Comparison of different substances for subureteric injection in the management of vesicoureteric reflux in children.

INTRODUCTION: Endoscopic techniques are becoming increasingly accepted for treatment of vesicoureteric reflux as alternatives to open surgical reimplantation. However, there is some debate about the ideal injectable material. Since we have accumulated experience with several substances, an opportunity existed to compare them. MATERIALS AND METHODS: From 1991 to 2003, 101 children with vesicoureteric reflux were treated by endoscopic subureteric injection either once (74) or twice (27) by either of two pediatric urologists. There were a total of 165 ureteral injections, 83 with polytetrafluoroethylene (Teflon), 73 with polydimethylsiloxane (Macroplastique), and 9 with collagen. Each child was evaluated pre-operatively and 3 months post-operatively with a nuclear cystogram and renal ultrasonography. RESULTS: The polytetrafluoroethylene and polydimethylsiloxane groups were not significantly different with respect to sex, age, indication for surgery, severity of reflux or prior surgeries. The collagen group overall did very poorly with only 3 of 9 refluxing ureters cured. The other two substances had much more success with 61% of ureters in the polytetrafluoroethylene group cured on first injection and 75% with polydimethylsiloxane, plus another 19% and 11% cured on second attempt, respectively (total 80% and 86%). CONCLUSIONS: Subureteric injections of polytetrafluoroethylene and polydimethylsiloxane are very effective at curing vesicoureteric reflux in children with little morbidity. When comparing individual cases, ureters, and all grades of reflux, polytetrafluoroethylene and polydimethylsiloxane have similar success rates. Collagen injections were less successful, and patients with neurogenic bladders had poor results.

CI-1017, a functionally M1-selective muscarinic agonist: design, synthesis, and preclinical pharmacology.

The five muscarinic receptor subtypes (M1-M5) are characterized by seven helices that define a transmembrane cavity which serves as the binding pocket for agonists and antagonists. The five cavities appear to be topographically different enough to permit subtype selectivity among antagonists but not among classical agonists which tend to be smaller in size than antagonists. It was reasoned that synthesis of muscarinic agonists longer/larger than their classical counterparts might result in subtype selectivity. M1 subtype selectivity was found in a class of 1-azabicyclo[2.2.1]heptan-3-one, O-(3-aryl-2-propynyl) oximes. One of these, CI-1017, improved spatial memory of hippocampally deficient mice and nbM-lesioned rats at doses of 1.0-3.2 and 0.1-0.3 mg/kg, respectively, while producing parasympathetic side effects only at very high doses (100-178 mg/kg). Additionally, CI-1017 inhibited production of amyloidogenic A beta and increased secretion of soluble APP. Thus, CI-1017, besides treating AD symptomatically, may also retard its progression. CI-1017 has recently completed phase I clinical trials.

Evidence for seeding of beta -amyloid by intracerebral infusion of Alzheimer brain extracts in beta -amyloid precursor protein-transgenic mice.

Many neurodegenerative diseases are associated with the abnormal sequestration of disease-specific proteins in the brain, but the events that initiate this process remain unclear. To determine whether the deposition of the beta-amyloid peptide (Abeta), a key pathological feature of Alzheimer's disease (AD), can be induced in vivo, we infused dilute supernatants of autopsy-derived neocortical homogenates from Alzheimer's patients unilaterally into the hippocampus and neocortex of 3-month-old beta-amyloid precursor protein (betaAPP)-transgenic mice. Up to 4 weeks after the infusion there was no Abeta-deposition in the brain; however, after 5 months, the AD-tissue-injected hemisphere of the transgenic mice had developed profuse Abeta-immunoreactive senile plaques and vascular deposits, some of which were birefringent with Congo Red. There was limited deposition of diffuse Abeta also in the brains of betaAPP-transgenic mice infused with tissue from an age-matched, non-AD brain with mild beta-amyloidosis, but none in mice receiving extract from a young control case. Abeta deposits also were not found in either vehicle-injected or uninjected transgenic mice or in any nontransgenic mice. The results show that cerebral beta-amyloid can be seeded in vivo by a single inoculation of dilute AD brain extract, demonstrating a key pathogenic commonality between beta-amyloidosis and other neurodegenerative diseases involving abnormal protein polymerization. The paradigm can be used to clarify the conditions that initiate in vivo beta-amyloidogenesis in the brain and may yield a more authentic animal model of Alzheimer's disease and other neurodegenerative disorders.

The M1 muscarinic agonist CI-1017 facilitates trace eyeblink conditioning in aging rabbits and increases the excitability of CA1 pyramidal neurons.

The M1 muscarinic agonist CI-1017 was administered intravenously to aging rabbits on a daily basis before and during hippocampally dependent trace eyeblink conditioning sessions. Circulating levels of CI-1017 were significantly related to the drug dose. The drug was found to significantly increase the rate and amount of learning in a dose-dependent manner with no significant effects on the amplitude, area, or latency of conditioned responses. There was no evidence of pseudoconditioning at the highest drug concentration, and the minimally effective dose produced only mild and temporary hypersalivation as a side effect. CI-1017 (10 microM) was also found to increase the excitability of CA1 pyramidal neurons recorded from hippocampal slices from young and aging naive rabbits as measured by changes in spike-frequency adaptation and the postburst afterhyperpolarization. These biophysical changes were reversed with either atropine (1 microM) or pirenzepine (1 microM). These results suggest that M1 agonists ameliorate age-related learning and memory impairments at least in part by reducing the afterhyperpolarization and spike-frequency adaptation of hippocampal pyramidal neurons and that M1 agonists may be an effective therapy for reducing the cognitive deficits that accompany normal aging and/or Alzheimer's disease.

Milameline (CI-979/RU35926): a muscarinic receptor agonist with cognition-activating properties: biochemical and in vivo characterization.

Milameline (E-1,2,5,6-tetrahydro-1-methyl-3-pyridinecarboxaldehyde, O-methyloxime monohydrochloride, CI-979, PD129409, RU35926) was characterized in vitro and evaluated for effects on central and peripheral cholinergic activity in rats and rhesus monkeys. In muscarinic binding studies, milameline displayed nanomolar affinity with an agonist ligand and micromolar affinity with antagonist ligands, with approximately equal affinities determined at the five subtypes of human muscarinic receptors (hM(1)-hM(5)) with whole cells or membranes from stably transfected Chinese hamster ovary (CHO) cells. On binding, milameline stimulated phosphatidylinositol hydrolysis in hM(1) and hM(3) CHO cells and inhibited forskolin-activated cAMP accumulation in hM(2) and hM(4) CHO cells. Additionally, it decreased K(+)-stimulated release of [(3)H]acetylcholine from rat cortical slices. Responses were not caused by the inhibition of acetylcholinesterase, and there was no significant binding to approximately 30 other neurotransmitter binding sites. In rats, milameline decreased spontaneous and scopolamine-induced swimming activity, improved water-maze performance of animals impaired by basal forebrain lesions, increased cortical blood flow, decreased core body temperature, and increased gastrointestinal motility. Electroencephalogram activity in both rats and monkeys was characterized by a predominance of low-voltage desynchronized activity consistent with an increase in arousal. Milameline also reversed a scopolamine-induced impairment of attention on a continuous-performance task in monkeys. Thus, milameline possesses a pharmacological profile consistent with that of a partial muscarinic agonist, with central cholinergic actions being produced in rats and monkeys at doses slightly lower than those stimulating peripheral cholinergic receptors.

Design and synthesis of novel quinoxaline-2,3-dione AMPA/GlyN receptor antagonists: amino acid derivatives.

PNQX (1,4,7,8,9,10-hexahydro-9-methyl-6-nitropyrido[3, 4-f]quinoxaline-2,3-dione) is a potent AMPA (IC50 = 0.063 microM) and GlyN (IC50 = 0.37 microM) receptor antagonist that was developed in our laboratories. While possessing a desirable in vitro and in vivo activity profile, this compound suffers from low aqueous solubility. In an effort to improve its potency and physical properties, we have designed and synthesized novel ring-opened analogues 4, 6, 9, and 11. Modeling analyses demonstrated that, while the 5-substituent in these analogues was forced to adopt an out-of-plane conformation due to steric contacts with neighboring substituents, the overall structure retained a good fit to a previously described AMPA pharmacophore model. This nonplanar orientation may lessen efficient packing in the solid state, compared to PNQX, leading to increased water solubility. Indeed, several nonplanar analogues containing appropriate functionalities, for example, the sarcosine analogue 9, were found to retain AMPA (IC50 = 0.14 microM) and GlyN (IC50 = 0.47 microM) receptor affinity and possess improved aqueous solubility compared to PNQX. The synthesis and the SAR of these compounds are discussed.

Inhibitors of V-type ATPases, bafilomycin A1 and concanamycin A, protect against beta-amyloid-mediated effects on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

The functional viability of cells can be evaluated using a number of different assay determinants. One common assay involves exposing cells to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which is converted intracellularly to a colored formazan precipitate and often used to assess amyloid peptide-induced cytotoxic effects. The MTT assay was employed to evaluate the role of endosomal uptake and lysosomal acidification in amyloid peptide-treated differentiated PC12 cell cultures using selective vacuolar-type (V-type) ATPase inhibitors. The macrolides bafilomycin A1 (BAF) and concanamycin A (CON) block lysosomal acidification through selective inhibition of the V-type ATPase. Treating nerve growth factor-differentiated PC12 cells with nanomolar concentrations of BAF or CON provides complete protection against the effects of beta-amyloid peptides Abeta(1-42), Abeta(1-40), and Abeta(25-35) and of amylin on MTT dye conversion. These macrolides do not inhibit peptide aggregation, act as antioxidants, or inhibit Abeta uptake by cells. Measurements of lysosomal acidification reveal that the concentrations of BAF and CON effective in reversing Abeta-mediated MTT dye conversion also reverse lysosomal pH. These results suggest that lysosomal acidification is necessary for Abeta effects on MTT dye conversion.

Identification and characterization of m1 selective muscarinic receptor antagonists1.

A series of esters of 1,4-disubstituted tetrahydropyridine carboxylic acids (I) has been synthesized and characterized as potential m1 selective muscarinic receptor antagonists. The affinity of these compounds for the five human muscarinic receptor subtypes (Hm1-Hm5) was determined by the displacement of [3H]-NMS binding using membranes from transfected Chinese hamster ovarian cells. One of the most potent and selective compounds of this series is an analogue of I [11, R1 = (CH2)5CH3], which has an IC50 value of 27.3 nM at the m1 receptor and possesses 100-fold (m2), 48-fold (m3), 74-fold (m4), and 19-fold (m5) selectivities at the other receptors. Thus, this analogue appears to be more selective on the basis of binding than the prototypical m1 antagonist, pirenzepine. Functional data, such as the inhibition of carbachol-stimulated phosphatidylinositol hydrolysis, on selected analogues confirmed the muscarinic antagonistic properties of this chemical series.

Differential expression of group I metabotropic glutamate receptors (mGluRs) in the rat pheochromocytoma cell line PC12: role of nerve growth factor and ras.

Glutamate treatment of PC12 cells has been shown to result in the accumulation of intracellular inositol phosphates suggesting the presence of glutamate metabotropic receptors (mGluRs) positively coupled to phospholipase C. The present study examined the expression of group I mGluRs (mGluR1 and mGluR5) in PC12 cells. Undifferentiated PC12 cells were found to express both mGluR5 mRNA and receptor protein by reverse transcription polymerase chain reaction (RT-PCR) and western blot techniques. However, mGluR1 mRNA was not detected in these cells and western blot analysis showed only faint mGluR1alpha immunoreactivity suggesting a very low level of mGluR1 expression. Nerve growth factor-induced differentiation of PC12 cells resulted in the induction of mGluR1alpha and mGluR1beta mRNA and mGluR1alpha protein. PC12 cells overexpressing dominant negative ras revealed that NGF-induced mGluR1 induction, but not mGluR5 expression, is dependent on ras pathway activation in these cells. These results suggest PC12 cells may be a useful model for investigating the regulation and expression of group I mGluR isoforms and their role in neuronal processes in vitro.

Design and synthesis of m1-selective muscarinic agonists: (R)-(-)-(Z)-1-Azabicyclo[2.2.1]heptan-3-one, O-(3-(3'-methoxyphenyl)-2-propynyl)oxime maleate (CI-1017), a functionally m1-selective muscarinic agonist.

The synthesis and SAR of a series of (Z)-(+/-)-1-azabicyclo[2.2. 1]heptan-3-one, O-(3-aryl-2-propynyl)oximes are described. The biochemistry and pharmacology of 24Z (PD 142505) and its enantiomers are highlighted. 24Z is functionally an m1-selective muscarinic agonist. Efficacy and m1 selectivity reside in the R enantiomer, (R)-24Z (CI-1017).

Identification and characterization of m4 selective muscarinic antagonists.

Our interest in the area of m4 muscarinic antagonists had led us to study a series of benzoxazine isoquinolines. One of the most potent and selective compounds of this series is example 1 with an IC50 value of 90.7 nM at m4 receptors, and 72-fold (m1), 38-fold (m2), 10-fold (m3), and 82-fold (m5) more selective compared to the other receptors. The synthesis and receptor binding affinity of analogs of 1 are reported.

Acute epididymitis in boys: are antibiotics indicated?

OBJECTIVES: To report the results of using supportive therapy only, rather than antibiotics, in managing boys with acute sterile epididymitis. PATIENTS AND METHODS: From 1991 to 1995, 48 boys presented with acute epididymitis. The diagnosis was confirmed by radionuclide scan in 43 cases, ultrasonography in one, surgical exploration in one and physical examination in three. Urine was collected for microscopy and culture: if pyuria was detected, antibiotics were prescribed. If the urine analysis was normal, the patient was advised to minimize physical activity and analgesics were prescribed. RESULTS: Of the 48 boys, five (10%) had pyuria; seven patients with either no urine tested or negative urine culture were given antibiotics. The remaining 36 were managed with supportive therapy only. The mean follow-up was 87 days (with three patients lost to follow-up). No boys showed any evidence of testicular atrophy or other complications. CONCLUSION: Only a minority of boys with acute epididymitis, as defined by increased flow on radionuclide scanning of the scrotum, have a bacterial aetiology. For those without pyuria or positive urine culture, the condition is self-limiting and does not lead to testicular atrophy. We recommend that for boys with acute epididymitis who have no urinary abnormalities, antibiotics are not indicated. The aetiology of acute sterile epididymitis in boys remains obscure.

A structure-activity relationship study of novel phenylacetamides which are sodium channel blockers.

A structure-activity relationship study of a series of novel Na(+) channel blockers, structurally related to N-[3-(2,6-dimethyl-1-piperidinyl)propyl]-alpha-phenylbenzeneacetamide (1, PD85639) is described. The diphenylacetic acid portion of the molecule was left unchanged throughout the study, while structural features in the amine portion and the amide alkyl linkage of the molecule were modified. The compounds were tested for inhibition of veratridine-stimulated Na(+) influx in CHO cells expressing type IIA Na(+) channels. Several derivatives show a trend toward more potent Na+ channel blockade activity with increasing lipophilicity of the amine portion of the molecule. The presence of a phenyl ring near the amine increases inhibitory potency. A three-carbon spacer between the amide and amine is optimal, and a secondary amide linkage is preferred.

Preclinical and phase 1 clinical characterization of CI-979/RU35926, a novel muscarinic agonist for the treatment of Alzheimer's disease.

In vitro and in vivo characterization in rodents and monkeys shows that CI-979/RU35926 is a partial muscarinic agonist with equal affinity for the five subtypes of muscarinic receptors. It activates central cholinergic receptors as shown by its ability to decrease body temperature, enhance local cortical blood flow and increase cortical arousal measured by QEEG. Further, it reverses spatial memory deficits in rats with ibotenic acid-induced lesions of forebrain cholinergic neurons. Signs of peripheral cholinergic stimulation appear at doses higher or equal to those necessary to produce central activity. In a single-dose tolerance study in young, healthy human volunteers, CI-979/RU35926 was well tolerated at doses of 0.002-1.0 mg with cholinergic symptoms such as hypersalivation and sweating, observed at 2-4 mg. It demonstrated linear pharmacokinetic behavior over a dose range of 0.1 to 4 mg and elimination half-life varied from 2-5 hours. Measurement of unchanged drug in urine suggests that the drug was extensively metabolized. Thus, the safety profile supported further clinical evaluation and CI-979/RU35926 is currently in Phase II clinical trials.

Mutations of aspartate 103 in the Hm2 receptor and alterations in receptor binding properties of muscarinic agonists.

Aspartate 103 (D103) in the third transmembrane domain of the Hm2 receptor was mutated to glutamate (D103E), asparagine (D103N), or alanine (D103A). As measured by [3H]-NMS, no significant binding was observed in D103A, while a 2-fold decrease in ligand affinity was seen in D103E and a 32-fold decrease in affinity was found in the D103N mutant. Examination of reference agonists showed greater loss of affinity in D103N than in D103E with the rank order of change being: L-607,207>carbachol>arecoline>pilocarpine>oxotremorine>McN-A-343. Of the novel 1-azabicyclo[2.2.1]-heptan-3-one oxime agonists examined, arylacetylene oximes showed little alteration in binding in either the D103E or D103N mutants, while the geometric isomers of several bicyclic aryl-ene-yne oximes showed significant changes in affinity, especially in the D103N mutant. Thus, overall size of the agonist and/or spatial orientation of the molecule within the binding pocket contribute to changes measured in binding.

Substituted 2-benzothiazolamines as sodium flux inhibitors: quantitative structure-activity relationships and anticonvulsant activity.

Thirty-two aryl-substituted 2-benzothiazolamines have been tested for their ability to modulate sodium flux in rat cortical slices. A QSAR analysis, applied to these derivatives, showed a trend toward increasing potency as sodium flux inhibitors with increasing lipophilicity, decreasing size, and increasing electron withdrawal of the benzo ring substituents. Additionally, 4- or 5-substitution of the benzo ring was found to decrease potency. The combination of increased lipophilicity, small size, and electron withdrawal severely limited which groups were tolerated on the benzo ring, thus suggesting that the optimal substitution patterns have been prepared within this series. Nine of these compounds were potent inhibitors of veratridine-induced sodium flux (NaFl). These nine compounds also proved to be anticonvulsant in the maximal electroshock (MES) assay. Fourteen additional 2-benzothiazolamines demonstrated activity in the MES screen, yet exhibited no activity in the NaFl assay. These derivatives may be interacting at the sodium channel in a manner not discernible by the flux paradigm, or they may be acting by an alternative mechanism in vivo.

PD 142676 (CI 1002), a novel anticholinesterase and muscarinic antagonist.

Inhibition of brain acetylcholinesterase (AChE) can provide relief from the cognitive loss associated with Alzheimer's disease (AD). However, unwanted peripheral side effects often limit the usefulness of the available anticholinesterases. Recently, we identified a dihydroquinazoline compound, PD 142676 (CI 1002) that is a potent anticholinesterase and a functional muscarinic antagonist at higher concentrations. Peripherally, PD 142676, unlike other anticholinesterases, inhibits gastrointestinal motility in rats, an effect consistent with its muscarinic antagonist properties. Centrally, the compound acts as a cholinomimetic. In rats, PD 142676 decreases core body temperature. It also increases neocortical arousal, as measured by quantitative electroencephalography, and cortical acetylcholine levels, measured by in vivo microdialysis. The compound improves the performance of C57/B10j mice in a water maze task and of aged rhesus monkeys in a delayed match-to-sample task involving short-term memory. The combined effect of AChE inhibition and muscarinic antagonism distinguishes PD 142676 from other anticholinesterases, and may be useful in treating the cognitive dysfunction of AD and produce fewer peripheral side effects.

Synthesis and pharmacological evaluation of phenylacetamides as sodium-channel blockers.

The synthesis and structure-activity relationships of a series of phenylacetamides related to N-[3-(2,6-dimethyl-1-piperidinyl)propyl]-alpha-phenylbenzeneacetamide (1) (PD85639) acting at the voltage-dependent Na+ channel are described. All structural variations for this study were made in the phenylacetic acid portion of these molecules, and the compounds were synthesized by coupling the appropriately substituted phenylacetic acid derivative with 3-[1-(2,6-dimethyl)piperidinyl]-propanamine using standard methods of amide formation. Compounds were tested as inhibitors of [3H]batrachtoxinin binding in rat neocortical membranes and also as inhibitors of veratridine-induced Na+ influx in Chinese hamster ovary cells expressing type IIA Na+ channels. Diphenylacetic acid derivatives with halogenated aromatic rings (12-15) were very potent in both assays, while alkoxy and alkyl substitution did not affect activity (16 and 17). Selected compounds were tested as potential neuroprotective agents in two cell culture assays involving inhibition of veratridine-induced and hypoxia-induced lactate dehydrogenase release. Compound 15 was equipotent with flunarizine, a reference compound in both neuroprotection assays.

Specific binding of the novel Na+ channel blocker PD85,639 to the alpha subunit of rat brain Na+ channels.

The local anesthetic-like Na+ channel-blocking drug [3H]PD85639 [alpha-([4-3H]phenyl)-N-[3-(2,6-dimethyl-1-piperizinyl)-alpha-prop yl] [4-3H]benzeneacetamide] binds specifically to receptor sites on Na+ channels in intact synaptosomes and synaptosomal membranes, purified and reconstituted Na+ channels, and type IIA Na+ channel alpha subunits expressed in the transfected Chinese hamster ovary cell line CNaIIA-1. No specific binding was observed in nontransfected CHO-K1 cells, confirming the specificity of binding to Na+ channels. Two classes of binding sites that differed in affinity and dissociation rate were observed in all three preparations. In synaptosomes, the high affinity sites had Kd values of 3-20 nM and a Bmax of approximately 0.2 pmol/mg, whereas the low affinity sites had Kd values of 0.4-20 microM and a Bmax of approximately 5 pmol/mg. Binding of PD85,639 was inhibited by the local anesthetics tetracaine, bupivacaine, and mepivacaine at concentrations in the same range as those that inhibit Na+ channels. Tetracaine did not affect the dissociation rate of PD85,639, consistent with competitive binding of these two drugs at the same receptor site. In contrast, binding of PD85,639 was unaffected by the anticonvulsants phenytoin and carbamazepine, which also inhibit Na+ channels. Veratridine and batrachotoxin, which bind at neurotoxin receptor site 2 on Na+ channels, inhibited specific PD85,639 binding completely. PD85,639 accelerated dissociation of specifically bound batrachotoxin, consistent with an indirect allosteric interaction between these two compounds. Thus, like local anesthetics, PD85,639 inhibits binding of batrachotoxin by an allosteric mechanism. The results indicate that PD85,639 binds specifically to a local anesthetic receptor site on the Na+ channel alpha subunit that is allosterically linked to neurotoxin receptor site 2. PD85,639 may be a useful molecular probe of this important drug receptor site on the Na+ channel.