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Odanacatib (ODN) is a selective and reversible inhibitor of cathepsin K (CatK) currently being developed as a once-weekly treatment for osteoporosis. In this study, we evaluated the effects of ODN on bone turnover, bone mineral density (BMD), and bone strength in the lumbar spine of estrogen-deficient, skeletally mature rhesus monkeys. Ovariectomized (OVX) monkeys were treated in prevention mode for 21 months with either vehicle, ODN 6 mg/kg, or ODN 30 mg/kg (p.o., q.d.) and compared with intact animals. ODN treatment persistently suppressed the bone resorption markers (urinary NTx [75% to 90%] and serum CTx [40% to 55%]) and the serum formation markers (BSAP [30% to 35%] and P1NP [60% to 70%]) versus vehicle-treated OVX monkeys. Treatment with ODN also led to dose-dependent increases in serum 1-CTP and maintained estrogen deficiency-elevated Trap-5b levels, supporting the distinct mechanism of CatK inhibition in effectively suppressing bone resorption without reducing osteoclast numbers. ODN at both doses fully prevented bone loss in lumbar vertebrae (L₁ to L₄) BMD in OVX animals, maintaining a level comparable to intact animals. ODN dose-dependently increased L₁ to L₄ BMD by 7% in the 6 mg/kg group (p < 0.05 versus OVX-vehicle) and 15% in the 30 mg/kg group (p < 0.05 versus OVX-vehicle) from baseline. Treatment also trended to increase bone strength, associated with a positive and highly significant correlation (R = 0.838) between peak load and bone mineral content of the lumbar spine. Whereas ODN reduced bone turnover parameters in trabecular bone, the number of osteoclasts was either maintained or increased in the ODN-treated groups compared with the vehicle controls. Taken together, our findings demonstrated that the long-term treatment with ODN effectively suppressed bone turnover without reducing osteoclast number and maintained normal biomechanical properties of the spine of OVX nonhuman primates.

Odanacatib (ODN) a selective and reversible cathepsin K inhibitor, inhibits bone resorption, increases bone mass and reduces fracture risk in women with osteoporosis. A 16-month (~7-remodeling cycles) study was carried out in treatment mode to assess the effects of ODN versus ALN on bone mass, remodeling status and biomechanical properties of lumbar vertebrae (LV) and femur in ovariectomized (OVX) rabbits. This study also evaluated the impact of discontinuing ODN on these parameters. Rabbits at 7.5months post-OVX were dosed for 16-months with ODN (7.5μM·h0-24, in food) or ALN (0.2mg/kg/wk, s.c.) and compared to vehicle-treated OVX- (OVX+Veh) or Sham-operated animals. After 8months, treatment was discontinued in half of the ODN group. ODN treatment increased in vivo LV aBMD and trabecular (Tb) vBMD until reaching plateau at month 12 by 16% and 23% vs. baseline, respectively, comparable levels to that in Sham and significantly above OVX+Veh. LV BMD was also higher in ALN that plateaued around month 8 to levels below that in ODN or Sham. ODN treatment resulted in higher BMD, structure and improved biomechanical strength of LV and central femur (CF) to levels similar to Sham. ALN generally showed less robust efficacy compared to ODN. Neither ODN nor ALN influenced material properties at these bone sites following ODN or ALN treatment for 7 remodeling cycles in rabbits. ODN and ALN persistently reduced the bone resorption marker urinary helical peptide over study duration. While ALN reduced the bone formation marker BSAP, ODN treatment did not affect this marker. ODN also preserved histomorphometry-based bone formation indices in LV trabecular, CF endocortical and intracortical surfaces, at the levels of OVX+Veh. Discontinuation of ODN returned bone mass, structure and strength parameters to the comparable respective levels in OVX+Veh. Together, these data demonstrate efficacy and bone safety profile of ODN and suggests the potential long-term benefits of this agent over ALN with respect to accrued bone mass without long-term effects on bone formation.

The objectives here were to evaluate the effects of odanacatib (ODN) at doses exceeding the clinical exposure on biomechanical properties of lumbar vertebrae (LV), hip and central femur (CF), and compare ODN to alendronate (ALN) on bone remodeling/modeling in ovariectomized (OVX) monkeys. Ten days post-surgery, animals were treated with vehicle (VEH), ODN-L (2mg/kg/day, p.o.), ODN-H (8/4mg/kg/day), or ALN (30μg/kg/week, s.c.) for 20months. An intact group was also included. ODN-L provided systemic exposures of 1.8-fold of clinical exposure. ODN-H started at 20-fold for 5.5months, and then reduced to 7.8-fold of clinical exposure, compared to ALN at approximated clinical exposure. From cross sectional analyses, LV density and peak load in ODN at both doses or ALN were not different from VEH or Intact. However, cortical thickness of femoral neck (FN) and CF in ODN were higher (21-34%, p

Enaminones, enamines of beta-dicarbonyl compounds, have been known for many years. Their early use has been relegated to serving as synthetic intermediates in organic synthesis and of late, in pharmaceutical development. Recently, the therapeutic potential of these entities has been realized. This review provides the background and current research in this area with emphasis of these agents as potential anticonvulsants, their proposed mechanisms of action, and as potential modulators of multidrug resistance (MDR).

The enaminones represent potentially useful agents for the clinical treatment in generalized tonic-clonic seizures (Epilepsia, 1993, 34(6), 1141-1145, Biopharm. Drug Disp. 2003, 397-407). A regression analysis was performed to provide a quantitative structure-activity relationship (QSAR) correlation model for prediction of activity for the anticonvulsant enaminones. Molecular modeling was performed to determine the molecular confluence of the Unverferth model (J. Med. Chem. 1998, 41, 63-73) to the enaminones. Conclusions related to the sodium channel model were assessed.

An improved method for the synthesis of benzamides from 3-amino-2-cyclohexenones is presented. Using sodium hydride, a base-catalyzed N-benzoylation provided significantly higher yields (71-79%) for the reported compounds. This novel protocol was applied in the solution-phase parallel synthesis of a 12-member library of vinylic benzamide derivatives of 3-amino-2-cyclohexenones in 63-90% yield, using a Radley's Carousel Reaction Station.

A simple high performance liquid chromatography (HPLC) method has been developed for the stability study of an anticonvulsant enaminone (E139). Using a Chiral HSA column and a mobile phase of n-octanoic acid (5 mM) and isopropyl alcohol and disodium hydrogen phosphate solution 1:9 v/v at a flow rate of 1 mL/min., the chromatograms exhibited well resolved peaks at retention times of < 5 min. for the predominant diastereoisomer. The stability study for E139 was carried out in acid, alkaline, neutral solution, and in a phosphate buffer solution of physiological pH. The results confirmed that the hydrolysis of E139 was fastest in acid medium, indicating that protonation of the enaminone system enhanced hydrolysis at a degradation rate constant (Kdeg) of 0.044 min.−1 and a degradation half-life (t½) of 15.75 min. at room temperature (25°C). Deprotonation of E139 in alkaline solution also resulted in hydrolysis, but at a slower rate (Kdegof 0.017 min.−1) and longer degradation half-life (t½: 40.76 min.) at 25°C...

In a continuing evaluation of the aniline-substituted enaminones, the synthesis of additional para-substituted analogs has been made in an attempt to further quantify the electronic (sigma) and lipophilic (pi) requirements for anticonvulsant activity in this series. In addition, meta- and ortho-substituted and polysubstituted compounds have been synthesized and evaluated for anticonvulsant activity. In the para-substituted series, 4-cyano analogs (32 and 33) (+ sigma, - pi), which were highly active via intraperitoneal (ip) injection in mice, were inactive on oral (po) administration in rats. The para-substituted trifluoromethoxy (+ sigma, + pi) analog (8) had significant potency by both routes. Meta substitution limited the activity due to steric factors. Bromo and iodo substituents produced active para-substituted analogs (5 and 17) but were inactive when substituted in the meta position (37 and 41, respectively). Ortho substitution provided no clear relationship due to nonparametric deviations. Neither 1, the prototype enaminone, nor 2, the putative metabolite, produced significant nephrotoxicity or hepatotoxicity. Sodium channel binding of 1 and 8 indicated that 8 displayed relatively potent sodium channel binding but 1 showed weaker effects with IC50 values of 489 and 170 microM respectively against [3H]batrachotoxinin A 20 alpha-benzoate ([3H]BTX-B).

Antihyperglycaemic therapy on bone was evaluated in the ovariectomized (OVX), non-diabetic adult rat. Animals were treated daily for 12 weeks with various doses of sitagliptin, pioglitazone, rosiglitazone, combinations of sitagliptin with pioglitazone or vehicle alone. Sitagliptin target engagement was confirmed by assessing inhibition of plasma dipeptidyl peptidase-4 (DPP-4) and oral glucose tolerance. Parameters related to bone health were evaluated in femur and vertebrae by dual-energy X-ray absorptiometry and histomorphometry. Bone mineral density (BMD) generally did not differ significantly between OVX-sitagliptin-treated animals and OVX-vehicle controls. In lumbar vertebrae, however, there was significantly less BMD loss with increasing sitagliptin dose. Thiazolidinedione (TZD) treatment generally resulted in lower BMD; OVX-TZD-treated (but not OVX-sitagliptin-treated) animals also had lessened cortical thickness in central femur and profoundly greater bone marrow adiposity in lumbar vertebrae. These findings support prior findings with TZDs and suggest a neutral or beneficial impact of DPP-4 inhibition on bone health.

Enaminones, enamines of B-dicarbonyl compounds have been known for many years. Their main therapeutic utility was as prodrugs for various amines, stabilizing these active compounds principally in basic media. Our laboratory discovered a series of enamines of cyclic 1,3-diketo esters which were stable and possessed significant anticonvulsant activity. The most notable compound was methyl 4- [(p­ chlorophenyl)amino)-6-methyl-2-oxocyclohex-3-en-1-oate, 15, desig­ nated ADD 196022 by the Anti-convulsant Drug Development (ADD) Program of the National Institutes of Health. Compound 15 displayed an intraperitoneal (ip) EDso of 26.2 mg/kg and a TDso of 254.8 mg/kg in the mouse and an oral (po) EDso of 5.8 mg/kg and a TDso of >380 mg/kg in the rat. The protective indices (TDso/EDso) were 9.7 in the mouse and >65.6 in the rat, respectively. This data compared favorably to phenytoin and carbamazepine. In addition, 15 demonstrated oral effectiveness in the rat corneal kindling model, providing an EDso of 34.2 mg/kg. Quantitative structure-activity studies in this series successfully utilized the Craig Plot method (Craig, P. N.J. Med Chern. 1971, 14, 680; Craig, P. N.J. Med Chern. 1972, 15, 144). High field NMR , molecular modeling studies and CLOGP were also employed to assess the potential interactions of these compounds and phenytoin in its relationship to potential anticonvulsant activity. Each of these factors is discussed.

1H nuclear magnetic resonance (NMR) spectra of enaminones were determined and compared to the anticonvulsant activity of the compounds. Although the precise employment of the NMR data to predict anticonvulsant activity of the enaminones could not be established, general inferences were made. The NMR data confirmed that the enaminones existed predominantly in the amino tautomer, and no evidence was found for the imino tautomer. The ketamine form of the enaminones was supported by the observed spin-spin splittings of the NH with the alpha-protons on certain enaminones. The NH of secondary enaminones was very important in conferring anticonvulsant activity to the enaminones. The peak for the NH proton which could be seen between delta (ppm) 4.50 and 9.70 was present in all of the active enaminones. The tertiary enaminones, which were devoid of the NH proton, were uniformly inactive. It appeared that a combination of steric and electronic effects, lipophilicity, and hydrogen bonding were necessary for the anticonvulsant activity of the enaminones. The cyclic enaminones existed in the trans-S-trans fixed conformation, and the NMR data supported our hypothesis that enantioselectivity is retained in synthesizing enaminones from cyclic, diasteriomeric 1,3-diketones. In addition, the AB system and many unique features were observed in some enaminones. The para, meta, and ortho substituted patterns were observed for monosubstituted phenyl protons, and the NMR patterns for di- and trisubstituted phenyl groups were elucidated.

Parathyroid hormone (PTH) is the anabolic standard of care for patients with severe osteoporosis. The CaSR allosteric antagonist JTT-305/MK-5442, a PTH secretagogue, could offer an oral osteoanabolic treatment alternative for postmenopausal women with osteoporosis. Here we disclose the pharmacokinetic profile of JTT-305/MK-5442 and its activity on bone remodeling in ovariectomized (OVX) osteopenic rats. Daily treatments (0.3 to 2.4 mg/kg/d) for 12 weeks resulted in plateaued BMD increases (3.8 to 5.3%) at axial and appendicular skeletal sites. However, treatment effects were not statistically significant, in agreement with effects seen in animals treated with low dose PTH (1-84) (5 μg/kg/d). In a consecutive study we tested JTT-305/MK-5442 effects on bone formation in OVX-rats challenged with combined alendronate (ALN) treatment paradigms. At 7 month, JTT-305/MK-5442 treatment significantly increased BMD in lumbar vertebrae (LV), while no change in BMD was observed in femora or tibiae. ALN add-on co-treatment produced incremental increases in LV, distal femur (DF) and proximal tibia (PT) BMD over the respective ALN control. Histological analyses confirmed modest increases in mineralized surface (MS/BS) and bone formation rate (30.5±1.9%) on trabecular surfaces by JTT-305/MK-5442. As expected, ALN administration profoundly reduced bone formation, however, JTT-305/MK-5442 significantly stimulated MS/BS and BFR in ALN treated groups. In summary, JTT-305/MK-5442 acts as a PTH secretagogue in the osteopenic OVX-rat, eliciting consistent, though modest effects on remediation of BMD due to estrogen depletion. Induction of bone formation by JTT-305/MK-5442 at trabecular bone surfaces appears to be resilient to ALN-mediated suppression of bone formation. This study provides for the first time, a mechanistic evaluation of combination treatment of a PTH secretagogue with ALN.

Genetic studies have linked both osteoporotic and high bone mass phenotypes to low-density lipoprotein receptor-related proteins (LRP4, LRP5, and LRP6). LRPs are receptors for inhibitory Dickkopf-1 (DKK1) protein, and treatment modalities that modulate LRP/DKK1 binding therefore may act as stimulators of bone mass accrual. Here, we report that RH2-18, a fully human monoclonal anti-DKK1 antibody elicits systemic pharmacologic bone efficacy and new bone formation at endosteal bone surfaces in vivo in a mouse model of estrogen-deficiency-induced osteopenia. This was paralleled by partial-to-complete resolution of osteopenia (bone mineral density) at all of the skeletal sites investigated in femur and lumbar-vertebral bodies and the restoration of trabecular bone microarchitecture. More importantly, testing of RH2-18 in adult, osteopenic rhesus macaques demonstrated a rate-limiting role of DKK1 at multiple skeletal sites and responsiveness to treatment. In conclusion, this study provides pharmacologic evidence for the modulation of DKK1 bioactivity in the adult osteopenic skeleton as a viable approach to resolve osteopenia in animal models. Thus, data described here suggest that targeting DKK1 through means such as a fully human anti-DKK1-antibody provides a potential bone-anabolic treatment for postmenopausal osteoporosis.

Summary: The anticonvulsant and toxic properties of methyl 4-[(p-chlorophenyl)amino]-6-methyl-2-oxo-cyclohex-3-en-l-oate (ADD 196022), were compared with those of phenytoin (PHT), carbamazepine (CBZ), and valproate (VPA). These compounds were evaluated in mice and rats using well-standardized anticonvulsant testing procedures. Results indicate that ADD 196022 is a very potent anticonvulsant in the maximal electroshock seizure (MES) model. The compound was effective in nontoxic doses after intraperitoneal (i.p.) administration in mice and oral administration in rats. In mice, i.p. administration of ADD 196022 resulted in an ED50 value of 26.2 mg/kg as compared with a value of 6.48 mg/kg for PHT in the same assay. ADD 196022 was more potent that PHT in the oral rat model, having an ED50 value of 5.79 mg/kg as compared to 23.2 mg/kg for PHT. ADD 196022 was ineffective in nontoxic doses against all other seizure models evaluated and thus has a pharmacologic profile similar to that of PHT.

This report continues the in-depth evaluation of methyl 4-[(p-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-en-1-oate , 1 (ADD 196022), and methyl 4-(benzylamino)-6-methyl-2-oxocyclohex-3-en-1-oate, 2, two potent anticonvulsant enaminones. These compounds were evaluated employing the amygdala kindling model. Neither 1 nor 2 was active against amygdala kindled seizures, further supporting the corneal kindled model as a definitive tool for antielectroshock seizure evaluation as previously reported. Additional intraperitoneal (ip) data on 1 revealed toxicity at 24 h at 100 mg/kg. Several active analogs have been prepared with the view to minimizing toxicity. In a special ip rat screen developed by the Antiepileptic Drug Development (ADD) Program, these newer analogs were evaluated for protection against maximal electroshock seizures (MES) at 10 mg/kg and neurotoxicity at 100 mg/kg. From this screen, several compounds were shown to be safer alternatives, the most notable was methyl 4-[(p-bromophenyl)amino]-6-methyl-2-oxocyclohex-3-en-1-oate, 13. Compound 13 had an ip ED50 of 4 mg/kg in the rat and a TD50 of 269 mg/kg, providing a protective index (TD50/ED50) of > 67. By variation in the ring size, additional aromatic substitutions and the synthesis of acyclic analogs, these newer compounds provide a more definitive insight into the structure-activity correlation. CLOGP evaluation and molecular modeling studies are also provided to further elaborate the molecular characteristics of potential anticonvulsant enaminones.

This study is concerned with the development of a carbamazepine (CBZ):phospholipid (PL) solid dispersion formulation with improved dissolution characteristics. CBZ powders were blended with PL to produce CBZ:PL physical mixtures or made into solid dispersions with PL by the solvent method. CBZ exhibited significantly improved dissolution rates in PL coprecipitate (coppt) as compared to the physical mixtures or CBZ alone. Dissolution studies suggested that less than 10:1 ratio of CBZ to PL (9.1% PL) was required to disperse amorphous CBZ completely in the carrier. The coppt yielded a 3.6-fold greater initial dissolution rate (computed over the 5 min of dissolution) than the pure CBZ. Also, the total amount dissolved after 60 min was 2.1-fold greater at a CBZ:dimyristoylphosphatidylglycerol (DMPG) of 10:1 ratio (9.1% DMPG) than for the corresponding pure drug. Increasing the DMPG concentration to 4:1 (20% DMPG) compositions resulted in only a further 10% increase in the initial dissolution rate and 8.5% increase in the limiting concentration. Thus, a small amount of PL improved the dissolution of CBZ to a significant level. Some effect was also observed by changing the composition of the PLs. The coppts were formulated into tablets in order to compare the dissolution profile with that of TegretolR, a commercially available tablet of CBZ. It was found that the total amount dissolved after 60 min was two-fold higher in our tablet formulation than the commercial product, TegretolR. Powder X-ray diffraction spectra showed no changes in the diffraction patterns of CBZ in the coppt. It is concluded that CBZ:PL solid dispersions may have clinical advantages of quick in-vivo release to yield better bioavailability than existing commercial formulations.

Wnt/LRP5 signaling is a central regulatory component of bone formative and resorptive activities, and the pathway inhibitor DKK1 is a suppressor of bone formation and bone mass accrual in mice. In addition, augmented DKK1 levels are associated with high bone turnover in diverse low bone mass states in rodent models and disease etiologies in human. However, examination of the precise role of DKK1 in the normal skeleton and in higher species requires the development of refined DKK1-specific pharmacological tools. Here, we report the strategy resulting in isolation of a panel of fully human anti-DKK1 antibodies applicable to studies interrogating the roles of mouse, rhesus, and human DKK1. Selected anti-DKK1 antibodies bind primate and human DKK-1 with picomolar affinities yet do not appreciably bind to DKK2 or DKK4. Epitopes mapped within the DKK1 C-terminal domain necessary for interaction with LRP5/6 and consequently effectively neutralized DKK1 function in vitro. When introduced into naive normal growing female mice, IgGs significantly improved trabecular bone volume and structure and increased both trabecular and cortical bone mineral densities in a dose-related fashion. Furthermore, fully human DKK1-IgG displayed favorable pharmacokinetic parameters in non-human primates. In summary, we demonstrate here a rate-limiting function of physiologic DKK1 levels in the regulation of bone mass in intact female mice, amendable to specific pharmacologic neutralization by newly identified DKK1-IgGs. Importantly the fully human IgGs display a profile of attributes that recommends their testing in higher species and their use in evaluating DKK1 function in relevant disease models.

Previous results of anticonvulsant activity in several imidooxy carboxylates related to (aminooxy)acetic acid in young chicks, prompted an in-depth reinvestigation of these analogues in mice. A series of 22 succinimidooxy, phthalimidooxy, and naphthalimidooxy carboxylates were synthesized and evaluated for anticonvulsant activity by the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS). Methyl (succinimidooxy)acetate (2d), ethyl (succinimidooxy)acetate (2e), methyl (phthalimidooxy)acetate (3d), ethyl (phthalimidooxy)acetate (3e), and ethyl 2-(phthalimidooxy)propionate (3g), which were initially found to be active as anticonvulsants in young chicks were uniformly inactive in the Phase I seizure tests involving maximal electroshock (MES), pentylenetetrazol (scMet), or neurologic toxicity toxicity (Tox). Several newer analogues, ethyl (succinimidooxy)formate (2c) and methyl 3-(phthalimidooxy)-2-methylacrylate (4h) were found to be active in the scMet (3a) or both (4h) evaluations. Most interesting was the anticonvulsant results of N-(benzyloxy)-2-azaspiro[4,4] nonane-1,3-dione (5b), which displayed anti-MES activity and a protective index (TD50/ED50) of greater than 4.5.

A new series of novel enaminones has been synthesized from cyclic beta-dicarbonyl precursors which were condensed with morpholine, pyrrolidine, phenethylamine, hydrazines, substituted benzyl amines, and substituted anilines. These compounds were subsequently evaluated for anticonvulsant activity in a variety of anticonvulsant models by the National Institute of Neurological and Communicative Disorders and Stroke and in our laboratory. Several of these compounds exhibited potent anticonvulsant activity with a remarkable lack of neurotoxicity. The most active analog, methyl 4-[(p-chlorophenyl)amino]-6-methyl-2-oxo-cyclohex-3-en-1-oate++ + (27), was protective in the maximal electroshock (MES) seizure test in the rat with an oral ED50 of 5.8 mg/kg with no toxicity noted at doses up to 380 mg/kg, thus providing a protective index (TD50/ED50) of greater than 65.5. A similar protective index for 27 was noted upon intraperitoneal (ip) administration in mice. The anticonvulsant effect of 27 occurred within 15 min of administration and the compound remained active beyond 4 h. Compound 27 was also active in the rat corneal kindled model. The application of Free-Wilson analysis to structure-activity correlation in this series is discussed.

Summary: The pharmacokinetic and pharmacodynamic properties of the spiro carboxylic acid, spiro[4.6]un-decane-2-carboxylic acid (SUCA, ADD 93024), were investigated in rats and compared with those of the standard anticonvulsant carboxylic acid, valproate (VPA). The clearance of SUCA was dose-dependent, although the observed nonlinearity did not appear to be due to classical saturable elimination. The change in clearance across doses was consistent with end-product inhibition or cosubstrate depletion. The volume of distribution of the spiro compound also evidenced nonlinearity, possibly due to concentration-dependent binding to serum proteins. In contrast, the dose-dependent clearance displayed by VPA was composed of both saturable and non-saturable components. Furthermore, the disposition of VPA was characterized by a significant enterohepatic recirculation, whereas no such recirculatory process was apparent for SUCA. Both compounds afforded significant protection from pentylenetetrazol (PTZ)-induced seizures, and the time course of anticonvulsant effect did not correspond to that of drug concentrations in serum for either anticonvulsant. The apparent dissociation between the pharmacokinetics and pharmacodynamics of VPA may be a function of the mechanism of antiepileptic action and not due to the presence of active metabolites of the drug RESUME Les auteurs ont etudie chez le rat les caracteristiques pharma-cocinetiques et pharmacodynamiques de l'acide spiro(4–6) un-decane-2-carboxilique (SUCA, ADD 93024) et ont compare ces parametres a ceux de l'acide valproique (VPA). La clairance du SUCA a ete; dose-dependante, bien que l'absence de linearite observee ne semble pas etre due a une Elimination saturable classique. Les modifications de clairance entre les differentes doses semblaient correspondre a une inhibition du produit terminal ou a une depletion du co-substrate. Le volume de distribution du SUCA semble egalement avoir ete non lineaire, peutetre en raison d'une liaison concentration-dependante aux pro-twines seriques. En revanche, la clairance dose-dependante mise en evidence pour le VPA pouvait etre attribuee a la fois a des composantes saturables et non saturables. De plus, la disposition du VPA etait caracterisee par une re-circulation enterohepatique significative, alors qu'il n'y avait pas de tel processus de re-circulation pour le SUCA. L'un et l'autre de ces composes ont fourni une protection significative vis a vis des crises induites par pentylenetetrazol et, pour ces deux medicaments, l'evolution temporelle de l'efficacitE anticonvulsivante n'a pas correspondu aux taux sanguins seriques. Cette dissociation apparente entre pharmacocinetique et pharmacodynamique du VPA peut etre une fonction du mecanisme de l'action antiepileptique, et ne pas etre due a la presence de metabolite actif du medicament. RESUMEN Las propiedades farmacocineticas y farmacodinamicas del acido spiro carboxflico, del acido spiro [4.6] undecano-2-car-boxflico (SUCA, ADD 93024), han sido investigadas en la rata y los resultados comparados con el acido carboxflico anticonvulsivante standard, el acido valproico (VPA). El aclaramiento del SUCA era dosis˜dependiente a pesar de que la no-linearidad ob-servada no parece haber sido debida a la eliminacion saturable clasica. El cambio en el aclaramiento a travEs de las dosis fue consistente con una inhibicidn del fin del producto o con una depleci6n del co-sustrato. El volumen de la distribucion del compuesto spiro tambien evidenceo nolinearidad, posiblemente de-bido a un acoplamiento concentracion-dependiente a las pro-temas sericas. En contraste, el aclaramiento dosis-dependiente que mostro el VPA estaba compuesto de ambos componentes, el saturable y el nosaturable. Ademas la disposition del VPA se caracterizo por una recirculacion entero-hepatica significativa mientras que este proceso reciclatorio no era aparente en el caso del SUCA. Ambos compuestos ofrecen una proteccicm significativa con respecto a los ataques inducidos por el penlilentetra-zol y la evolucion en el tiempo del efecto anticonvulsivante no corresponde a la derivada de la concentracidn de las medica-ciones en el suero de ninguno de los dos anticonvulsivantes. La aparente disociacion entre la farmacodinamica y la farmacodinam-ica del VPA puede ser funcion de los mecanismos de accion anti-epileptica y no debida a la presencia de metabolitos activos de la citada droga. ZUSAMMENFASSUNG Pharmakokinetik und–dynamik von Spiro-Carboxylsaure, Spiro (4.6) Undecane-2-CarboxyUc Acid (SUCA, ADD 93024), wurden bei der Ratte untersucht und mit der Carboxylsaure Val-proat (VPA) verglichen. Die Clearance von SUCA war dosisab-hangig, wenngleich die beobachtete Nichtlinearitat nicht auf-grund einer klassischen Sattigungseliminationskinelik verursacht schien. Die Anderung der Clearance stand in Ubereinstimmung mit einer Endproduktinhibition oder einer Co-Substrat-verminderung. Das Verteilungsvolumen von SPIRO zeigte eben-falls cine Nichtlinearitat, moglicherweise aufgrund einer konzen-trations-abhangigen Bindung an Serum-Protein. Demgegenuber bestand die dosisabhangige Clearance von Valproat aus einer gesattigten und nichtgesattigten Komponente. Zusatzlich war die Verfugbarkeit von Valproat durch einen signifikanten, enterohe-patischen Kreislauf gekennzeichnct, der fur SUCA nicht ersich-tlich ist. Beide Substanzen boten einen signifikanten Schutz vor Pentylentetrazol-induzierten Anfallen, der zeitliche Verlauf der antikonvulsiven Wirkung stimmte far beide Substanzen nichl mit der Serum-Konzentration ilberein. Die offenskhtlicne Ditsozia-tion zwischen Pharmakokinetik und–dynamik von Valproat ist moglicherweise ein Ausdruck der antikonvulsiven Wirkung und nicht durch die Anwesenheit von aktiven Metaboliten bedingt.

Activation of parathyroid hormone 1 (PTH-1) receptors on vascular smooth muscle cells causes relaxation and decreases blood pressure in rats and humans. However, when PTH(1-84) and PTH(1-34) were injected in anesthetized rats, PTH(1-34) produced a greater decrease in blood pressure. This study quantified the dose-response relationship of the hypotensive response to intravenously injected PTH(1-84) and PTH(1-34) in conscious rats and assessed the role that the C-terminal region of PTH(1-84) played in the differences. Mean arterial pressure (MAP) decreased rapidly following injection of both peptides (0-100 nmol/kg) and reached a nadir at 1-2 minutes before increasing at a rate that was dose- and time-dependent. PTH(1-34) produced a greater hypotensive effect than PTH(1-84) at most doses tested and was significantly different from PTH(1-84) at 1-10 nmol/kg. The greatest difference in MAP decrease between PTH(1-84) and PTH(1-34) (24 and 35 mm Hg, respectively) occurred at 10 nmol/kg. Median effective dose (ED50) values for PTH(1-84) and PTH(1-34) were significantly different (5.9 and 1.3 nmol/kg, respectively). The C-terminal PTH fragments PTH(7-84), PTH(39-84), and PTH(53-84) did not affect MAP when injected alone (10 nmol/kg), nor did they influence the hypotensive response when given at a 10-fold molar excess in combination with PTH(1-84) or PTH(1-34) (1.4 nmol/kg). In conclusion, PTH(1-84) is a less potent but, because it induced the same maximum response, not a less efficacious hypotensive agent than PTH(1-34) when administered by bolus intravenous injection in conscious rats. We found no evidence to support the concept that the C-terminal region of PTH is responsible for this difference in potency.

The sulfate conjugate 2 of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide, a potential metabolite of the nephrotoxicant N-(3,5-dichlorophenyl)succinimide, is prepared from the 2-hydroxysuccinimide (1) by the reaction with chlorosulfonic acid in chloroform and ether mixture at −78°.

Due to the exceptional anticonvulsant activity displayed by substituted aniline enaminones, related pyridine derivatives and phenothiazines synthesised in our laboratories, the further investigation of various aromatic heterocycles was undertaken. Condensation of cyclic 1,3-diketo esters with 3-, and 5-aminoisoxazole derivatives led to a series of potent anti-maximal electroshock (MES) analogues, three of which occurred in the 3-amino series: ethyl ester (10), orally (po) active in rats [ED(50) 68.9 mg kg(-1), TD(50)500 mg kg(-1), protective index (PI = TD(50)/ED(50))49.6]; methyl ester (9), ED(50) 68.9 mg kg(-1) intraperitoneally (ip) in mice, TD(50)500 mg kg(-1), PI7.3, and tert-butyl ester (8), ED(50) 28.1 mg kg(-1) po in rats, TD(50)500 mg kg(-1), PI17.8. Sodium channel binding studies, as well as evaluations against pentylenetetrazol, bicuculline, and picrotoxin on isoxazole 10 were all negative, leading to an unknown mechanism of action. X-ray diffraction patterns of a representative of the 3-amino series (isoxazoles 6-11) unequivocally display the existence of intramolecular hydrogen bonding of the nitrogen to the vinylic proton in the cyclohexene ring, providing a pseudo three ring structure which was also shown previously with the vinylic benzamides. Physicochemical-permeability across the BBB suggested an efflux mechanism for the previously synthesised aniline enaminones, but not with isoxazole 10.

Previous in vitro studies evaluating the permeability of enaminones suggested that their blood–brain barrier (BBB) transport might be influenced by the presence of an efflux mechanism. Therefore, transport mechanisms responsible for these anticonvulsants across the BBB were examined. The transport of enaminones (1×10 −4 M) were evaluated over 120 min with verapamil (50 μM) and probenecid (100 μM) using bovine brain microvessel endothelial cells (BBMECs) to assess the role of multidrug resistant (MDR) transport proteins [i.e., P ‐glycoprotein (Pgp) and MDR protein 1 (MRP1)] on efflux, respectively. Uptake studies in the presence and absence of rhodamine 123 (R123; 3.2 and 5.0 μM) were also performed in a Pgp overexpressing cell line, MCF‐7/ Adr . Select enaminone esters (12.5 mg/kg) were administered intravenously to mdr 1 a/b (+/+), mdr 1 a/b (−/−) knockout and probenecid pretreated mice (20 ± 5g). Enaminones and R123 were assayed with validated ultraviolet and fluorescence high‐performance liquid chromatography methods, respectively. Verapamil and probenecid significantly ( p >0.05) inhibited the transport of select enaminone esters across BBMECs. Two enaminones caused a statistically significant increase in the uptake of R123 in MCF‐7/ Adr cells. Concentrations of select enaminones in mdr 1 a/b (−/−) mice brains were significantly higher ( p mdr 1 a/b (+/+) mice brains; however, no differences were observed in probenecid pretreated animals. Taken together, these results strongly suggest that Pgp may influence enaminone transport at the BBB and hence affect epilepsy treatment with these agents. © 2001 Wiley‐Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1540–1552, 2001

In a continuing evaluation of the aniline-substituted enaminones, the synthesis of additional para-substituted analogs has been made in an attempt to further quantify the electronic (sigma) and lipophilic (pi) requirements for anticonvulsant activity in this series. In addition, meta- and ortho-substituted and polysubstituted compounds have been synthesized and evaluated for anticonvulsant activity. In the para-substituted series, 4-cyano analogs (32 and 33) (+ sigma, - pi), which were highly active via intraperitoneal (ip) injection in mice, were inactive on oral (po) administration in rats. The para-substituted trifluoromethoxy (+ sigma, + pi) analog (8) had significant potency by both routes. Meta substitution limited the activity due to steric factors. Bromo and iodo substituents produced active para-substituted analogs (5 and 17) but were inactive when substituted in the meta position (37 and 41, respectively). Ortho substitution provided no clear relationship due to nonparametric deviations. Neither 1, the prototype enaminone, nor 2, the putative metabolite, produced significant nephrotoxicity or hepatotoxicity. Sodium channel binding of 1 and 8 indicated that 8 displayed relatively potent sodium channel binding but 1 showed weaker effects with IC50 values of 489 and 170 microM respectively against [3H]batrachotoxinin A 20 alpha-benzoate ([3H]BTX-B).

Valproic acid, an antiepileptic drug, is extensively metabolized in humans. Two putative metabolites, 2-n-propyl-3-aminopentanoic acid (3-aminovalproic acid, 3-amino-VPA; 2a) and 2-n-propyl-4-aminopentanoic acid (4-amino-valproic acid, 4-amino-VPA; 4a), which may result from the transamination of the respective keto acids 1a and 3a may explain the unusual extended seizure protection elicited by valproic acid. The title compounds were synthesized as their diasteriomeric ethyl esters 2b and 4b and submitted for anticonvulsant evaluation by the Antiepileptic Drug Development Program of the National Institute of Neurological and Communicative Disorders and Stroke. The results verified our hypothesis, as 4b was active in the subcutaneous pentylenetetrazol (scMet) evaluation at 30 mg/kg. Both compounds were highly toxic at 300 mg/kg.

The anticonvulsant and toxic properties of methyl 4-[(p-chlorophenyl)amino]-6-methyl-2-oxocyclohex-3-en-1-oate (ADD 196022), were compared with those of phenytoin (PHT), carbamazepine (CBZ), and valproate (VPA). These compounds were evaluated in mice and rats using well-standardized anticonvulsant testing procedures. Results indicate that ADD 196022 is a very potent anticonvulsant in the maximal electroshock seizure (MES) model. The compound was effective in nontoxic doses after intraperitoneal (i.p.) administration in mice and oral administration in rats. In mice, i.p. administration of ADD 196022 resulted in an ED50 value of 26.2 mg/kg as compared with a value of 6.48 mg/kg for PHT in the same assay. ADD 196022 was more potent that PHT in the oral rat model, having an ED50 value of 5.79 mg/kg as compared to 23.2 mg/kg for PHT. ADD 196022 was ineffective in nontoxic doses against all other seizure models evaluated and thus has a pharmacologic profile similar to that of PHT.

A new series of novel enaminones has been synthesized from cyclic beta-dicarbonyl precursors which were condensed with morpholine, pyrrolidine, phenethylamine, hydrazines, substituted benzyl amines, and substituted anilines. These compounds were subsequently evaluated for anticonvulsant activity in a variety of anticonvulsant models by the National Institute of Neurological and Communicative Disorders and Stroke and in our laboratory. Several of these compounds exhibited potent anticonvulsant activity with a remarkable lack of neurotoxicity. The most active analog, methyl 4-[(p-chlorophenyl)amino]-6-methyl-2-oxo-cyclohex-3-en-1-oate++ + (27), was protective in the maximal electroshock (MES) seizure test in the rat with an oral ED50 of 5.8 mg/kg with no toxicity noted at doses up to 380 mg/kg, thus providing a protective index (TD50/ED50) of greater than 65.5. A similar protective index for 27 was noted upon intraperitoneal (ip) administration in mice. The anticonvulsant effect of 27 occurred within 15 min of administration and the compound remained active beyond 4 h. Compound 27 was also active in the rat corneal kindled model. The application of Free-Wilson analysis to structure-activity correlation in this series is discussed.

Vertically oriented suborbicular nerve fibers are frequently encountered during upper eyelid surgery. It has not been well established whether these fibers are terminal motor branches of the facial nerve (VII) or sensory branches of the ophthalmic nerve (V-1). To investigate the origin of these nerve fibers, three different techniques were used: (1) intraoperative nerve stimulation; (2) cadaver dissection; and (3) immunohistochemical analysis. The results of all three investigative methods are in agreement and conclusively demonstrate that these fibers represent sensory branches of the ophthalmic nerve (V-1) and not motor branches of the facial nerve (VII). Anatomical dissection showed that the sensory fibers to the upper eyelid do not travel solely within the suborbicular fascial plane, but also course in the preorbicular plane and within the orbicularis muscle itself. The terminal branches of the facial nerve to the upper eyelid innervate the orbicularis oculi from the undersurface and in a horizontal orientation. Clinical correlation of these findings is discussed.

A positive temporal artery biopsy is required to conclusively establish the diagnosis of temporal arteritis. The temporal artery biopsy technique we describe is based on the anatomical branching patterns of the superficial temporal artery, the various fascial layers, and the location of the temporal branches of the facial nerve. Special emphasis is placed on avoiding facial nerve trauma during a biopsy of the frontal branch of the superficial temporal artery. Proper surgical technique combined with a working knowledge of the anatomy of the temporalis region enhances the safety of a temporal artery biopsy procedure.

Previous results of anticonvulsant activity in several imidooxy carboxylates related to (aminooxy)acetic acid in young chicks, prompted an in-depth reinvestigation of these analogues in mice. A series of 22 succinimidooxy, phthalimidooxy, and naphthalimidooxy carboxylates were synthesized and evaluated for anticonvulsant activity by the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS). Methyl (succinimidooxy)acetate (2d), ethyl (succinimidooxy)acetate (2e), methyl (phthalimidooxy)acetate (3d), ethyl (phthalimidooxy)acetate (3e), and ethyl 2-(phthalimidooxy)propionate (3g), which were initially found to be active as anticonvulsants in young chicks were uniformly inactive in the Phase I seizure tests involving maximal electroshock (MES), pentylenetetrazol (scMet), or neurologic toxicity toxicity (Tox). Several newer analogues, ethyl (succinimidooxy)formate (2c) and methyl 3-(phthalimidooxy)-2-methylacrylate (4h) were found to be active in the scMet (3a) or both (4h) evaluations. Most interesting was the anticonvulsant results of N-(benzyloxy)-2-azaspiro[4,4] nonane-1,3-dione (5b), which displayed anti-MES activity and a protective index (TD50/ED50) of greater than 4.5.

Summary: Inheritance of resistance to simazine was investigated in reciprocal pair crosses of resistant and susceptible genotypes of Senecio vulgaris. The simazine resistant genotypes had been selected in a screening experiment and were unaffected by 2.8 kg ha−1˜’a.i. simazine. Reciprocal differences for resistance and susceptibility to simazine in F2 families showed that resistance is maternally inherited. The practical implication of maternal inheritance is that a resistant field population will build up very rapidly from a very low initial frequency of the resistant genotype. Resume: Transmission hereditaire, par la voie maternelle, de la resistance â la simazine dans une population de Senecio vulgaris La transmission hereditaire de la resistance a la simazine a eteetudiee dans des paires de croisements reciproques entre genotypes resistants et sensibles de Senecio vulgaris. Les genotypes resistants avaient ete selectionnes experimentalement et n'etaient pas endommages par 2.8 kg/ha m.a. de simazine. Les differences reciproques quant a la resistance et a la sensibilitea la simazine dans les familles F2 ont montre que la resistance etait transmise par heredite maternelle. L'application pratique de cette transmission par la voie maternelle est qu'une population resistante se constitue tres rapidement au champ, a partir d'une tres faible frequence initiale du genotype resistant. Zusammenfassung Maternale Vererbung der Simazinresistenz in einer Population von Senecio vulgaris Die Vererbung der Simazinresistenz wurde durch reziproke Parchenkreuzung mit resistenten und empfindlichen Genotypen von Senecio vulgaris untersucht. Die gegenuber Simazin resistenten Genotypen wurden in einem Screening-Verfahren selektiert und blieben durch 2.8 kg/ha Aktivsubstanz Simazin unbeeinflusst. Reziproke Unterschiede in der Resistenz oder Empfindlichkeit gegenuber Simazin in den F2-Generation zeigten, dass die Resistenz maternal vererbt wird. Die praktische Konsequenz maternaler Vererbung ist, dass sich selbst bei sehr geringem Vorkommen resistenter Genotypen sich rasch eine resistente Feldpopulation aufbaut.

In a four-tank recirculation system serviced by a single biological filter, hydrocyclone prefilters removed over 87% of the particulate matter greater than 77 μm (3 x 10-3 in) in diameter. Biological nitrification occurred on a substrate of 25 mm (1 in) diameter, Actifil (R) polypropylene biorings contained in a 0.29-m3 (10-ft3) fiberglass tank. The four 0.9 m3 (32-ft3) tanks were stocked with 1200 rainbow trout (Salmo gairdneri), averaging 66.9 g (0.148 lb) each. Fish were thinned every 2 weeks to maintain approximately constant density. During the first 62 days of the 248-day test when the filter was being conditioned, the fish had essentially no growth. Over the last 186 days the average weight increased from 71.4 to 294.1 g (0.157 to 0.648 lb) resulting in a specific growth rate of 0.76%/day. Recirculation and makeup water rates were 130 and 5 L/minute (34 and 1.3 gal/minute), respectively. Chlorinated makeup water was passed through a small ultraviolet irradiator where chlorine was oxidized ...

Particulate filtration was achieved with hydrocyclones and biological nitrification occurred on a substrate of polypropylene biorings (40 mm in diameter) in a 175-L fiberglass tank. Recirculation and makeup water rates were 150 and 5.0 L/min, respectively. In a 1,700-L tank, 261 fish increased in average weight from 92.1 to 330.0 g over a 23-week test period, resulting in a compound weekly weight gain of 5.5%. At the end of the test, total weight of fish in the tank was 84.5 kg for a loading density of 49.7 kg/m³. Cumulative feed conversion ratio was 1.41 g of feed per gram of weight gain. Ammonium-nitrogen and nitrite-nitrogen, temperature, dissolved oxygen, and pH were satisfactorily controlled. Hydrocyclone prefliters proved highly effective in removing particulates, thereby overcoming a major complication of earlier systems by lengthening the biological filter backwash intervals to more than 3 weeks, and requiring only 300 L of water per backwash.

A coldroom complex recently installed at the Fisheries Research Board of Canada, Freshwater Institute laboratory at Winnipeg features several design aspects that are considered novel. These include foamed-in-place urethane insulation, two alternating R-502 refrigeration systems incorporating automatic safety switch-over and adjustable defrost, "straight-line" pneumatic temperature control, hot gas bypass control, and a master panel. The facility combines a cold laboratory at +2 C, a long-term storage room at −37 C, a small anteroom at −26 C, and a room containing eight 10.0-ft3 precise temperature cabinets at −40 C. Room temperature variation is ±0.25 degrees C during steady state conditions. Temperature rise during daily defrosting is less than 2 degrees C for a duration of 1 hr.

The use of a compact recirculation system for rearing rainbow trout is described. An 8-month experiment showed that up to 75 kg offish could be maintained in a healthy condition in a 1600-liter tank using a make-up water rate of only 1.0 liter/min. Temperature, oxygen, pH, ammonia nitrogen, and nitrate nitrogen levels were satisfactorily controlled. An average weight gain of 8% per week was achieved with a feed rate of 2% of body weight per day. Fish mortality was insignificant.

A controlled environment cabinet for the study of insects or plant–insect interactions which has a novel air velocity control mechanism is described. The useful working space is 0.56 × 0.84 × 1.83 m long and the temperature in this area can be selected from 18° to 35°C at air velocities ranging from 1.5 to 18.0 km/hr. Fluorescent lamps provide a light intensity of 32,300 lm/m2, 0.32 m below the lamps.Temperatures were controlled within ±0.67°C and the air velocity was uniform within 20% throughout the working area.

Efficiencies of aeration of five different combinations of Venturis, air stones, and sprays were measured in a 1.8-m3 tank. Results showed that the venturi method discharging through a nozzle near the tank bottom had the highest efficiency. Air stones were 53% as effective and sprays directed at the surface 41% as effective as the venturi method. Details are given for fabrication of the PVC (polyvinyl chloride) venturi. The unit is reliable, inexpensive, and eliminates the need for a separate air supply.

not available

A peripunctal nevus is a rare lesion of the eyelid margin, six examples of which are included in this report. The lesions all involved the lower punctum and had been present for many years without producing epiphora. In addition to their variable clinical pigmentation and translucent appearance on biomicroscopy, all shared several other diagnostically useful clinical features: an overall dome-shape with a fine micronodularity; a prominent ramifying vascularity; and, most importantly, circumferentially swollen punctal lips that created a slit-like punctal orifice. Histopathologically, the lesions were predominantly or exclusively subepithelial melanocytic nevi and were demonstrated not to compress the punctum or canaliculus. Infiltration of the nevus cells within the orbicularis striated muscle fibers was a common finding owing to the latter's superficial location in the eyelids. If patients request excision for cosmetic reasons, these lesions are best managed by a horizontal circular shave excision with the temporary placement of a silicon punctual plug. The differential diagnosis includes cyst, papilloma, melanoma, and basal cell carcinoma.