Tough combinatorial poly(urethane-isocyanurate) polymer networks and hydrogels synthesized by the trimerization of mixtures of NCO-prepolymers.

The development of tough hydrogels is an essential but challenging topic in biomaterials research that has received much attention over the past years. By the combinatorial synthesis of polymer networks and hydrogels based on prepolymers with different properties, new materials with widely varying characteristics and unexpected properties may be identified. In this paper, we report on the properties of combinatorial poly(urethane-isocyanurate) (PUI) type polymer networks that were synthesized by the trimerization of mixtures of NCO-functionalized poly(ethylene glycol) (PEG), poly(propylene gylcol) (PPG), poly(ε-caprolactone) (PCL) and poly(trimethylene carbonate) (PTMC) prepolymers in solution. The resulting polymer networks showed widely varying material properties. Combinatorial PUI networks containing at least one hydrophilic PEG component showed high water uptakes of >100 wt%. The resulting hydrogels demonstrated elastic moduli of up to 10.1 MPa, ultimate tensile strengths of up to 9.8 MPa, elongation at break values of up to 624.0% and toughness values of up to 53.4 MJ m-3. These values are exceptionally high and show that combinatorial PUI hydrogels are among the toughest hydrogels reported in the literature. Also, the simple two-step synthesis and wide range of suitable starting materials make this synthesis method more versatile and widely applicable than the existing methods for synthesizing tough hydrogels. An important finding of this work is that the presence of a hydrophobic network component significantly enhances the toughness and tensile strength of the combinatorial PUI hydrogels in the hydrated state. This enhancement is the largest when the hydrophobic network component is crystallizable in nature. In fact, the PUI hydrogels containing a crystallizable hydrophobic network component are shown to be semi-crystalline in the water-swollen state. Due to their high toughness values in the water-swollen state together with their water uptake values, elastic moduli and ultimate tensile strengths, the developed hydrogels are expected to be promising materials for biomedical coating- and adhesive applications, as well as for tissue-engineering. STATEMENT OF SIGNIFICANCE: The development of tough hydrogels is a challenging topic that has received much attention over the past years. At present, double network type hydrogels are considered state-of-the-art in the field, demonstrating toughness values of several tens of MJ m-3. However, in terms of ease and versatility of the synthesis method, the possibilities are limited using a double network approach. In this work, we present combinatorial poly(urethane-isocyanurate) type polymer networks and hydrogels, synthesized by the trimerization of mixtures of NCO-functionalized prepolymers. The resulting hydrogels demonstrate exceptionally high toughness values of up to 53 MJ m-3, while the synthesis method is versatile and widely applicable. This new class of hydrogels is therefore considered highly promising in the future development of load-bearing biomaterials.

Carbamylation reduces the capacity of IgG for hexamerization and complement activation.

Carbamylation is a post-translational modification that can be detected on a range of proteins, including immunoglobulin (Ig)G, in several clinical conditions. Carbamylated IgG (ca-IgG) was reported to lose its capacity to trigger complement activation, but the mechanism remains unclear. Because C1q binds with high affinity to hexameric IgG, we analyzed whether carbamylation of IgG affects binding of C1q, hexamerization and complement-dependent cytotoxicity (CDC). Synovial tissues of rheumatoid arthritis (RA) patients were analyzed for the presence of ca-IgG in vivo. Synovial tissues from RA patients were analyzed for the presence of ca-IgG using mass spectrometry (MS). Monomeric or hexameric antibodies were carbamylated in vitro and quality in solution was controlled. The capacity of ca-IgG to activate complement was analyzed in enzyme-linked immunosorbent (ELISAs) and cellular CDC assays. Using MS, we identified ca-IgG to be present in the joints of RA patients. Using in vitro carbamylated antibodies, we observed that ca-IgG lost its capacity to activate complement in both solid-phase and CDC assays. Mixing ca-IgG with non-modified IgG did not result in effective inhibition of complement activation by ca-IgG. Carbamylation of both monomeric IgG and preformed hexameric IgG greatly impaired the capacity to trigger complement activation. Furthermore, upon carbamylation, the preformed hexameric IgG dissociated into monomeric IgG in solution, indicating that carbamylation influences both hexamerization and C1q binding. In conclusion, ca-IgG can be detected in vivo and has a strongly reduced capacity to activate complement which is, in part, mediated through a reduced ability to form hexamers.

[Basophilic granulocytes and autoimmune diseases. Can basophilic granulocytes modulate B-cell functions in systemic lupus erythematosus?]. / Basophile Granulozyten und Autoimmunerkrankungen. Können basophile Granulozyten die Funktion von B-Zellen beim systemischen Lupus erythematodes beeinflussen?

BACKGROUND: B-cells play an important role in the pathogenesis of systemic lupus erythematosus (SLE); however, many other cell types are also involved in disease development. In a murine lupus model it was demonstrated that basophils are indispensable for the development of lupus symptoms. AIM: This study investigated whether there is evidence for a relevant interaction between B-cells and basophils under physiological and pathological conditions. MATERIAL AND METHODS: A selective review of the literature was performed and some preliminary data about the interaction of basophils and B-cells are reported in this article. For the experiments, isolated B-cells were cultured in vitro in the presence or absence of basophils and B-cell survival, proliferation, plasma cell development and antibody production were determined. RESULTS: Data from the literature show that there is evidence for an interaction between basophils and B-cells in a murine model. Our investigations confirmed that human basophils also support the survival and proliferation of B-cells. Furthermore, plasma cell differentiation and antibody production, most importantly IgG secretion, are enhanced. First experimental ex vivo analyses of basophils from SLE patients demonstrate that these cells exhibit a higher activation level compared to basophils from healthy controls. DISCUSSION: In summary, previously published data and our own data demonstrate that there is an interaction between human basophils and B-cells. A better understanding of the role of basophils in the pathogenesis of SLE could lead to the development of new therapeutic strategies.

Clogging of microchannels by nano-particles due to hetero-coagulation in elongational flow.

We have investigated the phenomenon of flow-induced aggregation in highly concentrated colloidal dispersions exposed to strongly converging flow fields. This phenomenon is relevant not only for classical technical operations like coating, pumping or filtration, but also for the application of concentrated suspensions in upcoming processing technologies based on microfluidic devices. A ring-slit device (gap height 10-25 µm), which allows for a variation of flow kinematics in a wide range, has been developed in order to investigate this phenomenon. Various polymer dispersions with different particle surface properties have been used as model systems. Our experiments exclude, that channel clogging is due to retention of pre-existing aggregates, fouling or hydrodynamic bridging. Instead, we demonstrate that clogging of the microchannel is induced by hetero-coagulation between primary colloidal particles and micron-sized impurities present at concentrations on the order of 100-1000 ppm. Clogging can occur even if the diameter of these impurities is less than a tenth of the gap height. Aggregation takes place in the converging flow field at the channel entrance, but not in the shear field within the slit. It can be suppressed by appropriate stabilization of the primary particles.

The in vitro and in vivo evaluation of new synthesized prodrugs of 5-OH-DPAT for iontophoretic delivery.

The feasibility of transdermal iontophoretic transport of 4 novel ester prodrugs of 5-OH-DPAT (glycine-, proline-, valine- and beta-alanine-5-OH-DPAT) was investigated in vitro and in vivo. Based on the chemical stability of the prodrugs, the best candidates were selected for in vitro transport studies across human skin. The pharmacokinetics and pharmacodynamic effects of the prodrug with highest transport efficiency, were investigated in a rat model. The in vitro transport, plasma profile and pharmacological response were analyzed with compartmental modeling. Valine- and beta-alanine-5-OH-DPAT were acceptably stable in the donor phase and showed a 4-fold and 14-fold increase in solubility compared to 5-OH-DPAT. Compared to 5-OH-DPAT, valine- and beta-alanine-5-OH-DPAT were transported less and more efficiently across human skin, respectively. Despite a higher in vitro transport, lower plasma concentration was observed following 1.5h current application (250 microAcm(2)) of beta-alanine-S-5-OH-DPAT in comparison to S-5-OH-DPAT. However the prodrug showed higher plasma concentrations post-iontophoresis, explained by a delayed release due to hydrolysis and skin depot formation. This resulted in a pharmacological effect with the same maximum as 5-OH-DPAT, but the effect lasted for a longer time. The current findings suggest that beta-alanine-5-OH-DPAT is a promising prodrug, with a good balance between stability, transport efficiency and enzymatic conversion.

Murine and human Langerhans cells express a functional histamine H4 receptor: modulation of cell migration and function.

BACKGROUND: Histamine is an important mediator of allergic reactions, and recent studies indicated that the function of different types of antigen presenting cells (APC) can be modulated by histamine, in particular via the newly described histamine H(4) receptor (H(4)R). Therefore, we investigated possible interactions of histamine via the H(4)R on Langerhans cells (LC), which represent the professional APC in the skin and therefore have an important role in the initiation and maintenance of allergic skin diseases. METHODS: The expression of the H(4)R was evaluated by real-time PCR, flow cytometry and immunofluorescence staining. The function of the H(4)R was determined by intracellular flow cytometric measurement of chemokine production and LC migration assays. RESULTS: Here, we show H(4)R expression on in vitro generated monocyte-derived LC (mRNA and protein) and on primary LC from murine and human skin samples (protein). The immunofluorescence staining in murine and human skin samples clearly proved that LC express the H(4)R in situ. Stimulation with histamine or a H(4)R agonist downregulated the chemokine (C-C motif) ligand 2 (CCL2) in human monocyte-derived LC and primary LC. Prestimulation with a selective H(4)R antagonist abolished this effect. Moreover, migration of LC from the epidermis was increased after H(4)R agonist stimulation in ex vivo migration assays using human epidermis and murine in vivo assays. CONCLUSION: Our findings show that LC express a functional H(4)R and point towards a possible pathogenic relevance of the H(4)R in inflammatory and allergic diseases.

Histamine H4 receptors modulate dendritic cell migration through skin--immunomodulatory role of histamine.

BACKGROUND: Dendritic cells (DC) are the major antigen-presenting cells and play a key role in adaptive immunity as they are able to activate naive T cells. It was recently described, that the histamine H(4) receptor (H4R) is present on human monocyte-derived DC and that chemotaxis and T-helper (Th)1-Th2 polarization is mediated by this receptor. However, the distribution of histamine receptors on murine DC has not been studied yet. METHODS: The histamine receptor expression on murine bone marrow (BM)-derived DC and effects of histamine and H4R agonism on DC migration through skin were studied. As it was demonstrated in scratching experiments that NMRI mice are more susceptible to H4R-mediated itch than BALB/c mice, DC function of NMRI and BALB/c mice was compared. RESULTS: The mRNA of the H1R, H2R and H4R could be detected in murine BM-derived DC, while mRNA of the H3R was found to be low or undetectable. There were no distinct differences in mRNA expression and in H4R protein level (flow cytometry) between NMRI compared with BALB/c mice indicating, that a higher susceptibility is not associated with a generally higher H4R expression in all cell types. Histamine as well as the H4R agonist clobenpropit induced an enhanced chemotaxis in the skin DC migration assay. The enhanced chemotaxis was blocked by the H4R antagonist JNJ7777120. This finding was confirmed by in vitro migration experiments with BM-derived DC. CONCLUSION: Referring to DC migration, blocking the H4R on inflammatory cells might be a promising anti-inflammatory, immunomodulatory strategy.

Protection against cocaine toxicity in mice by the dopamine D3/D2 agonist R-(+)-trans-3,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol [(+)-PD 128,907].

Cocaine abuse is a public health concern with seizures and death being one consequence of overdose. In the present study, dopamine D(3/)D(2) receptor agonists dose dependently and completely prevented the convulsant and lethal effects of cocaine. The D(3)-preferring agonists R-(+)-trans-3,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol) [(+)-PD 128,907], (+)-7-hydroxy-dipropylaminotetralin, and the mixed D(3/)D(2) agonists quinpirole and quinelorane were all effective against cocaine toxicity in mice. The anticonvulsant effects of these compounds occurred at doses below those that produced motor impairment as assessed in the inverted screen test. Protection against the convulsant effects of the selective dopamine uptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy] ethyl]-4-[3-phenyl-propyl]piperazine (GBR 12909) was also conferred by (+)-PD 128,907. The possible selectivity of the effects of (+)-PD 128,907 (3 mg/kg) for these dopaminergic compounds was demonstrated by its general lack of protective efficacy against a host of convulsants acting through other neural mechanisms [pentylenetetrazol, (+)-bicuculline, and picrotoxin, 4-aminopyridine, and t-butylbiclyclophosphoorothionate, N-methyl-d-aspartate, kainate, pilocarpine, nicotine, strychnine, aminophylline, threshold electric shock, and 6-Hz electrical stimulation]. Direct and correlational evidence suggests that these effects were mediated by D(3) receptors. Protection was stereospecific and reversible by an antagonist of D(3) receptors [3-[4[1-(4-[2[4-(3-diethyamino-propoxy)-phenyl]-benzoimidazol-l-yl]-butyl)-1H-benzoimidazol-2-yl]-phenoxy]-propyl)-diethyl-amine; PD 58491] but not D(2) receptors [3[[4-(4-chlorophenyl)-4 hydroxypipeidin-1-yl]methyl-1H-indole; L-741,626]. Anticonvulsant potencies were positively associated with potencies in a functional assay of D(3) but not D(2) receptor function. Together, these findings suggest that the prevention of cocaine convulsions and lethality by (+)-PD 128,907 may be due to D(3) receptor-mediated events.

Modification of behavioral effects of drugs in mice by neuroactive steroids.

RATIONALE: Neuroactive steroids represent a novel class of potential therapeutic agents (epilepsy, anxiety, migraine, drug dependence) thought to act through positive allosteric modulation of the GABA(A) receptor. A synthetically derived neuroactive steroid, ganaxolone (3alphahydroxy-3beta-methyl-5alpha-pregnan-20-one), is in phase-II clinical trials for epilepsy. Unlike traditional anticonvulsants such as diazepam and phenobarbital, ganaxolone shows equipotent suppression of both the seizure activity and the behavioral effects of pentylenetetrazol (PTZ) administration. OBJECTIVES: The present study explored possible reversal by ganaxolone and related neuroactive steroids of some behavioral effects of additional pharmacological challenges. METHODS: Direct behavioral observation and photocell-counted locomotor activity of male, Swiss-Webster mice were made with various compounds alone and in conjunction with ganaxolone. RESULTS: Ganaxolone both prevented and reversed PTZ-induced locomotor depression in mice. Further, ganaxolone reversed the locomotor depression induced by other convulsant/anxiogenic stimuli: bicuculline, picrotoxin and, to a lesser extent, yohimbine. Ganaxolone failed to reverse the locomotor stimulation induced by cocaine, methamphetamine, dizocilpine, and phencyclidine. In addition to ganaxolone, the endogenous neuroactive steroids allopregnanolone and pregnanolone and the synthetic neuroactive steroid Co 2-1068 also reversed observed behaviors and locomotor depression induced by PTZ. CONCLUSIONS: The present findings support the unique pharmacological effects of neuroactive steroids as a novel class of positive allosteric modulators of GABA.

Synthesis and pharmacological evaluation of thiopyran analogues of the dopamine D3 receptor-selective agonist (4aR,10bR)-(+)-trans-3,4,4a,10b-tetrahydro-4-n-propyl-2H,5H [1]b enzopyrano[4,3-b]-1,4-oxazin-9-ol (PD 128907).

Benzopyranoxazine (+)-7 (PD 128907) is the most dopamine (DA) D3 receptor-selective agonist presently known. The only structural feature which distinguishes 7 from the analogous nonselective naphthoxazines is an oxygen atom in the 6-position. To extend this series of tricyclic DA agonists we used a classic bioisoster approach and synthesized thiopyran analogues of 7, which have a sulfur atom in the 6-position. We prepared trans-4-n-propyl-3,4,4a,10b-tetrahydro-2H,5H-[1]benzothiopyrano[4, 3-b]-1,4-oxazin-9-ol (9, trans-9-OH-PTBTO), its enantiomers ((+)-9 and (-)-9), the racemic cis-analogue (10), and the racemic trans-sulfoxide (11) and studied the potency and selectivity for DA receptors of these compounds. As with other rigid DA agonists, the highest affinity for DA receptors resided in one of the enantiomers, in this case the (-)-enantiomer of 9. On the basis of a single-crystal X-ray analysis of a key intermediate, the absolute configuration of (-)-9 was found to be 4aS,10bR, which is homochiral with (+)-(4aR,10bR)-7. In contrast to (+)-7 however, (-)-9 displayed no selectivity for any of the DA receptors. In addition, it has affinity for 5HT1A receptors. (+/-)-cis-4-n-Propyl-3,4,4a,10b-tetrahydro-2H,5H-[1]benzothiopyrano++ +[4,3-b]-1,4-oxazin-9-ol (10), which was expected to be inactive, displayed affinity and selectivity for the DA D3 receptor, whereas the sulfoxide 11 displayed some DA D3 selectivity, but with a lower affinity. Further pharmacological evaluation revealed that (-)-9 is a very potent full agonist at DA D2 receptors and a partial agonist at DA D3 receptors. The cis-analogue (+/-)-10 displayed the same profile, but with lower potency. These findings were confirmed in vivo: in reserpinized rats (-)-9 displayed short-acting activation of locomotor activity (DA D2 agonism) and also lower lip retraction and flat body posture, (5HT1A agonism). Compound (+/-)-10 had no effect on locomotor activity. In unilaterally 6-OH-DA lesioned rats, (-)-9 gave short-acting locomotor activation. Furthermore, in microdialysis studies in rat striatum, (-)-9 potently decreased DA release, confirming its activation of presynaptic DA D2 receptors.

Thiophene analogs of naphthoxazines and 2-aminotetralins: bioisosteres with improved relative oral bioavailability, as compared to 5-OH-DPAT.

In the present study, a series of thiophene analogs of 2-aminotetralins and hexahydronaphthoxazines were studied in vivo for their ability to decrease striatal dopamine release, their effects on locomotor activity, and their behavioral characteristics in reserpinized rats, in order to investigate whether a thiophene moiety can act as a bioisostere for the phenol moiety. In general, the new compounds showed lower in vivo activities than 5-hydroxy-2-(N,N,-di-n-propylamino)tetralin (5-OH-DPAT). However, the introduction of the thiophene moiety gave a significant improvement of the relative oral bioavailability, compared to 5-OH-DPAT. Our results suggest that the thiophene moiety can act as a bioisostere for a phenol group in hydroxylated 2-aminotetralins. For the thianaphthoxazines it was not possible to discriminate between bioisosterism for a phenyl or a phenol moiety. The tetrahydrobenzo[b]thiophenes could be used as lead compounds for the development of novel dopamine receptor ligands with improved relative oral bioavailability.

Dopamine D(2) activity of R-(-)-apomorphine and selected analogs: a microdialysis study.

In the present study, R-(-)-apomorphine and three of its analogs were studied for their potency in decreasing the release of dopamine in the striatum after subcutaneous administration and for their oral bioavailability using the microdialysis technique in freely moving rats. The analogs R-(-)-N-n-propylnorapomorphine and R-(-)-11-hydroxy-N-n-propylnoraporphine displayed a higher potency than R-(-)-apomorphine in decreasing the release of dopamine in the striatum. A high dose of R-(-)-11-hydroxyaporphine, a dopamine D(2) receptor partial agonist, had a small effect on the release of dopamine in the striatum. The catechols R-(-)-N-n-propylnorapomorphine and R-(-)-apomorphine displayed a comparable oral bioavailability (1%), while the mono-hydroxy analog R-(-)-11-hydroxy-N-n-propylnoraporphine displayed a slightly higher oral bioavailability (3%). In conclusion, R-(-)-N-n-propylnorapomorphine and R-(-)-11-hydroxy-N-n-propylnoraporphine did not show a substantial improvement in bioavailability. However, due to the clear difference in their efficacy in decreasing dopamine release, in spite of the similar agonist binding affinities to the dopamine D(2) receptor of the two analogs compared to R-(-)-apomorphine, they could be useful alternatives for apomorphine in the treatment of Parkinson's disease.

Homocysteine and methionine metabolism in ESRD: A stable isotope study.

BACKGROUND: Hyperhomocysteinemia has a high prevalence in the end-stage renal disease (ESRD) population, which may contribute to the high cardiovascular risk in these patients. The cause of hyperhomocysteinemia in renal failure is unknown, and therapies have not been able to normalize plasma homocysteine levels. Insight into methionine-homocysteine metabolism in ESRD is therefore necessary. METHODS: Using a primed, continuous infusion of [2H3-methyl-1-13C]methionine, we measured whole body rates of methionine and homocysteine metabolism in the fasting state in four hyperhomocysteinemic hemodialysis patients and six healthy control subjects. RESULTS: Remethylation of homocysteine was significantly decreased in the hemodialysis patients: 2.6+/-0.2 (SEM) vs. 3.8+/-0.3 micromol. kg(-1)x hr(-1) in the control subjects (P = 0.03), whereas transsulfuration was not 2.5+/-0.3 vs. 3.0+/-0.1 micromol. kg(-1) x hr(-1) (P = 0.11). The transmethylation rate was proportionally and significantly lower in the ESRD patients as compared with controls: 5.2+/-0.4 vs. 6.8+/-0.3 micromol. kg(-1) x hr(-1) (P = 0.02). Methionine fluxes to and from body protein were similar. CONCLUSIONS: The conversion of homocysteine to methionine is substantially (approximately 30%) decreased in hemodialysis patients, whereas transsulfuration is not. Decreased remethylation may explain hyperhomocysteinemia in ESRD. This stable isotope technique is applicable for developing new and effective homocysteine-lowering treatment regimens in ESRD based on pathophysiological mechanisms.

[Difficult weaning from mechanical ventilation: a graduated approach and teamwork]. / Moeizaam ontwennen van de beademing: maatwerk en teamwerk.

Weaning from mechanical ventilation in patients with chronic obstructive pulmonary disease (COPD) or left ventricular failure may be difficult. At the time of intubation and initiation of mechanical ventilatory support, this treatment is usually life-saving in the short term. Only later on, the condition which necessitated such support may prove irreversible. COPD patients often require positive end-expiratory pressure to enable them to trigger the ventilator comfortably. Patients with left ventricular failure need pharmacological support to reduce the circulating volume and to reduce left ventricular afterload because the ventilatory support itself reduces both left ventricular preload and afterload. Gradual withdrawal of pressure support and gradually increasing periods of T-piece weaning are probably equally effective. New methods have been described but have not yet been tested in randomized controlled trials. More important than the method of weaning is the presence of qualified and dedicated nursing support. Not all patients can be weaned; for most of those who cannot, prospects are grim. These patients require optimal palliative support with recognition of their autonomy.

Risk factors for malaria: a microepidemiological study in a village in Sri Lanka.

Environmental and socioeconomic risk factors for malaria were studied in a village in Sri Lanka. Over a period of one year, all 49 households in the village were visited every alternate day to obtain information on malaria episodes. Information on risk factors was obtained through questionnaires and direct observations. Age below 17 years (relative risk [RR] = 1.66, 95% confidence interval [95% CI] 1.18-2.35), use of bed nets (RR = 0.16, 95% CI 0.05-0.45) and traditional fumigants (RR = 0.58, 95% CI 0.37-0.93) were independent predictors of malaria. People using anti-mosquito pyrethrum coils had a higher risk for malaria than people living in houses where they were not used (RR = 1.46, 95% CI 1.03-2.07). The build-up of Anopheles culicifacies populations before the start of the transmission season had taken place in a stream near the village. Living close to the stream was a risk factor for malaria early in the transmission season, although this did not reach statistical significance (comparing < 250 m with > 500 m, RR = 2.13, 95% CI 0.96-4.71).

Central administration of dopamine D3 receptor antisense to rat: effects on locomotion, dopamine release and [3H]spiperone binding.

A 15-mer, all-phosphorothioate-modified antisense oligodeoxynucleotide (ASO) targeted against rat dopamine D3 receptor mRNA (4 microM, 5 days) significantly reduced (28%) the amount of binding sites labelled with [3H]spiperone in monolayer cultured Chinese hamster ovary (CHO) cells transfected with the complementary desoxyribonucleic acid (cDNA) for the rat D3 receptor. In contrast, D3-ASO treatment did not reduce the amount of bound [3H]spiperone in CHO cells transfected with D2 receptor cDNA. Intracerebroventricular infusion of D3-ASO (osmotic minipump, 10 microg/microl/h, 7 days) influenced dopamine receptor density in the limbic forebrain such that the upper part of the dopamine/[3H]spiperone displacement curve--tentatively representing the D3 receptor--was altered significantly. Spontaneous locomotor activity of non-habituated rats was increased significantly in D3-ASO-treated animals; in addition, in vivo microdialysis revealed a moderate increase in dopamine release in the nucleus accumbens in these animals. In all experiments, an oligodeoxynucleotide comprising the same nucleotides as the antisense sequence, but in random order, was used as control. It is concluded that the antisense strategy is useful for investigating the functional role of dopamine D3 receptors and that the dopamine D3 receptor is involved in rat locomotor behaviour.

Atypical antipsychotic-like effects of the dopamine D3 receptor agonist, (+)-PD 128,907.

Anti-schizophrenia agents with improved efficacy and side-effect profiles are required. A dopamine D3 receptor agonist, R-(+)-trans-3,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3- b]-1,4-oxazin-9-ol HCl ((+)-PD 128,907), displayed an atypical antipsychotic profile comparable to that of clozapine. (+)-PD 128,907 blocked stereotypy produced by dizocilpine (MK-801) at 12-fold lower doses than those affecting apomorphine-induced stereotypes in mice and did not produce catalepsy. These effects of (+)-PD 128,907 were stereospecific and were blocked by a D3 antagonist. These data suggest a role for D3 receptors in antipsychotic drug action.

Reversal of behavioral effects of pentylenetetrazol by the neuroactive steroid ganaxolone.

Neuroactive steroids are naturally occurring or synthetically derived compounds many of which have anticonvulsant, anesthetic, anxiolytic, analgesic or hypnotic properties. The major site of neuronal activity appears to be with a specific steroid-sensitive site on the gamma-aminobutyric acidA receptor/chloride ionophore complex. Ganaxolone (3 alpha-hydroxy-3 beta-methyl-5 alpha-pregnan-20-one) is a synthetic neuroactive steroid protected from metabolic attack of the 3 alpha position. Ganaxolone is an efficacious anticonvulsant agent in a variety of acute seizure models, as well as in electrical and chemical kindling models, and is currently under Phase II clinical investigation for epilepsy. A prior observation that ganaxolone appeared to reverse the marked behavioral changes induced by the convulsant pentylenetetrazol (PTZ) was systematically examined in the present study. A model to quantify PTZ-induced behaviors is described and used to evaluate ganaxolone in comparison with the anticonvulsants valproate, ethosuximide, clonazepam, diazepam and phenobarbital. All compounds were compared using dose equivalents based on their respective ED50 values in preventing convulsions induced by 70 mg/kg PTZ. The ED50 and lower doses of ganaxolone prevented the observed behavioral effects of PTZ as well as its depressant effects on locomotor activity and rearing of mice. In contrast, the other anticonvulsants, if effective, were much less potent. Strikingly, most of the other anticonvulsants were incapable of preventing all the behavioral effects of PTZ. Only phenobarbital prevented all the behavioral effects of PTZ and only at doses 4 to 8 times the anticonvulsant ED50. Rather than normalizing behavior as ganaxolone did, however, phenobarbital resulted in supranormal behavioral responses (e.g., increases in activity). Repeated administration of PTZ did not decrease the protective efficacy of ganaxolone. The results document the unique pharmacological profile of ganaxolone and suggest additional potential benefits from its use as an antiepileptic. Furthermore, because behavioral effects of PTZ have been used to model anxiety and anxiety associated with withdrawal from drugs of abuse, ganaxolone may find additional therapeutic application in those areas.

Determination of dopaminergic prodrugs by high-performance liquid chromatography followed by post-column ion-pair extraction.

One possibility to optimize the therapeutic application of dopaminergic compounds with a catechol function is the reversible protection of this moiety using a prodrug approach. Important features in this respect are a proper chemical stability in the gastrointestinal tract, an adequate release rate after arrival in the blood stream or the possibility to cross the blood-brain barrier. A HPLC method was developed to measure the hydrolysis of prodrugs of dopamine and epinine directly. The method is based on reversed-phase separation followed by post-column ion-pair extraction with a fluorescent counter-ion. The separation of di-isobutyryl esters of dopamine and epinine is obtained within 10 min while the more hydrophobic dopaminergic esters, di-benzoyl and di-pivaloyl dopamine, are retained for 30 min. The precision of the assay measuring 160 ng dibudop and 100 ng ibopamine was 1.2 and 1.0%, respectively. The detection limit of all prodrugs tested was approximately 10 ng.