 inhibitor metyrapone lack any effects. The rates of reduction to the hydroxylamines and secondary amines were dependent on the lipid solubility of the nitroxides. Therefore, it can be assumed that the nitroxides must enter the cells for their bioreduction. The mostly discussed intracellular nitroxide reducing substances ascorbic acid and glutathione were unable to form the secondary amines. In conclusion, our results suggest that the secondary amine represents one of the major metabolites of nitroxides besides the hydroxylamine inside keratinocytes formed via the flavoenzyme thioredoxin reductase most probably. Further metabolic conversions were detected with 4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl and the benzoate of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl as substrates.

Direct and continuous determination of pH values in nontransparent w/o systems by means of EPR spectroscopy

Abstract The change of the pH value induced by the decomposition of hydrolyzable drugs with ester structure was monitored directly and continuously in nontransparent w/o systems by means of EPR spectroscopy using the pH sensitivity of the spectral parameters of imidazoline-derived spin probes. This nondestructive method makes it possible to perform measurements in nontransparent w/o systems without the need of any preparatory steps. While the hydrolysis of acetylsalicylic acid caused a rapid decrease of the pH value during 5 h at room temperature (25°C) within the emulsion, benzocain exhibited a relatively higher stability.

Metabolism of the stable nitroxyl radical 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPONE)

The formation of new metabolites of the stable nitroxyl radical 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPONE) inside the isolated perfused rat liver was examined. The paramagnetic 4-hydroxy derivative (TEMPOL) and the diamagnetic 1,4-dihydroxy derivative were found to be the major metabolites besides the well-known corresponding hydroxylamine of TEMPONE. No reoxidation of the hydroxyl group in the 4-position was observed. The conversion of nitroxides to the sterically hindered secondary amines remains speculative. A redox cycle of nitroxide and hydroxylamine including the secondary amines is discussed. For the first time the biotransformation of the stable nitroxyl radical TEMPONE detected by means of GC and GC-MS has been examined and new metabolites have been described, i.e. the newly discussed metabolites have to be considered for the interpretation of electron paramagnetic resonance (EPR), magnetic resonance imaging (MRI) and dynamic nuclear polarization (DNP) measurements on using the spin probe TEMPONE.

In vivo ESR studies on subcutaneously injected multilamellar liposomes in living mice

AbstractPurpose. An innovative, noninvasive, low-frequency electron spin resonance (ESR) spectroscopy method was applied and adapted to investigate the integrity of multilamellar liposomes from hydrogenated phospholipids after subcutaneous injection in living mice. Moreover, the fate of the injected liposomal preparations was examined, as well as the possibility to achieve a depot effect. Methods. Highly concentrated solutions of the spin probe 2,2,6,6-tetramethyl-4-trimethylammoniumpiperidine-1-oxyl-iodide (CAT-1; 138 mM) were encapsulated in liposomes. They were characterized by laser diffraction, and the liberation of spin probe was investigated by ESR spectroscopy. Results. Line shape changes allowed the differentiation between encapsulated and released CAT-1 after subcutaneous injection of liposomes. Multilamellar liposomes form a local depot at the site of injection. A sustained release of the spin probe from the depot was monitored by means of ESR. Whereas 40% of the spin probe was released within the first 96 h after administration, 60% remained in intact liposomes under the skin. No depot formation could be observed after injection of CAT-1 solutions, but a fast signal decrease due to systemic distribution and bioreduction of the nitroxide spin probe. Conclusions. Noninvasive analysis of liposomal integrity in living animals was successfully accomplished using a new L-Band ESR spectroscopy method. The liberation of CAT-1 from liposomes in vitro and in vivo was monitored by changes in the lineshape of ESR spectra and Heisenberg spin exchange. The significance of liposomal integrity for the formation of a localized drug depot effect was proved.

Detection of free radicals in living mice after inhalation of DTBN by X-band ESR

A new application of X-band electron spin resonance (ESR) is demonstrated for in vivo investigations on living mice. It is the first report on in vivo detection, identification, and localization of a free radical after uptake in the body of a living mammal by inhalation. The volatile di-tert-butyl-nitroxyl (DTBN) has been used as a suitable spin probe. After inhalation of DTBN, a superimposed ESR spectrum consisting of a polar (g = 2.0057; a = 1.65 mT) and a nonpolar paramagnetic species (g = 2.0061; a = 1.51 mT) was detected in mice tails. Collected blood exhibits only the polar spectrum. The decay of signals after removing of the DTBN-soaked swab follows first-order kinetics with k = 0.08 min-1.