A comparative investigation of glycinebetaine and dimethylsulphoxide as liposome cryoprotectants

The release of streptomycin from lecithin liposomes following a freeze-thaw cycle was used to measure the cryoprotective activities of glycinebetaine and dimethylsulphoxide (DMSO). At concentrations between 4 and 8% w/v in the external solution, glycinebetaine was superior to DMSO at freezing rates faster than 50 degrees C min-1. At lower rates their activities were similar, and drug loss ranged between 10 and 20% depending upon freezing rate and cryoprotectant concentration. The pattern of streptomycin loss when the concentrations of cryoprotectants inside and outside the liposome were varied indicated that glycinebetaine, in contrast to DMSO, does not diffuse across the liposome membrane. The activity of glycinebetaine was not impaired by the presence in the membrane of cholesterol or charged lipids.     

Prevention of strychnine-induced seizures and death by the N-methylated glycine derivatives betaine, dimethylglycine and sarcosine

Betaine (N,N,N-trimethylglycine) and N,N-dimethylglycine have been reported to have anticonvulsant properties in animals. The purpose of the present study was to determine whether these compounds can antagonize strychnine-induced seizures when administered intraperitoneally and to compare their effects with those of sarcosine (N-methylglycine) and glycine. Betaine, N,N-dimethylglycine and sarcosine were equipotent in decreasing the incidence of seizures and death, causing a 38 to 72 percent decrease in the incidence of seizures and death at a dosage of 5 mmole/kg. Glycine had no effect. Thus anticonvulsant activity is conferred to glycine by a single N-methylation.     

Effects of sarcosine,a glycine transporter type 1 inhibitor,in two mouse seizure models

Sarcosine, a natural amino acid found in muscles and other body tissues, is an endogenous glycine transporter type 1 inhibitor that increases the glycine concentration, resulting in an indirect potentiation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. Sarcosine, similar to other NMDA receptor-activating agents, is an effective adjuvant in the treatment of schizophrenia. It is widely accepted that increased glutamatergic neurotransmission is involved in the initiation and propagation ofseizures. Because sarcosine facilitates NMDAreceptor function, it may affect the seizure threshold. Therefore, we examined the effects of sarcosine on the seizure threshold in two different mouse seizure models: the timed intravenous (iv) pentylenetetrazol (PTZ) infusion test and the maximal electroshock seizure threshold test. In the iv PTZ test, sarcosine did not exert a significant effect on the seizure threshold at any of the doses tested (100,200,400 and 800 mg/kg, ip). However, at doses of400 and 800 mg/kg, sarcosine significantly raised the threshold for electroconvulsions (p < 0.01). The present findings indicate that sarcosine did not lower the seizure threshold. Conversely, sarcosine showed weak anticonvulsant properties by increasing the threshold current for the induction of tonic seizures. Therefore, sarcosine may be considered as a safe adjuvant treatment for schizophrenia without proconvulsant risk. In addition, the compound may serve as an interesting addition to epilepsy treatment.     

GABA and hypothalamic feeding systems. II. A comparison of GABA,glycine and acetylcholine agonists and their antagonists

Microinjections of various compounds into the paraventricular hypothalamic nucleus (PVH) were made and the effects on feeding observed. During the light phase of the lighting cycle, injections of 0.3 μl of muscimol (100 ng) and flurazepam diHCl (20 μg) increased feeding. Similar injections of glycine (500 ng) did not influence feeding during the light phase. During the dark phase, 0.3 μl injections of bicuculline methiodide (30 ng) and picrotoxin (160 ng) suppressed feeding. Similar injections of carbachol increased drinking during the dark phase. Injections of strychnine during this phase were without effect. Tilt box activity levels were not altered by injection of picrotoxin (160 ng) into the PVH.     

Synthesis and immunological properties of N-modified GM3 antigens as therapeutic cancer vaccines

The problem of immunotolerance to GM3, an important tumor-associated trisaccharide antigen, seriously hinders its usage in cancer vaccine development. To solve this problem, the keyhole limpet hemocyanin (KLH) conjugates of a series of GM3 derivatives were synthesized and screened as therapeutic cancer vaccines. First, the beta-linked anomeric azides of differently N-acylated GM3 analogues were prepared by a highly convergent procedure. Next, a pentenoyl group was linked to the reducing end of the carbohydrate antigens following selective reduction of the azido group. The linker was thereafter ozonolyzed to give an aldehyde functionality permitting the conjugation of the antigens to KLH via reductive amination. Finally, the immunological properties of the resultant glycoconjugates were studied in C57BL/6 mice by assessing the titers of specific antibodies induced by the GM3 analogues. While KLH-GM3 elicited low levels of immune response, the KLH conjugates of N-propionyl, N-butanoyl, N-iso-butanoyl, and N-phenylacetyl GM3s induced robust immune reactions with antibodies of multiple isotypes, indicating significantly improved and T-cell dependent immune responses that lead to isotype switching, affinity maturation, and the induction of immunological "memory". It was suggested that GM3PhAc-KLH is a promising vaccine candidate for glycoengineered immunotherapy of cancer with GM3 as the primary target.     

Availability of glycine and coenzyme A limits glycine conjugation in vivo.

Using benzoic acid as substrate, this study tested the hypothesis that capacity limitation of glycine conjugation in vivo is due to substrate-induced depletion of hepatic cosubstrates (i.e., ATP, coenzyme A, and glycine) utilized in the conjugation reaction. Benzolyglycine formation was investigated by following the disappearance of benzoic acid from blood and appearance of benzoylglycine in blood and urine after administration of sodium benzoate (0.2-2 mmol/kg, iv) to anesthetized rats whose urine formation was stimulated by mannitol administration. Capacity limitation of glycine conjugation is indicated by (a) the gradual dose-dependent reduction of benzoate blood clearance from 39 ml/min/kg at 0.2 mmol/kg benzoate to 3.7 ml/min/kg at 2 mmol/kg, and (b) the tendency to attain maximal blood levels and urinary excretion rates of benzoylglycine after administration of 0.5-1 mmol/kg benzoate. The maximal urinary excretion rate of benzoylglycine after benzoylglycine administration exceeded the maximal excretion rate of endogenously formed benzoylglycine (approximately 5 mumol/kg/min) 5-fold. This suggests that the urinary excretion rate of endogenously formed benzoylglycine reflects the rate of its formation. Benzoate depleted hepatic glycine (to 40%) and coenzyme A (to 14%) in a dose-dependent fashion; however, it did not change ATP levels in liver. The pattern of this dose-dependent cosubstrate depletion suggests that benzoate primarily causes consumption of hepatic glycine which, at high substrate dosage, leads to marked depletion of coenzyme A in the liver. Thus, these observations indicate that capacity-limited glycine conjugation may be due to limited availability of glycine and coenzyme A for the conjugation process.     

N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS) is a selective persistent inhibitor of glycine transport.

1. The regulation of glycine concentrations within excitatory synapses is poorly understood and it has been proposed that the GLYT1 subtypes of glycine transporters play a critical role in determining resting concentrations of glycine. Selective GLYT1 inhibitors may provide pharmacological tools to probe the dynamics of synaptic glycine concentrations, which may influence the activation properties of NMDA receptor activity. 2. We have characterized the selectivity and mechanism of action of the glycine transport inhibitor N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS). The glycine transporters, GLYT1a, b and c and GLYT2a were expressed in Xenopus laevis oocytes and two electrode voltage clamp techniques and radiolabelled (3)H-glycine flux measurements were used to characterize the effects of NFPS on glycine transport. 3. NFPS inhibits glycine transport by the GLYT1a, b and c subtypes of glycine transporters, but has no effect on the GLYT2a subtype of transporter. We show that NFPS does not attain its specificity via an interaction with the Na(+), Cl(-) or glycine site, nor does it act at an intracellular site. NFPS inhibition of glycine transport is time and concentration dependent and inhibition of transport by NFPS persists after washout of NFPS from the bath solution, which suggests that inhibition by NFPS is long lasting.     

Filter extrusion of liposomes using different devices: comparison of liposome size, encapsulation efficiency, and process characteristics.

Liposomes were prepared by stepwise extrusion through 5, 1, 0.4, 0.2, 0.1 and 0.05 microm pore sizes using two different filter-extruders, the continuous high pressure device Dispex Maximator (CE) or alternatively the discontinuous Avestin LiposoFast (DE). The liposome dispersions obtained were compared in terms of particle size, lamellarity and encapsulation efficiency of calcein. The liposomes were smaller with CE than DE at all stages due to higher flow rates and pressure drops, except for final filter pore size (0.05 microm) where both preparations had similar sizes. The particle size analysis technique itself had a strong influence on the liposome sizes measured. For bigger liposomes (extruded through 0.4 microm filters) the Nicomp 370 revealed bigger volume-based mean particle sizes along with more stringent differences between volume-based and number-based diameters than the Malvern Zetasizer. In contrast, for small liposomes extruded through 0.05 microm filters, similar liposome sizes were found no matter which of the two PCS techniques or cryo-transmission electron microscopy was used. In congruence to the liposome sizes measured, encapsulation efficiencies were smaller for CE than DE at all filter stages except the final (0.05 microm). No lipid loss occurred and lyso-phosphatidylcholine formation was negligible irrespective of which extrusion technique was used.     

N,N'-〔2,2'-二硫双(苯甲酰)〕-双 N-环己烷甘氨酸的降压作用

用麻醉大鼠观察了Ｎ，Ｎ′－〔２，２′－二硫双（苯甲酰）〕－双Ｎ－环己烷甘氨酸（ＤＧ－Ｉ）对血流动力学的影响、对肾血管性高血压的抗高血压作用及对ＡＩ、ＢＫ血压反应影响。ＤＧ－Ｉ２ｍｇ·ｋｇ－１ｉｖ可降低正常麻醉大鼠之ＳＡＰ、ＤＡＰ，不伴有ＬＶＰ、±ｄＰ／ｄｔｍａｘ下降，亦无反射性ＨＲ增快。ＤＧ－Ｉ２，４ｍｇ·ｋｇ－１ｉｖ可使肾血管性高血压大鼠血压下降，４ｍｇ·ｋｇ－１组伴有ＨＲ减慢。ＤＧ－Ｉ依剂量抑制ＡＩ之升压效应并增强ＢＫ之降压反应。结果表明ＤＧ－Ｉ具有血管紧张素转化酶抑制剂之特点，其降压作用与此有关。     

Antimicrobial and diffusional correlation of N-alkyl betaines and N-alkyl-N,N-dimethylamine oxides from semisolids.

Previous studies have shown that two classes of amphoteric surfactants, N-alkyl betaines and N-alkyl-N,N-dimethylamine oxides, exhibit pronounced antimicrobial activity in combination and have potential for use in a semisolid formulation for topical or vaginal delivery. In this work, several potential delivery systems were prepared and evaluated for antimicrobial activity and diffusional properties. A novel antimicrobial test for semisolids was proposed that determined the contact time needed to kill microorganisms. The unformulated agents in solution exhibited the faster kill within 60 min, followed by the hydroxyethylcellulose gel formulation in 90 min, and the poloxamer gel and a cream that required several hours. Diffusion from the dosage form utilized a Slide-A-Lyzer diffusion cassette with a 10,000 MWCO membrane with (14)C-labeled active species added to the aforementioned antimicrobial formulations. Diffusion of the individual betaine and amine oxide derivatives were tracked over time to determine the diffusion rates and profiles of the components in each formulation and in solution. The betaine derivative diffused up to three times faster than the amine oxide derivative within the first 2 h, but the amount diffused was approximately equivalent at 24 h. The formulations delayed release in the same rank order as the contact time kill analysis: hydroxyethylcellulose gel > poloxamer gel > cream.     

Characterization of the neuropsychological phenotype of glycine N-methyltransferase-/- mice and evaluation of its responses to clozapine and sarcosine treatments

Glycine N-methyltransferase (GNMT) affects cellular methylation capacity through regulating the ratio between S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). The product of its enzymatic reaction-sarcosine has antipsychotic effect in patients with schizophrenia. In this study, through RT-PCR and immunohistochemical staining, we demonstrated that GNMT expressed in various neurons located in the cerebral cortex, hippocampus, substantia nigra and cerebellum. Compared to the wild-type mice, Gnmt-/- mice had significantly lower level of sarcosine in the cerebral cortex. Real-time PCR identified genes involved in the methionine metabolism (Dnmt1 and Dnmt3a), ErbB (Nrg1 and ErbB4) and mTOR (Akt2, S6, S6k1 and S6k2) signaling pathways were dysregulated significantly in the cortex of Gnmt-/- mice. Acoustic startle reflex test demonstrated that Gnmt-/- mice had significantly lower level of prepulse inhibition and the deficit was ameliorated through clozapine or sarcosine treatment. Furthermore, liver-specific-human-GNMT transgenic with Gnmt-/- (Tg-GNMT/Gnmt-/-) mice were used to rule out that the phenotype was due to abnormal liver function. In summary, the neuropsychological abnormalities found in Gnmt-/- mice may represent an endophenotype of schizophrenia. GNMT plays an important role in maintaining normal physiological function of brain and Tg-GNMT/Gnmt-/- mice are useful models for development of therapeutics for patients with schizophrenia.     

A comparison of dimaprit, nordimaprit, methylamine and chloroquine as inhibitors of mitogen-induced lymphocyte activation.

Methylamine and chloroquine both 'lysosomotropic' agents (i.e. agents which sequester in lysosomes) caused a dose-related inhibition of mitogen-induced lymphocyte activation in the concentrations which have previously been shown to increase the pH of lysosomes. The dose-response curves of inhibition of mitogen-induced lymphocyte activation for chloroquine and methylamine are very steep and are similar to the dose-response curves obtained with dimaprit and nordimaprit, but very different from the flat dose-response curves previously described for histamine. Approximate IC50 values were methylamine 6.4 mM, dimaprit 0.13 mM, nordimaprit 0.03 mM and chloroquine 18 microM. It is suggested that the mechanism of action of methylamine and chloroquine may be related to their lysosomotropic action and consequent interference with ligand-receptor processing, and that dimaprit and nordimaprit but not histamine may act by a similar mechanism.     

N-modified analogues of cocaine: synthesis and inhibition of binding to the cocaine receptor.

Cocaine methiodide (2), N-norcocaine (1b), N-benzyl-N-norcocaine (1c), and N-nor-N-acetylcocaine (1d) were synthesized and evaluated for their ability to inhibit binding of [3H]-3 beta-(4-fluorophenyl)tropane-2 beta-carboxylic acid methyl ester (WIN 35,428) to the cocaine receptor. The study showed that removal of the N-methyl group to give 1b, or replacement with the larger N-benzyl group to give 1c, has a relatively small effect on binding potency. In contrast, replacement of the N-methyl group by the acetyl moiety to give 1d, or the addition of a methyl group to give 2, reduces affinity for the receptor by a large factor. In order to gain preliminary information concerning the importance of the nitrogen location on the tropane ring system, the receptor binding affinity of 8-methyl-8-azabicyclo[3.2.1]octan-3 beta-ol benzoate (5, beta-tropacocaine) was compared to that of the isomeric 6-methyl-6-azabicyclo[3.2.1]octan-3 beta-ol benzoate (4d). The fact that both compounds have similar binding affinities for the cocaine receptor suggests that 3 beta-(benzoyloxy)-6-methyl-6-azabicyclo[3.2.1] octane-2-carboxylic acid methyl ester, which is isomeric with cocaine, may possess binding potency similar to cocaine.     

Cross-conjugated and pseudo-cross-conjugated mesomeric betaines, XVIII: Bicyclic mesoionic pyrimidines with cardiovascular activity.

Reaction of alpha-Anilino-azines 1a-i with activated malonates (magic malonates) 2a-e leads to bicyclic mesoionic systems 3-7. Out of these, pyrimido[1,2-alpha]pyrimidines 3b,d are active as cardiotonics, whereas pyrimido[1,2-alpha]pyrimidines 4a-g show significant anti-anginal and anti-hypertensive activity.     

A comparison of gag, pol and rev antisense oligodeoxynucleotides as inhibitors of HIV-1.

Sequences from the gag, pol and rev regions of the RF strain of HIV-1 (HIV-1RF) were chosen as targets for antisense phosphorothioate oligodeoxynucleotides (S-oligos). These sequences were the p18/p24 junction in gag, the active site of HIV protease in pol; a sequence from the first exon of the rev gene and S-oligodeoxycytidylic acid controls. Compounds were tested against HIV-1 in both acutely and chronically infected cells. The results show that these phosphorothioate analogues tested in acutely infected cells were active in the 0.1-2 microM range, were dependent on chain length but had no sequence specificity. To study the mechanism of action, the time of addition of S-oligos to acutely infected cells was delayed for up to 48 h post-infection. It was found that antiviral activity was lost when compounds were added to the cultures later than 10 h post-infection. With chronically infected cells only the antisense rev sequence showed activity at 30 microM and neither of the gag or pol antisense sequences has a significant effect on HIV replication at 50 microM. These results are consistent with previous in vitro studies which demonstrate that antisense S-oligodeoxynucleotides have several modes of action.     

N,N-dialkylaminosubstituted chromones and isoxazoles as potential anti-inflammatory agents.

The ability of some N,N-dialkylaminosubstituted chromones and isoxazoles to inhibit the protein kinase C (PKC) dependent signal transduction pathway was tested. As a cellular model, human neutrophils stimulated with either phorbol myristate acetate (PMA) or formylmethionine-leucine-phenylalanine (f-MLF) were used. The efficiency of the compounds was established by their capacity to reduce the O2- production by activated human neutrophils. Compounds carrying a 3-bis(2-methoxyethyl)amino group, a substituent found active in previously tested tricyclic compounds, do not show significant anti-PKC activity in this study. On the other hand, substitution with a 1-piperidinyl group leads all tested compounds to a high biological activity against stimulated neutrophils.     

A comparison of nicotine and structurally related compounds as discriminative stimuli.

1 Of seven nicotine-like compounds tested as discriminative stimuli in the rat, only 3-pyridyl-methylpyrollidine (3-PMP) generalized to the stimulus effects of nicotine. 2 3-PMP caused equivalent nicotine-like responding at a dose (800 microgram/kg) approximately 4 times that used for the original nicotine discrimination (200 microgram/kg). The ED50 for 3-PMP was about 5 times that for nicotine. 3 Testing of the compounds as possible antagonists of the nicotine-elicited cue were negative. 4 The nicotine-like cue produced by an 800 microgram/kg injection of 3-PMP was effectively blocked by mecamylamine but not by hexamethonium or atropine. Thus, 3-PMP appears to produce generalization to the nicotine cue via action on central nicotinic-cholinoceptors as has been previously reported for the nicotine discriminative stimulus. 5 Mecamylamine blocked the stimulus-effects of 3-PMP (800 microgram/kg) and of nicotine (200 microgram/kg) with an ED50 of 0.32 and 0.20 microgram/kg respectively.