Synthesis and evaluation of a prototypal artificial red cell.

A new process allows microencapsulation of purified human hemoglobin and 2,3-diphosphoglycerate to form neohemocytes. The microcapsule membrane is composed of phospholipids and cholesterol. Neohemocytes are substantially smaller than erythrocytes, contain 15.1 grams per decaliter of hemoglobin, and have a P50 value (the partial pressure of oxygen at which the hemoglobin is half-saturated) of 24.0 torr. All rats given 50-percent exchange transfusions survived with only limited evidence of reversible toxicity. Normal serum glutamate-pyruvate-transaminase values at 1, 7, and 30 days after transfusion were consistent with minimal hepatotoxicity. The concentration of blood urea-nitrogen was elevated by 35 percent after 1 day but returned to normal by day 7. However, histopathology revealed normal kidneys on day 1 as well as on days 7 and 30. Neohemocytes cleared from the circulation of transfused rats with an apparent half-life of 5.8 hours.

The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein.

In CNS synapses, the synaptic junctional complex with associated postsynaptic density is presumed to contain proteins responsible for adhesion between pre- and postsynaptic membranes and for postsynaptic signal transduction. We have found that a prominent, brain-specific protein (PSD-95) enriched in the postsynaptic density fraction from rat brain is highly similar to the Drosophila lethal(1)discs-large-1 (dlg) tumor suppressor protein. The dlg protein is associated with septate junctions in developing flies and contains a guanylate kinase domain that is required for normal control of cell division. The sequence similarity between dlg and PSD-95 suggests that molecular mechanisms critical for growth control in developing organisms may also regulate synapse formation, stabilization, or function in the adult brain.

Credibility, Replicability, and Reproducibility in Simulation for Biomedicine and Clinical Applications in Neuroscience.

Modeling and simulation in computational neuroscience is currently a research enterprise to better understand neural systems. It is not yet directly applicable to the problems of patients with brain disease. To be used for clinical applications, there must not only be considerable progress in the field but also a concerted effort to use best practices in order to demonstrate model credibility to regulatory bodies, to clinics and hospitals, to doctors, and to patients. In doing this for neuroscience, we can learn lessons from long-standing practices in other areas of simulation (aircraft, computer chips), from software engineering, and from other biomedical disciplines. In this manuscript, we introduce some basic concepts that will be important in the development of credible clinical neuroscience models: reproducibility and replicability; verification and validation; model configuration; and procedures and processes for credible mechanistic multiscale modeling. We also discuss how garnering strong community involvement can promote model credibility. Finally, in addition to direct usage with patients, we note the potential for simulation usage in the area of Simulation-Based Medical Education, an area which to date has been primarily reliant on physical models (mannequins) and scenario-based simulations rather than on numerical simulations.

Metabolic disposition of N,N-dibenzyldaunorubicin in the rat.

The more efficacious and less cardiotoxic analogue of daunorubicin, N,N-dibenzyldaunorubicin (B2D), was found to be metabolized in rats by stepwise debenzylation that was superimposed on the known anthracycline metabolism via 13-ketone reduction and deglycosidation. Using high-pressure liquid chromatography for resolution and fluorescence for detection, we observed a series of metabolites in plasma, liver, heart, muscle, and lungs of rats receiving 10 mg B2D per kg, i.v., i.p., and p.o. Rats receiving 40 mg B2D per kg, i.v., died immediately, but this dose given p.o. was not lethal during 24 hr. Patterns of B2D and metabolites varied quantitatively with tissue and route of administration. Rat liver perfusion studies indicated extensive metabolism of B2D compared with limited metabolism of doxorubicin. These observations were consistent with an observed major first-pass effect on B2D in intact rats given B2D p.o. The predominant metabolites of B2D were the glycosidic derivatives, N-benzyldaunorubicin, daunorubicin, and their 13-dihydro derivatives. These metabolites of B2D had exhibited activity against mouse leukemia P388 as did B2D and were active in in vitro tests in which B2D was essentially inactive. These results indicate that B2D acts as a prodrug for a series of active metabolites. Conversion of B2D to these metabolites was relatively more efficient after p.o. administration than following i.v. or i.p. treatments.

Liposomes disposition in vivo. V. Liposome stability in plasma and implications for drug carrier function.

The kinetics of [14C]sucrose release from multilamellar liposomes of fixed diameter (approx. 0.23 micron) incubated in human plasma (serum and blood) were quantified. Composition was various ratios of phosphatidylcholine, phosphatidic acid and cholesterol with alpha-tocopherol included as antioxidant. Considerable intra-individual variability was noted for liposome stability in blood and its derived fluids, yet reproducible results were obtained for pooled samples. The destabilizing effects of plasma decreased with increasing lipid concentrations. Results of fitting a kinetic model to the data showed that four of five model parameters were linearly related to liposome cholesterol content. Liposomes depleted plasma of its destabilizing factors, and when pre-incubated with plasma were partially stabilized to the effects of a subsequent plasma addition. Plasma caused a rapid rise in liposome membrane permeability which then declined non-linearly, presumably because of a rearrangement of membrane lipids and adsorbed proteins to form their most stable configuration. The therapeutic availability of drugs administered encapsulated in liposomes, which can be governed by the kinetics of their in vivo extracellular release, may be directly proportional to--and predictable from--the time-course and extent of release in plasma. The kinetic model was used in conjunction with simple pharmacokinetic assumptions to show that the effectiveness of a liposome drug carrier cannot be predicted based simply on its plasma stability; more stable liposomes may not be more effective drug carriers. Interestingly, plasma-induced solute release from liposomes serendipitously mimics an important facet of ideal carrier behavior.

Liposome disposition in vivo. III. Dose and vesicle-size effects.

The effect of lipid dose (4,3-512.8 mumol total lipid/kg body weight), administered intravenously as liposomes encapsulating radioactive inulin, upon the ability of mouse organs to bind and/or take-up the radioactive label has been studied in vivo. Three different liposome diameters were investigated: 0.46 micrometers (L), 0.16 micrometers (M) and 0.058 micrometers(S). All liposomes were negatively charged with lipid composition of phosphatidylcholine/phosphatidic acid/cholesterol/alpha-tocopherol in the molar ration 4 : 1 : 5 : 0.1 or 4 : 1 : 1 : 0.05. Overall radioactive label disposition after 2 h was consistent with localization predominantly in the reticuloendothelial system. A saturation of liver with increasing lipid dose was demonstrated for all three sizes, together with a corresponding increase in blood levels. Spleen radioactivity increased with increasing dose of L- and M-liposomes, but decreased for increasing doses of S-liposomes. Levels in residual carcass exhibited no trend. It was noted that by adjusting liposomal lipid dose and vesicle diameter the percentage of administered dose present in blood could be varied 733-fold, that in spleen 9-fold, liver 4-fold. Stability in vivo was ranked L greater than M greater than S-liposomes. Correction for differences of in vivo stability reduced the differences in organ accumulation between the three liposome sizes. The organ accumulation pattern suggested a dose- and diameter-dependent mechanism for liposome disposition. It was expected that when doses of fixed liposome composition were expressed as number of liposomes or their total surface area, organ saturation patterns would be similar. However, re-plotting the percent dose values for liver and spleen versus the number of liposomes administered revealed a saturation pattern for L-, M- and S-liposomes which was different in each case. Plotting the data versus the total surface area of the dose revealed a similar disposition pattern for L-, M- and S-liposomes in liver and L- and M-liposomes in spleen. The data indicate that in addition to composition, the lipid dose, total liposomal surface area and effective mean diameter are important pharmacokinetic variables. Further, the optimization of the therapeutic index of an encapsulated agent or target-tissue delivery via liposomes will require consideration of both the surface area and diameter of the liposome doses together with liposome composition.

Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes.

Liposomes of defined size and homogeneity have been prepared by sequential extrusion of the usual multilamellar vesicles through polycarbonate membranes. The process is easy, reproducible, produces no detectable degradation of the phospholipids, and can double the encapsulation efficiency of the liposome preparation. Multilamellar vesicles extruded by this technique are shown by both negative stain and freeze-fracture electron microscopy to have mean diameters approaching the pore diameter of the polycarbonate membrane through which they were extruded. When sequentially extruded down through a 0.2 micron membrane, the resulting vesicles exhibit a very homogeneous size distribution with a mean diameter of 0.27 micron while maintaining an acceptable level of encapsulation of the aqueous phase.

Screening and characterization of estrogenic activity from a hydroxystilbene library.

BACKGROUND: Compounds that either inhibit or induce an estrogen response in vivo are important as potential drugs and biochemical tools. Non-steroidal stilbene analogs such as tamoxifen are known to function as both estrogen agonists and antagonists depending upon the analog structure. This family of compounds is amenable to parallel-manifold synthesis because stilbene analogs are easily synthesized using a single-step olefination reaction. RESULTS: We have prepared a small 23-component hydroxystilbene library using a solid phase synthesis approach. The library was screened for estrogenic and antiestrogenic activity using a cell-based bioassay that measures estrogen receptor-mediated transcription of a reporter gene. Three of the analogs proved to have dose-dependent estrogenic activity with EC50 values between 5 microM and 15 microM. Further characterization of the hydroxystilbene-mediated estrogenic activity suggests that the agonist activity results from direct binding to the steroid site on the estrogen receptor with IC50 values of 1-10 microM. CONCLUSIONS: The results of this study show that classic olefination chemistry can be adapted to a solid-phase format for parallel synthesis of analog libraries. Although yields varied for the individual analogs, sufficient quantity of pure material was obtained directly from the resin for structural characterization and biological evaluation. This study further validates solid-phase organic synthesis as a useful approach for rapid parallel-manifold library synthesis to augment both lead compound discovery and optimization.

Characteristics of oligonucleotide uptake in human keratinocyte cultures.

Oligodeoxyribonucleotides have the potential to interfere selectively with cellular protein synthesis by sequence-specific hybridization to DNA or RNA molecules. We have investigated the properties of uptake and intracellular localization of fluorescently labeled oligonucleotides in cultured human keratinocytes using confocal laser scanning microscopy. Unlike many other cell types studied, keratinocytes can internalize oligonucleotides without apparent sequestration in endosomes or cell surface accumulation. Uptake is primarily nuclear and unaltered by sodium azide, monensin, or chloroquine pretreatment. We have verified our results with two different fluorophores, fluorescein and Bodipy, and found similar uptake and distribution patterns in both live and fixed cell populations. Surprisingly, we have found uptake to be heterogeneous within a population, with 15-30% of cells internalizing the oligonucleotides. This percentage is drastically increased to roughly 80% at cell population margins, and after release from M phase arrest. These results on uptake and intracellular localization suggest that keratinocytes may have increased sensitivity as target cells for oligonucleotide based gene regulation strategies.

Inhibition of transcription factor binding to the HER2 promoter by triplex-forming oligodeoxyribonucleotides.

We have identified a 28-bp homopurine/homopyrimidine sequence capable of triple helix (triplex) formation with G+T-rich oligodeoxyribonucleotides (oligos) within the critical proximal promoter of the HER2/neu/c-erbB2 (HER2) proto-oncogene. To investigate the possible therapeutic potential of triplex-forming oligos in HER2 overexpressing breast cancers, we have studied the ability of triplex formation to compete with and to inhibit the binding of a transcription factor to its consensus sequence at an adjacent site. Competition binding assays demonstrate that a triplex-forming oligo can inhibit transcription factor binding in a sequence-specific manner. Moreover, we find that the addition of both nucleotide and non-nucleotide 'tails' to triplex-forming oligos do not confer any enhancement of binding affinity, but provide additional inhibition of transcription factor binding, potentially by steric hindrance.

Paradoxical production of target protein using antisense RNA expression vectors.

We used antisense RNA in a protocol designed to reduce estrogen receptor (ER) content in human breast cancer cells and observed paradoxical increases in ER levels. ER protein activity was measured using a highly sensitive reporter gene assay that relies on the ability of functional ER to bind a consensus estrogen response element (ERE) and drive the production of chloramphenicol acetyl-transferase (CAT). Upon transient transfection of ER-positive cell lines with three different vectors containing the full-length ER cDNA cloned in an antisense orientation, we observed unexpected increases in ER-driven CAT activity. To further investigate this phenomenon, expression from the antisense ER vectors was studied in an ER-negative breast tumor cell line, MDA-MB-453. ER activity was observed in these ER-negative cells upon transient transfection with each of three antisense ER vectors, but not from control vectors. Expression of ER from antisense constructs was 30-100-times less efficient than ER expression from isogenic sense constructs. The paradoxical ER activity was consistent with expected ER behavior in that it exhibited characteristic binding to the natural ligand, 17 beta-estradiol (E2), and it was inhibited by the antiestrogens, 4-hydroxy-tamoxifen (OHT) and ICI 164384 (ICI). Control vectors containing a truncated antisense ER cDNA produced no ER activity. Although the mechanism for this ER expression has not been determined, it appears likely that it is due to transcription off the opposite strand of the antisense construct.(ABSTRACT TRUNCATED AT 250 WORDS)

3-substituted thieno[2,3-b][1,4]thiazine-6-sulfonamides. A novel class of topically active carbonic anhydrase inhibitors.

3-Aminoalkyl derivatives of thieno[2,3-b][1,4]thiazine-6-sulfonamide were prepared for evaluation as topically active ocular hypotensive agents. The compounds described were found to be excellent in vitro inhibitors of carbonic anhydrase II and in vivo to lower intraocular pressure in three rabbit models of ocular hypertension. Compounds 20A, 20B, and 20C met the requirement of formulation as a 1% solution at pH 5.2, but none of the compounds described exhibited greater activity in the normotensive albino rabbit, the alpha-chymotrypsin-treated albino rabbit, or the normotensive pigmented rabbit than MK-927 or MK-507, the present clinical candidates.

Adrenoceptor and tetrabenazine antagonism activities of some pyridinyltetrahydropyridines.

A series of pyridinyltetrahydropyridine derivatives was synthesized and evaluated as adrenoceptor and tetrabenazine antagonists. 4-(3-Fluoro-2-pyridinyl)-1,2,5,6-tetrahydropyridine proved to be the most potent and selective alpha 2-adrenoceptor antagonist of the series as measured in vitro by displacement of [3H]clonidine and [3H]prazosin from membrane binding sites of calf cerebral cortex and by antagonism of the effects of clonidine and methoxamine in the rat isolated, field-stimulated vas deferens. In addition, this compound, and the corresponding desfluoro derivative, blocked tetrabenazine-induced ptosis in the mouse.

(+)- and (-)-3-Methoxycyproheptadine. A comparative evaluation of the antiserotonin, antihistaminic, anticholinergic, and orexigenic properties with cyproheptadine.

The synthesis and resolution of (+/-)-3-methoxycyproheptadine [(+/-)-4] are described. As a peripheral serotonin antagonist, (+/-)-4 was found to be one-half as potent as cyproheptadine (1b). The peripheral anticholinergic and antihistaminic activities as well as the orexigenic property of (+/-)-4 are less than those of 1b. A further comparison of the enantiomers (+)-4 and (-)-4 shows that all of the anticholinergic activity of (+/-)-4 resides solely in the dextrorotatory enantiomer, (+)-4, while the antiserotonin activity, which is similar to that of 1b, resides in the levorotatory enantiomer, (-)-4. Antihistaminic and orexigenic activity also resides in (-)-4 but these properties are reduced compared to those of 1b.

Structure-affinity relationships of arylquinolizines at alpha-adrenoceptors.

Hexahydroaryl[a]quinolizines comprise a prominent structural element in several alpha 2-adrenoceptor antagonists. Eight hexahydroheteroarylquinolizines were prepared as minimal ligands to investigate the relationship between the nature of the aromatic ring and affinity of these molecules for alpha-adrenoceptors. Affinity for alpha 1-and alpha 2-adrenoceptors was assessed by displacement of [3H]prasozin and [3H]clonidine, respectively. Lipophilicity of the aryl portion of the molecules, reflected by their partition coefficient between octanol and pH 7.4 buffer, correlated well with affinity at both receptor subtypes. Although some compounds showed nanomolar affinity for alpha-adrenoceptors, no subtype selectivity was observed. These results suggest that the aromatic ring enhances binding at both receptors chiefly through hydrophobic interactions and contributes little to subtype selectivity.

Pyridinylpiperazines, a new class of selective alpha 2-adrenoceptor antagonists.

A series of 1-(2-pyridinyl)piperazine derivatives was synthesized and evaluated for adrenergic activity. In vitro activity was assessed through the antagonism of clonidine's effect in the rat, isolated, field-stimulated vas deferens and by the displacement of [3H]clonidine from membrane binding sites of calf cerebral cortex. Antagonism of clonidine-induced mydriasis in the rat was used as an in vivo assay. Several members of the series proved to be potent, selective alpha 2-adrenoceptor antagonists. 1-(3-Fluoro-2-pyridinyl)piperazine was more potent than either yohimbine or rauwolscine in displacement of [3H]clonidine and had a higher affinity for this binding site (alpha 2) than for the [3H]prazosin site (alpha 1). In vivo, the 3-F derivative was more potent than the reference standards in reversing clonidine-induced mydriasis. None of the members of this series was more selective or potent than rauwolscine in antagonizing clonidine in the rat vas deferens.

Synthesis and stereospecific antipsychotic activity of (-)-1-cyclopropylmethyl-4-(3-trifluoromethylthio-5H-dibenzo[a,d]cyclohepten-5-ylidene)piperidine.

The synthesis and resolution of 3-iodocyproheptadine [(+/-)-5a] and 1-cyclopropylmethyl-4-(3-iodo-5H-dibenzo-[a,d]cyclohepten-5-ylidene)piperidine [(+/-)-5b] are described. The resulting atropisomers undergo reaction with trifluoromethylthiocopper to give optically active products without extensive racemization. In this manner, optically pure (+)- and (-)-3-trifluoromethylthiocyproheptadine [(+)-6a and (-)-6a, respectively] and (+)- and (-)-1-cyclopropylmethyl-4-(3-trifluoromethylthio-5H-dibenzo[a,d]cyclohepten-5-ylidene)piperidine [(+)-6b and (-)-6b, respectively] have been prepared. The influence of a chiral europium shift reagent on the proton and fluorine resonance signals as a diagnostic tool for the determination of the optical purities of these atropisomers is discussed. The four compounds, (+)-6a, (-)-6a, (+)-6b, and (-)-6b, were studied in squirrel monkeys for their ability to block conditioned avoidance responding. All of the antiavoidance activity was found to reside solely in the levorotatory compounds (-)-6a and (-)-6b. Further comparison of the enantiomers (-)-6b and (+)-6b showed that the ability to antagonize apomorphine-induced stereotyped behavior is confined to the levorotatory isomer (-)-6b while weak central anticholinergic activity resides solely in the dextrorotatory isomer (+)-6b. Neither (-)-6b has significant peripheral anticholinergic activity.

Synthesis and receptor binding studies relevant to the neuroleptic activities of some 1-methyl-4-piperidylidene-9-substituted-pyrrolo[2,1-b][3]benzazepine derivatives.

The synthesis of a series of 1-methyl-4-(9-substituted-11H-pyrrolo[2,1-b]benzazepin-11-ylidene)piperidines (4a-f) and 1-methyl-4-(9-substituted-6,11-dihydro-5H-pyrrolo[2,1-b][3]benzazepin-11-ylidene)piperidines (4g-l) is described. As with th e 3-substituted cyproheptadine compounds 1b-e, atropisomerism exists in 4b-f, but unlike the enantiomers of 1b-e, the pyrrolobenzazepine enantiomers racemize at room temperature. Thus, the bromo compound (+)-4b has a half-life of 128 +/- 1 min at 25 degrees C, while the chloro compound (-)-4c has a half-life of 114 +/- 9 min at 25 degrees C. Compounds 4a-l have been examined for receptor binding affinities in assays that have been recognized as predictive for antipsychotic activity. The displacement of specifically bound tritiated ligands, comprising the dopamine antagonist [3H]spiperone, the dopamine agonist [3H]apomorphine, the muscarinic cholinergic antagonist [3H]quinuclidinyl benzilate (QNB), the alpha-adrenergic antagonist [3H]prazosin, the alpha-adrenergic agonist [3H]clonidine, the serotonin-1 binding agent [3H]serotonin, and the mixed serotonin agonist-antagonist [3H]lysergic acid diethylamide (LSD), by 4a-l has been measured utilizing membrane preparations of mammalian brain. Certain of the features of the receptor binding of these compounds have been shown to be common to several of the receptor sites. Data from these binding studies have been compared to corresponding data previously obtained for a series of chiral 3-substituted cyproheptadine analogues, and the receptor binding data of the two classes of compounds are discussed with respect to their molecular geometries.

Non-peptide glycoprotein IIb/IIIa inhibitors. 17. Design and synthesis of orally active, long-acting non-peptide fibrinogen receptor antagonists.

The synthesis and pharmacological evaluation of 5 (L-738, 167), a potent, selective non-peptide fibrinogen receptor antagonist is reported. Compound 5 inhibited the aggregation of human gel-filtered platelets with an IC50 value of 8 nM and was found to be > 33000-fold less effective at inhibiting the attachment of human endothelial cells to fibrinogen, fibronectin, and vitronectin than it was at inhibiting platelet aggregation. Ex vivo platelet aggregation was inhibited by > 85% 24 h after the oral administration of 5 to dogs at 100 micrograms/kg. The extended pharmacodynamic profile exhibited by 5 appears to be a consequence of its high-affinity binding to GPIIb/IIIa on circulating platelets and suggests that 5 is suitable for once-a-day dosing.

An oligonucleotide blocks interferon-gamma signal transduction.

Interferon (IFN)-gamma is an important mediator of transplant graft rejection. It induces endothelial cell expression of HLA-DR and intercellular adhesion molecule-1, which render transplant grafts more susceptible to rejection by the host. Oligonucleotide 5'-GGG GTT GGT TGT GTT GGG TGT TGT GT-RNH2 (oligo I) blocks multiple IFN-gamma effects in human K562 cell cultures. A systematic approach revealed that oligo I has a novel, and potentially important, mode of action--it blocks the binding of IFN-gamma to its receptor, thus preventing activation of the IFN-gamma signal transduction pathway. The results are consistent with an aptamer mechanism of action, because oligo I exerts its inhibitory effects by interacting with protein, not intracellular nucleic acid targets, such as mRNA or genomic DNA.