Localization of Purine Metabolizing Enzymes in Bovine Brain Microvessel Endothelial Cells: An Enzymatic Blood-Brain Barrier for Dideoxynucleosides?

Purpose. The specific activities of the purine and pyrimidine metabolizing enzymes, purine nucleoside phosphorylase (PNP), adenosine deaminase (ADA) and cytidine deaminase (CDA) were determined in bovine brain microvessel endothelial cells (BBMECs), whole cerebral tissue and erythrocytes. In addition, the substrate specificities (K_m and V_max) of purified calf spleen PNP for inosine and 2,3-dideoxyinosine (ddl) and of purified calf intestinal ADA for 2,3-dideoxyadenosine (ddA), 6-chloro-2,3-dideoxypurine (6-Cl-ddP), and 2--fluoro-2,3-dideoxyadenosine (F-ddA) have been explored. Methods. BBMECs were isolated from bovine cerebral cortex by a two step enzymatic dispersion treatment followed by centrifugation over 50% Percoll density gradients. Activities of alkaline phosphatase, -glutamyl transpeptidase, ADA, PNP and CDA were determined in various tissue homogenates (cerebral cortex, BBMECs and erythrocytes). Enzyme kinetic studies were also conducted using commercially available enzymes and several nucleoside analogs of interest. Results. The activities of ADA and PNP were 42-fold and 247-fold higher in the cerebral microvessels than in the cerebral cortex, respectively, while there was no detectable CDA activity in the microvessel fraction and very little overall activity in the cortex. Conclusions. ADA and PNP may serve as an enzymatic blood-brain barrier for some of the anti-HIV dideoxynucleosides. Simulations of brain availability for ddl, ddA, 6-Cl-ddP, and F-ddA demonstrated that the quantitative significance of enzyme localization may vary dramatically, however, depending on the membrane permeability of the drug and its bioconversion rate constant within the endothelial cell.     

<Emphasis Type="Italic">In Vitro</Emphasis>/<Emphasis Type="Italic">in Vivo</Emphasis> Correlation of Transdermal Naltrexone Prodrugs in Hairless Guinea Pigs

Purpose The purpose of this investigation was to evaluate the in vitro and in vivo percutaneous absorption of the following prodrugs of naltrexone (NTX): 2-ethylbutyryl-3-O-ester-NTX (ETBUT-ester), methyl-3-O-carbonate-NTX (ME-carbonate), ethyl-3-O-carbamate-NTX (ET-carbamate), and N,N-dimethyl-3-O-carbamate-NTX (DME-carbamate) in hairless guinea pigs.Methods In vitro fluxes of NTX and its prodrugs through guinea pig skin were determined using a flow-through diffusion cell system. The pharmacokinetics of NTX prodrugs were determined after topical application of transdermal patches in guinea pigs.Results All the prodrugs hydrolyzed to NTX on passing through the skin, and ME-carbonate provided the highest NTX flux and had the highest apparent permeability coefficient (K_p). ME-carbonate and ET-carbamate underwent the highest extent of bioconversion to NTX upon passing through the skin as compared to ETBUT-ester and DME-carbamate. The results of the in vivo studies indicated that a significant amount of NTX was delivered after the application of transdermal patches of NTX prodrugs. A mean steady-state plasma concentration of 7.1 ng/ml was obtained after the application of transdermal patches of ME-carbonate. A good correlation was obtained between the in vitro and in vivo results.Conclusions The results of the in vivo studies indicated that the ME-carbonate prodrug of NTX was the most promising drug candidate for transdermal delivery.     

Relationship of metabolism of 2′-, 3′- and 5′-adenine nucleotides to presynaptic inhibition of transmitter release in rat vas deferens

Summary In the isolated rat vas deferens stimulated at 0.2 Hz, a series of 2, 3-, and 5-substituted adenine nucleotides all inhibited the twitch responses, their actions being potentiated by the nucleoside transport inhibitors, HNBTGR, NBMPR and dipyridamole.The metabolism of these nucleotides was examined utilising HPLC analysis of the bathing medium after exposure to 30 M nucleoside or nucleotide for 5 min. 5-AMP, 5-ADP, 5-ATP, and NAD⁺ were all partially hydrolysed to adenosine, the relative extent of this being 5-AMP>5-ADP=5-ATPNAD⁺. However, the other nucleotides examined were not detectably converted to adenosine or to adenosine deamination products.These results indicate that the 2-, 3- and 5-substituted nucleotides studied act at a P₁-purinoceptor in rat vas deferens to inhibit neurotransmission and, with the exception of 5-AMP, 5-ADP, 5-ATP and NAD⁺, all appear to act directly at this receptor. However, the 5-adenine nucleotides (AMP, ADP and ATP) and NAD⁺ all appear to act at least partially indirectly subsequent to their hydrolysis to adenosine.Abbreviations. The following abbreviations are used ADA adenosine deaminase (EC 3.5.4.4) - 5-ADP adenosine 5-diphosphate - 2,5-ADP adenosine 2,5-diphosphate - 3 5-ADP, adenosine 3,5-diphosphate - 2-, 3 or 5-AMP adenosine 2-, 3-, or 5-monophosphate - 5-ATP adenosine 5-triphosphate - cNADP⁺ -nicotinamide dinucleotide 2,3-cyclic monophosphate - CoA coenzyme A - HNBTGR 6-(2-hydroxy-5-nitrobenzyl)-thioguanosine - NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ -nicotinamide adenine dinucleotide phosphate - NBMPR 6-(4-nitrobenzylthio)-purine riboside     

Carboxylic Ester Hydrolase Activity in Hairless and Athymic Nude Mouse Skin

The carboxylic ester hydrolase activity was compared in athymic nude mouse skin and hairless mouse skin with respect to hydrolytic ability, heat inactivation, pH optima, and substrate specificity. Five aliphatic 5-esters of 5-iodo-2-deoxyuridine (IDU) were incubated with skin homogenate preparations, and the effect of linear chain length and branching of the ester substituent on hydrolysis rate was evaluated. The ester hydrolase activity was three times higher in athymic mouse skin relative to hairless variety. In both mice skin preparations maximum hydrolysis rates were obtained with the valeryl ester followed by butryl, isobutyryl, propionyl and pivaloyl derivatives. Kinetic studies, however, revealed that higher ester hydrolase activity (V _max) in athymic mouse skin is also associated with higher K _m values, while the carboxylic ester hydrolases from these two different strains of mice have similar biochemical properties with respect to heat inactivation and pH optima. Athymic mouse skin resembled hairless mice skin in terms of cholinesterase content. A significant fraction (70–80%) of ester hydrolyzing activities in both strains of mice skin resulted from cholinesterases (true and/or pseudo). The remaining activity was attributed to different ester cleaving enzymes in the two strains of mice. Carbonic anhydrases and arylesterases contributed to the ester hydrolyzing activity of the athymic and normal hairless mice skins, respectively. Product inhibition by the regenerated hydrolytic product, free IDU, was also noticed which resulted in incomplete conversion of rapidly hydrolyzable 5-esters such as the valeryl and butyryl derivatives.     

Iontophoretic in Vivo Transdermal Delivery of β-Blockers in Hairless Rats and Reduced Skin Irritation by Liposomal Formulation

Purpose. To demonstrate the in vivo transdermal delivery and establish the comparative pharmacokinetics of five -blockers in hairless rat. Methods. Intravenous dosing was initially done via jugular cannula. For iontophoretic delivery, current (0.1 mA/cm²) was applied for 2 h through a drug reservoir patch containing the -blocker (10 mg/ml). Blood samples were collected and analyzed by stereoselective HPLC assays. Any irritation resulting from patch application was quantified by a chromameter. Multilamellar liposomal formulation was prepared by the thin-film hydration method and converted to unilamellar liposomes by extrusion. Results. With transdermal iontophoresis, therapeutically relevant amounts of propranolol (83.78 ± 7.4 ng/ml) were delivered within an hour and lasted for up to 4 h. C_max (185.1 ± 56.8 ng/ml) was reached at hour 3. A significantly higher amount (p < 0.05) of sotalol HCl was delivered compared to other -blockers. There was no significant difference in the S/R ratio of AUC_0-t for enantiomers after both intravenous and transdermal delivery. Skin irritation was significantly reduced (p < 0.05) when a liposomal formulation of the propranolol base was used rather than the base itself. Conclusions. The comparative pharmacokinetics of intravenous and transdermal iontophoretic delivery of five -blockers in hairless rats was established. It was shown that there is no stereoselective permeation.     

Interactions of Phosphodiester and Phosphorothioate Oligonucleotides with Intestinal Epithelial Caco-2 Cells

Purpose. Oral bioavailability for antisense oligonucleotides has recently been reported but the mechanistic details are not known. The proposed oral delivery of nucleic acids will, therefore, require an understanding of the membrane binding interactions, cell uptake and transport of oligonucleotides across the human gastro-intestinal epithelium. In this initial study, we report on the cell-surface interactions of oligonucleotides with human intestinal cells. Methods. We have used the Caco-2 cell line as an in vitro model of the human intestinal epithelium to investigate the membrane binding interactions of 20-mer phosphodiester (PO) and phosphorothioate (PS) oligonucleotides. Results. The cellular association of both an internally [³H]-labelled and a 5end [³²P]-labelled PS oligonucleotide (3.0% at 0.4 µM extracellular concentration) was similar and was an order of magnitude greater than that of the 5end [³²P]-labelled PO oligonucleotide (0.2%) after 15 minutes incubation in these intestinal cells. The cellular association of PS was highly saturable with association being reduced to 0.9% at 5 µM whereas that of PO was less susceptible to competition (0.2% at 5 µM, 0.1% at 200 µM). Differential temperature-dependence was demonstrated; PS interactions were temperature-independent whereas the cellular association of PO decreased by 75% from 37°C to 17°C. Cell association of oligonucleotides was length and pH-dependent. A decrease in pH from 7.2 to 5.0 resulted in a 2- to 3-fold increase in cell-association for both backbone types. This enhanced association was not due to changes in lipophilicity as the octanol:aqueous buffer distribution coefficients remained constant over this pH range. The ability of NaCl washes to remove surface-bound PS oligonucleotides in a concentration-dependent manner suggests their binding may involve ionic interactions at the cell surface. Cell-surface washing with the proteolytic enzyme, Pronase^®, removed approximately 50% of the cell-associated oligonucleotide for both backbone types. Conclusions. Binding to surface proteins seems a major pathway for binding and internalization for both oligonucleotide chemistries and appear consistent with receptor (binding protein)-mediated endocytosis. Whether this binding protein-mediated entry of oligonucleotides can result in efficient transepithelial transport, however, requires further study.     

New Cationic Lipid Formulations for Gene Transfer

Purpose. To develop appropriate dosage forms of DNA for gene delivery. Methods. 3[N-(N, N dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) was mixed either with Tween 80 alone, or with additional lipid components including castor oil and phosphatidylcholine (PC) or dioleoylphosphatidylethanolamine (DOPE) to make different lipid formulations. The particle size and the physical stability of the formulations upon mixing with plasmid DNA containing the luciferase cDNA were examined using laser light scattering measurement. The transfection activity of the DNA/lipid complexes was tested in presence or absence of serum using a cell culture system. Results. We demonstrated that many favorable properties as a gene carrier could be achieved by formulating DNA into new dosage forms using Tween 80 as the major emulsifier. Compared to the cationic liposomes, these new formulations transfected different cell lines with an equivalent or higher efficiency. Not only are they resistant to serum, but also form stable DNA complexes which could be stored for longer periods of time without losing transfection activity. Conclusions. Cationic lipids formulated into different lipid formulations using Tween 80 as a surfactant appeared to have more favorable physical and biological activities than traditional cationic liposomes as a carrier for gene delivery.     

Antitumor Agents. 125. New 4β-Benzoylamino Derivatives of 4′-O-Demethyl-4-desoxypodophyllotoxin and 4β-Benzoyl Derivatives of 4′-O-Demethylpodophyllotoxin as Potent Inhibitors of Human DNA Topoisomerase II

A series of 4-benzoylamino (5-17) derivatives of 4-O-demethyl-4-desoxypodophyllotoxin and 4-benzoyl (18-20) derivatives of 4-O-demethyl podophyllotoxin have been synthesized and evaluated for their inhibitory activity against the human DNA topoisomerase II as well as for their activity in causing cellular protein-linked DNA breakage. Compounds 5-13 and 15-17 are more potent than etoposide in causing DNA breakage, while compounds 9, 10, 13, 14, 16, and 20 are more active than etoposide in their inhibition of the human DNA topoisomerase II. The order for the enzyme inhibitory activity of the derivatives of 4-O-demethyl-4-desoxypodophyllotoxin is 4-arylamino > 4-benzylamino > 4-benzoylamino.     

Synthesis and Anti-inflammatory Effect of Chalcones and Related Compounds

Purpose. Mast cell and neutrophil degradations are the important players in inflammatory disorders. Combined with potent inhibition of chemical mediators released from mast cells and neutrophil degranulations, it could be a promising anti-inflammatory agent. 2,5-Dihydroxychalcone has been reported as a potent chemical mediator and cyclooxygenase inhibitor. In an effort to continually develop potent anti-inflammatory agents, a novel series of chalcone, 2- and 3-hydroxychalcones, 2,5-dihydroxychalcones and flavanones were continually synthesized to evaluate their inhibitory effects on the activation of mast cells and neutrophils and the inhibitory effect on phlogist-induced hind-paw edema in mice. Methods. A series of chalcones and related compounds were prepared by Claisen-Schmidt condensation of appropriate acetophenones with appropriate aromatic aldehyde and the anti-inflammatory activities of these synthetic compounds were studied on inhibitory effects on the activation of mast cells and neutrophils. Results. Some chalcones showed strong inhibitory effects on the release of -glucuronidase and histamine from rat peritoneal mast cells stimulated with compound 48/80. Almost all chalcones and 4-hydroxyflavanone exhibited potent inhibitory effects on the release of -glucuronidase and lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe (fMLP). Some chalcones showed potent inhibitory effects on superoxide formation of rat neutrophils stimulated with fMLP/cytochalasin B (CB) or phorbol myristate acetate (PMA). 2,3-Dihydroxy-, 2,5-dihydroxy-4-chloro-, and 2,5-dihydroxychalcone showed remarkable inhibitory effects on hind-paw edema induced by polymyxin B in normal as well as in adrenalectomized mice. Conclusions. These results indicated that the anti-inflammatory effects of these compounds were mediated, at least partly, through the suppression of chemical mediators released from mast cells and neutrophils.     

Trennung und Bestimmung der Nucleotide des Gehirns

Ohne ZusammenfassungFolgende Abkürzungen werden in der Arbeit verwendet AMP Adenosin-5-monophosphat - ADP Adenosin-5-diphosphat - ATP Adenosin-5-triphosphat - GMP Guanosin-5-monophosphat - GDP Guanosin-5-diphosphat - GTP Guanosin-5-triphosphat - IMP Inosin-5-monophosphat - UMP Uridin-5-monophosphat - UDP Uridin-5-diphosphat - UTP Uridin-5-triphosphat - UDPAG Uridin-5-diphosphat-N-acetylglucosamin - UDPG Uridin-5-diphosphat-glucose - DPN Diphosphopyridinnucleotid - TPN Triphosphopyridinnucleotid Mit 10 TextabbildungenMit Unterstütznng der Deutschen Forschungsgemeinschaft.     

Hepatic Distribution and Clearance of Antisense Oligonucleotides in the Isolated Perfused Rat Liver

Purpose. This study was conducted to investigate the impact of backbone modifications on the hepatobiliary disposition of oligonucleotides. Methods. The disposition of backbone-modified antisense oligonucleotides [phosphorothioate (PS) and methylphosphonate (MP)] of the same base-length and sequence (5-TAC-GCC-AAC-AGC-TCC-3), complementary to the codon 12 activating mutation of Ki-ras, was investigated in the isolated perfused rat liver. Livers were perfused for 2 hr; perfusate and bile concentrations were analyzed by HPLC. Hepatocellular distribution was examined by measuring the amount of radiolabeled PS oligonucleotide associated with hepatocytes and Kupffer cells. Protein binding of the PS and MP oligonucleotides was determined in rat serum by ultrafiltration. Results. MP oligonucleotide perfusate concentrations remained constant during the 2-hour perfusion. In contrast, PS oligonucleotide was eliminated slowly by the isolated perfused liver [Cl = 1.05 ± 0.21 mL/min; extraction ratio = 0.06 ± 0.01]. Uptake of PS oligonucleotide by Kupffer cells appeared to exceed uptake by hepatocytes, based on standard cell separation techniques as well as confocal microscopy. The degree of protein binding in rat serum was greater for the PS oligonucleotide (79.9 ± 2.2%) than for the MP oligonucleotide (53.0 ± 4.7%). Conclusions. Backbone modifications significantly influence the hepatic clearance of oligonucleotides. Uncharged MP oligonucleotides are not extracted by the isolated perfused rat liver, whereas the charged PS oligonucleotide is processed more readily.     

Amino Acid Ester Prodrugs of Floxuridine: Synthesis and Effects of Structure,Stereochemistry, and Site of Esterification on the Rate of Hydrolysis

Purpose. To synthesize amino acid ester prodrugs of floxuridine (FUdR) and to investigate the effects of structure, stereochemistry, and site of esterification of promoiety on the rates of hydrolysis of these prodrugs in Caco-2 cell homogenates. Methods. Amino acid ester prodrugs of FUdR were synthesized using established procedures. The kinetics of hydrolysis of prodrugs was evaluated in human adenocarcinoma cell line (Caco-2) homogenates and pH 7.4 phosphate buffer. Results. 3-Monoester, 5-monoester, and 3,5-diester prodrugs of FUdR utilizing proline, L-valine, D-valine, L-phenylalanine, and D-phenylalanine as promoieties were synthesized and characterized. In Caco-2 cell homogenates, the L-amino acid ester prodrugs hydrolyzed 10 to 75 times faster than the corresponding D-amino acid ester prodrugs. Pro and Phe ester prodrugs hydrolyzed much faster (3- to 30-fold) than the corresponding Val ester prodrugs. Further, the 5-monoester prodrugs hydrolyzed significantly faster (3-fold) than the 3,5-diester prodrugs. Conclusions. Novel amino acid ester prodrugs of FUdR were successfully synthesized. The results presented here clearly demonstrate that the rate of FUdR prodrug activation in Caco-2 cell homogenates is affected by the structure, stereochemistry, and site of esterification of the promoiety. Finally, the 5-Val and 5-Phe monoesters exhibited desirable characteristics such as good solution stability and relatively fast enzymatic conversion rates.     

Cutaneous Metabolism of Nitroglycerin in Vitro. II. Effects of Skin Condition and Penetration Enhancement

The effects of skin storage, skin preparation, skin pretreatment with a penetration enhancer, and skin barrier removal by adhesive tape-stripping on the concurrent cutaneous transport and metabolism of nitroglycerin (GTN) have been studied in vitro using hairless mouse skin. Storing the skin for 10 days at 4°C did not alter barrier function to total nitrate flux [GTN + 1,2-glyceryl dinitrate (1,2-GDN) + 1,3-glyceryl dinitrate (1,3-GDN)]. However, metabolic function was significantly impaired and suggested at least fivefold loss of enzyme activity. Heating skin to 100°C for 5 min appreciably damaged hairless mouse skin barrier function. The ability to hydrolyze GTN was still present, however, and remained constant over the 10-hr experimental period, in contrast to the control, which showed progressively decreasing enzymatic function with time. Pretreatment of hairless mouse skin in vivo (prior to animal sacrifice, tissue excision, and in vitro transport/metabolism studies) with 1-dodecylazacyclo-heptan-2-one (Azone), a putative penetration enhancer, significantly lowered the skin barrier to nitrate flux (relative to the appropriate control). Again, barrier perturbation resulted in essentially constant metabolic activity over the observation period. The ratio of metabolites formed (1,2-GDN/1,3-GDN) was increased from less than unity to slightly above 1 by the Azone treatment. Adhesive tape-stripping gradually destroyed skin barrier function by removal of the stratum corneum. The effects of 15 tape-strips were identical to those of Azone pretreatment: a greatly enhanced flux, a constant percentage formation of metabolites over 10 hr (once again), and an increase in the 1,2-GDN/1,3 GDN ratio. Overall, the experiments caution that, for transdermal drug delivery candidates susceptible to skin metabolism, the status of barrier function (enhancer pretreated, skin damage or disease, etc.) may significantly affect systemic availability.     

Immunomodulatory effect and quantitation of a cytotoxic glycosphingolipid from Murdannia loriformis

NMR signal reassignments for a cytotoxic glycosphingolipid compound, 2, -O-D-glucopyranosyl-2-(2-hydroxy-Z-6-enecosamide)sphingosine, isolated from an ethanolic extract of the herb Murdannia loriformis, have been achieved by use of FAB-MS, and 1D and 2D ¹H and ¹³C NMR. The amount of 2 in the herb juice was quantitatively determined by use of a validated HPLC method (RP-18, MeOH–H₂O, UV detection at 210 nm). The immunomodulatory effect of the herb juice and of 2 was proved by means of in vitro cellular immunological assays. Compound 2 at a concentration of 13 nmol L^–1 stimulated PBMC proliferation and increased the CD 3,4:CD 3,8 ratio in T lymphocytes.     

Freeze-Concentration Separates Proteins and Polymer Excipients Into Different Amorphous Phases

Purpose. To study the miscibility of proteins and polymer excipients in frozen solutions and freeze-dried solids as protein formulation models. Methods. Thermal profiles of frozen solutions and freeze-dried solids containing various proteins (lysozyme, ovalbumin, BSA), nonionic polymers (Ficoll, polyvinylpyrrolidone [PVP]), and salts were analyzed by differential scanning calorimetry (DSC). The polymer miscibility was determined from the glass transition temperature of maximally freeze-concentrated solute (T_g) and the glass transition temperature of freeze-dried solid (T_g). Results. Frozen Ficoll or PVP 40k solutions showed T_g at –22°C, while protein solutions did not show an apparent T_g. All the protein and nonionic polymer combinations (5% w/w, each) were miscible in frozen solutions and presented single T_gs that rose with increases in the protein ratio. Various salts concentration-dependently lowered the single T_gs of the proteins and Ficoll combinations maintaining the mixed amorphous phase. In contrast, some salts induced the separation of the proteins and PVP combinations into protein-rich and PVP-rich phases among ice crystals. The T_gs of these polymer combinations were jump-shifted to PVP's intrinsic T_g at certain salt concentrations. Freeze-dried solids showed varied polymer miscibilities identical to those in frozen solutions. Conclusions. Freeze-concentration separates some combinations of proteins and nonionic polymers into different amorphous phases in a frozen solution. Controlling the polymer miscibility is important in designing protein formulations.     

Effect of Coadministered Uridine on Intestinal First-Pass Metabolism of 5′-Deoxy-5-Fluorouridine in Conscious Rats—An Evaluation by Method of Portal-Systemic Concentration Difference

Purpose. The effect of uridine (UR) coadministration on the intestinal metabolism from 5-deoxy-5-fluorouridine (5-DFUR) to 5-fluorouracil (5-FU) was evaluated by a method of concentration difference between portal and systemic bloods in conscious rats (PS method). Methods. 5-DFUR (100 mg/kg) alone (Group A), or 5-DFUR + UR (100 mg/kg each) (Group B) was orally administered to conscious rats. The portal and arterial bloods were simultaneously withdrawn from two canulas at appropriate time intervals, and blood concentrations of 5-DFUR, 5-FU, UR and uracil (U) were assayed by HPLC. The concentration-time profiles of these drugs and its metabolites were analyzed by local moment analysis. Results. UR coadministration made the local absorption ratio (F_a) of 5-DFUR decrease significantly from 60.1 ± 10.5% to 38.0 ± 18.6% of dose. Though the local absorption ratios (F_a ^m) of the metabolite (5-FU) were the same between Group A and Group B (8.3 ± 1.9 and 8.7 ± 4.0% of 5-DFUR, respectively), AUC of arterial 5-FU in Group B was 5 times greater than that in Group A. UR was not detected in the portal blood, and F_a ^m of U was estimated to be 41.9 ± 26.8% of UR in Group B. Conclusions. It is predicted that a large portion of 5-FU generated from 5-DFUR is further degraded in the intestine in Group A, and U generated from UR blocks 5-FU degradation in the intestine and the systemic circulation in Group B.     

Constituents of Laurus nobilis L. inhibit recombinant human lanosterol synthase

Extracts from 37 kinds of foods and foodstuffs were tested for inhibitory activity against recombinant human lanosterol synthase. Among them, extracts from five samples showed significant inhibition. Potent activity (55%) was found in 95% ethanol extract of Laurus nobilis L. Therefore, large-scale methanol extraction of the plant was carried out, and the constituents were separated by partition and fractionation by silica gel chromatography and HPLC. Four flavonoids, kaemperol 3-O-[2,4-O-di-E-p-coumaroyl--l-pyranorhamnoside] (1); 3,3,4,5,6,7,8-heptamethoxyflavone (2); 3,4,5,6,7,8-hexamethoxyflavone (nobiletin) (3); and 4,5,6,7,8-pentamethoxyflavone (tangeretin) (4); and six sesquiterpens, eremanthine (5), dehydrocostus lactone (6), costunolide (7), zaluzanin C (8), zaluzanin D (9) and reynosin (10) were isolated. Eremanthine (5) showed the most potent activity, 70% inhibition, at the concentration of 500 M.     

Amine Prodrugs Which Utilize Hydroxy Amide Lactonization. I. A Potential Redox-Sensitive Amide Prodrug

Several amides of 3-(3,6-dioxo-2,4-dimethylcyclohexa-l,4-diene)-3,3-diniethylpropionic acid (2) have been synthesized and tested as model redox-sensitive pro-prodrugs of amines. The reduction of these model pro-prodrugs generated hydroxy amide intermediates 4a-4h, the lactonization of which resulted in amine release. The rates of lactonization of 4a-4h were investigated at pH 7.4 and 37°C. The half-lives for appearance of the product lactone la from these intermediates were found to range from 1.4 to 3.4 min. With such rapid lactonization rates, it is believed that reduction will be the rate-limiting step in the two-step conversion of the pro-prodrug to the amine.     

Transport of Alpha-Tocopherol and Its Derivatives Through Erythrocyte Membranes

Purpose. To investigate the transport of -tocopherol (T), tocopherol succinate (TS) and tocopherol succinate-3-glucose (a newly synthetized, less hydrophobic T ester; TSG) through bovine erythrocyte membranes. Methods. Our experiments were carried out on erythrocytes (obtained from heparinized fresh bovine blood), because they represent a suitable model for investigations of membrane transport. Results. T was shown to reside almost completely in the suspension medium, while the greater part of TS disappeared from the suspension medium and was mainly incorporated into erythrocyte membranes. In comparison with T, a larger amount of TSG was incorporated into erythrocyte membranes and taken up by cells; however the TSG intra-cellular accumulation was significantly lower than that observed with TS. Furthermore, the transport of TS and TSG was partially inhibited by p-chloromercuribenzenesulfonate (which inhibits monocarboxylate uptake; PCMBS) and by maltose (a competitive inhibitor of glucose transport) respectively, with a concomitant increase in drug membrane incorporation. No significant change in drug transport was observed in the presence of 4,4-diisothiocyanostilbene-2,2-disulfonate, a selective and irreversible blocker of band 3 protein (DIDS). Conclusions. Our results show 1) the existence of large differences in membrane incorporation of T, TS and TSG (very likely caused by differing abilities to fill spaces in the lipid bilayer) and 2) a specific contribution of the monocarboxylate transport protein and of the glucose transport protein in the cellular uptake of TS and TSG, respectively. A tempting suggestion is that the unique cytoprotective properties of TS may be related to the differences in the transmembrane mobility observed between T and its succinate ester. Furthermore, T conjugation to a monocarboxylate or glycoside moiety could provide suitable substrates for active membrane transport, thus appearing as a promising pharmaceutical strategy for the improved delivery of tocopherol derivatives.     

Bis(carbamoyloxymethyl) Esters of 2′,3′-Dideoxyuridine 5′-monophosphate (ddUMP) as Potential ddUMP Prodrugs

No HeadingPurpose. We previously reported the synthesis of bis(pivaloyloxymethyl) 2,3-dideoxyuridine 5-monophosphate (POM₂-ddUMP) (1a) as a membrane-transport prodrug formulation of the free parent nucleotide, ddUMP. Although successful at delivering ddUMP into cells in culture, POM₂-ddUMP was rapidly degraded by plasma carboxylate esterases after intravenous administration to experimental animals, and therefore has limited therapeutic potential as a systemically administered prodrug. We now report the synthesis of bis(N,N-dimethylcarbamoyloxymethyl)- and bis(N-piperidinocarbamoyloxymethyl) 2,3-dideoxyuridine 5-m onophosphate [DM₂-ddUMP (1b) and DP₂-ddUMP (1c), respectively], analogues of POM₂-ddUMP that were designed to be more resistant to degradation by plasma esterases..Methods. After entering cell by passive diffusion, it was anticipated that loss of one of the carbamoyloxymethyl groups of 1b and 1c would occur by spontaneous chemical hydrolysis to give the intermediate phosphodiesters, 2b and 2c. Cleavage of the remaining carbamoyloxymethyl groups by cellular phosphodiesterase I would generate ddUMP. 1b and 1c were prepared by condensation of 2,3-dideoxyuridine (ddU) with the appropriate bis(N-alkylcarbamoyloxymethyl) phosphate in DMA in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent).Results. The half-lives of 1b and 1c when incubated at a concentration of 10^–4 M in human plasma at 37°C were 3.5 h and 3.7 h, respectively, similar to the half-lives observed under the same temperature conditions in 0.05 M aqueous phosphate buffer, pH 7.4. By contrast, the half-life of the POM₂ prodrug, 1a, in plasma was only 5 min. The initial products of degradation of 1b and 1c were the phosphodiesters 2b and 2c. The latter compounds gave rise to ddUMP when incubated with snake venom phosphodiesterase I.Conclusions. These findings support the premise inherent in the design of 1b and 1c, namely that the carbamate prodrugs are far more resistant to hydrolysis by plasma carboxylate esterases than their POM counterparts and can revert to the free parent 5-mononucletides by successive chemical and enzymatic hydrolysis. Further studies of 1b and 1c as membrane-permeable prodrugs of ddUMP are in progress.