The effects of an oral preparation containing hyaluronic acid (Oralvisc®) on obese knee osteoarthritis patients determined by pain, function, bradykinin, leptin, inflammatory cytokines, and heavy water analyses.

The purpose of this study was to determine the effects of an oral preparation containing hyaluronic acid on osteoarthritic knee joint pain and function as well as changes in inflammatory cytokines, bradykinin, and leptin. We also used heavy water to determine the turnover rates of glycosaminoglycans in synovial fluid. This was a double-blind, randomized, placebo-controlled study of 40 subjects over a period of 3 months. Visual analog scale, Western Ontario McMaster pain, and WOMAC function scores were recorded. Serum and synovial fluid were measured by enzyme-linked immunosorbent assays for inflammatory cytokines, bradykinin, and leptin. In 20 subjects, terminal heavy water ingestion was used for spectral analyses of serum and joint fluid samples. There were statistically significant improvements in pain and function. Both serum and synovial fluid samples showed significant decreases for a majority of inflammatory cytokines, leptin, and bradykinin in the oral hyaluronic acid preparation group. Heavy water analyses revealed a significant decrease in hyaluronic acid turnover in the synovial fluid of the treatment group. A preparation containing hyaluronic acid and other glycosaminoglycans holds promise for a safe and effective agent for the treatment for patients with knee osteoarthritis and who are overweight. Further studies will be required to see whether this is a disease-modifying agent.

Oral bioavailability of P-glycoprotein substrate drugs do not differ between ABCB1-1Δ and ABCB1 wild type dogs.

Previous studies have indicated that intestinal P-glycoprotein (P-gp) limits the oral bioavailability of substrate drugs and alters systemic pharmacokinetics. In this study, dogs lacking functional P-gp were used to determine the contribution of P-gp to the oral bioavailability and systemic pharmacokinetics of several P-gp substrate drugs. The P-gp substrates quinidine, loperamide, nelfinavir, cyclosporin and the control (non P-gp substrate) drug diazepam were individually administered intravenously and per os to ABCB1-1Δ dogs, which have a P-gp null phenotype and ABCB1 wildtype dogs. ABCB1-1Δ dogs have been shown to have greater brain penetration of P-gp substrates, but limited information is available regarding oral bioavailability of P-gp substrate drugs in this animal model. Plasma drug concentration vs. time curves were generated and pharmacokinetic parameters were calculated for each drug. There were no differences in oral bioavailability between ABCB1-1Δ dogs and ABCB1 wildtype dogs for any of the drugs studied, suggesting that intestinal P-gp does not significantly affect intestinal absorption of these particular substrate drugs in ABCB1-1Δ dogs. However, small sample sizes and individual variability in CYP enzyme activity may have affected the power of the study to detect the impact of P-gp on oral bioavailability.

Understanding the clinical pharmacokinetics of a GABAA partial agonist by application of in vitro tools.

1. 4-Oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxylic acid (4-methylaminomethyl-phenyl)-amide (1), developed for general anxiety disorder, was discontinued from clinical development due to unsuitable oral pharmacokinetics. 2. In humans, (1) demonstrated an unacceptable high apparent oral clearance (Cl(p)/F) that also demonstrated a supraproportional dose-exposure relationship. Secondary peaks in the plasma concentration-time profile suggested possible enterohepatic recirculation of (1). A combination of in vitro mechanistic tools was applied to better understand the processes underlying these complex clinical pharmacokinetic profiles of (1). 3. In metabolism experiments, (1) was shown to be a substrate of monoamine oxidase A (MAO-A) as well as being metabolized by cytochrome P450. The former appeared to be a high K(M) process with a high capacity, while the latter showed saturation between 1 and 10 microM, consistent with the supraproportional dose-exposure relationship. 4. In a sandwich-cultured hepatocyte model, (1) was shown to be a substrate for both uptake and efflux into the canicular space, which is consistent with the observation of pharmacokinetics suggestive of enterohepatic recirculation. Finally, in human epithelial colon adenocarcinoma cell line (Caco-2) and Madin-Darby canine kidney cells transwell flux experiments, (1) was shown to have relatively low permeability and a basolateral-to-apical flux ratio consistent with the activity of P-glycoprotein. 5. In combination, a compounding of the contributions of MAO-A, hepatic uptake and efflux transporters, and P-glycoprotein to the disposition of (1) may underlie the low oral exposure, saturable clearance, and aberrant concentration versus time profiles observed for this compound in humans.

Preclinical characterization of selective phosphodiesterase 10A inhibitors: a new therapeutic approach to the treatment of schizophrenia.

We have recently proposed the hypothesis that inhibition of the cyclic nucleotide phosphodiesterase (PDE) 10A may represent a new pharmacological approach to the treatment of schizophrenia (Curr Opin Invest Drug 8:54-59, 2007). PDE10A is highly expressed in the medium spiny neurons of the mammalian striatum (Brain Res 985:113-126, 2003; J Histochem Cytochem 54:1205-1213, 2006; Neuroscience 139:597-607, 2006), where the enzyme is hypothesized to regulate both cAMP and cGMP signaling cascades to impact early signal processing in the corticostriatothalamic circuit (Neuropharmacology 51:374-385, 2006; Neuropharmacology 51:386-396, 2006). Our current understanding of the physiological role of PDE10A and the therapeutic utility of PDE10A inhibitors derives in part from studies with papaverine, the only pharmacological tool for this target extensively profiled to date. However, this agent has significant limitations in this regard, namely, relatively poor potency and selectivity and a very short exposure half-life after systemic administration. In the present report, we describe the discovery of a new class of PDE10A inhibitors exemplified by TP-10 (2-{4-[-pyridin-4-yl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-quinoline succinic acid), an agent with greatly improved potency, selectivity, and pharmaceutical properties. These new pharmacological tools enabled studies that provide further evidence that inhibition of PDE10A represents an important new target for the treatment of schizophrenia and related disorders of basal ganglia function.

Degradation of the cartilage collagen matrix associated with changes in chondrocytes in osteoarthrosis. Assessment by loss of background fluorescence and immunodetection of matrix components.

Articular cartilage damage and eventual loss is the primary pathological change seen in osteoarthrosis (OA). In this study we have investigated the link between turnover of the collagen matrix and changes in chondrocytes. The background fluorescence of articular cartilage, as indicated by its emission spectrum and resistance to extraction was generated by the slow non-enzymic modification of the collagen matrix by advanced glycation end products (AGEs). Assessment of changes in background fluorescence in sections of articular cartilage provided a narrative of collagen degradation. Patients without OA pathology typically had a uniform strong background fluorescence throughout the depth of the cartilage. Cartilage from OA patients showed a range of changes in background fluorescence dependent on depth from the articular surface and proximity to overt lesions. Loss of background fluorescence was centered on chondrocytes, more extensive near the surface and associated with detection of the proteoglycan epitope 7D4. Expression of type X collagen was seen in articular cartilage in the region of the interface of with subchondral bone in most OA patients but was not associated with prominent, pericellular, loss of background fluorescence. These observations are consistent with progressive cartilage damage in OA, whereby collagen turnover and loss of surface integrity is associated with chondrocyte activity similar to that seen in immature articular cartilage.

Iliac crest bone-muscle transfer in periacetabular osteotomy.

Ganz et al reported acetabular osteotomy for extensive acetabular reorientation without changing the acetabular diameter. This article reports our modification of this surgical technique, which preserves the integrity of the tensor fascia latea muscle as well as providing viable bone for bone graft at the anterior iliac osteotomy site. The interval between the sartorius and tensor fascia lata is developed distal to the anterosuperior iliac spine. After iliac osteotomy through the anterosuperior iliac spine, the distal portion of the anterosuperior iliac spine (acetabular segment), along with its slip of tensor fascia lata (1-2 cm), is separated and shaped to fit the anterior bone gap. Two small holes are drilled into that segment and also into the distal cut surface in the anterior portion of the remaining crest so that when the sutures are tightened, the tensor muscle is preserved anatomically and bone grafting is achieved.

Sulphation heterogeneity in the trisaccharide (GalNAcSbeta1, 4GlcAbeta1,3GalNAcS) isolated from the non-reducing terminal of human aggrecan chondroitin sulphate.

We report here the isolation and sulphation isomer analyses of trisaccharides GalNAcS(beta1,4)GlcA(beta1,3)GalNAcS (in which S indicates sulphate) derived from the non-reducing termini of aggrecan chondroitin sulphate. Rat chondrosarcoma and human aggrecans were digested for 1 h at 37 degrees C with 30 micro-units of endo-chondroitinase ABC per microgram of chondroitin sulphate, and trisaccharides were isolated from the digests by ToyoPearl HW40S gel-filtration chromatography. Four trisaccharide species were identified; their sulphation isomer compositions, as determined by digestion with chondroitinase ACII and fluorescence-based ion-exchange HPLC, were GalNAc4Sbeta1,4GlcAbeta1,3GalNAc4S, GalNAc4Sbeta1,4GlcAbeta1,3GalNAc6S, GalNAc4,6Sbeta1,4GlcAbeta1, 3GalNAc4S and GalNAc4,6Sbeta1,4GlcAbeta1,3GalNAc6S. The abundances of such sequences in chondroitin sulphate on aggrecan from normal (foetal to 72 years of age) and from osteoarthritic human knee cartilages were also established. The results showed that non-reducing terminal GalNAc4S or GalNAc4,6S can be linked to either a 4-sulphated or a 6-sulphated disaccharide, suggesting that the sulphation of the last disaccharide might not have a direct effect on the specificity of chondroitin sulphate terminal GalNAc sulphotransferases. Furthermore, for each aggrecan preparation examined, the 4S-to-6S ratio of all chain interior disaccharides was equivalent to that in the last repeating disaccharides at the non-reducing terminus, suggesting that neither chondroitin 4-sulphotransferase nor chondroitin 6-sulphotransferase shows preferential activity near the chain terminus.

Glycosaminoglycan sulfation in human osteoarthritis. Disease-related alterations at the non-reducing termini of chondroitin and dermatan sulfate.

Chondroitin lyase products of aggrecan and small proteoglycans from normal and osteoarthritic cartilages were analyzed for chain internal Deltadisaccharides and terminal mono- or disaccharides. Chondroitin and dermatan sulfate chains from arthritic cartilages were of essentially normal size and internal sulfation but had significantly altered sulfation of the terminal residues. Whereas in normal cartilage, approximately 60% of terminal GalNAc4S was 4, 6-disulfated, it was reduced to approximately 30% in osteoarthritic cartilage. This is most likely due to a lower terminal GalNAc4, 6S-disulfotransferase activity and reveals that metabolic changes in osteoarthritis can affect this distinct sulfation step during chondroitin and dermatan sulfate synthesis. GlcAbeta1,3GalNAc6S-, the mimotope for antibody 3B3(-), was present on approximately 8 and approximately 10% of chains from normal and osteoarthritic cartilages, respectively. 3B3(-) assayed by immunodot blot was within the normal range for most osteoarthritic samples, with only 5 of 24 displaying elevated reactivity. This resulted not from a higher content of mimotope, but possibly from other structural changes in the proteoglycan that increase mimotope reactivity. In summary, chemical determination of sulfation isomers at the non-reducing termini of chondroitin and dermatan sulfate provides a reliable assay for monitoring proteoglycan metabolism not only during normal growth of cartilage but also during remodeling of cartilage in osteoarthritis.

Tolerance of the planarian Dugesia tigrina (Tricladida: Turbellaria) to pesticides and insect growth regulators in a small-scale field study.

Two insect growth regulators, methoprene and a benzyl-1,3,benzodioxole (J-2931), had no detrimental effects on Dugesia tigrina under field conditions. Three other compounds, resmethrin, temephos, and cyromazine, had only minimal effects. Asexual multiplication among these planarian predators exceeded 68% when combined with Culex quinquefasciatus larvae and methoprene at different concentration levels. Also, this combined treatment with D. tigrina and methoprene resulted in high level (98.9%) reduction of Cx. quinquefasciatus populations through the 7-wk field study.

Disposition of acrivastine in the male beagle dog.

Three male beagle dogs were given 10 mg/kg iv and oral doses of [14C]acrivastine, a novel nonsedating antihistaminic agent, in a nonrandomized crossover experiment. Urine and feces were collected for 72 hr after dosing. After iv dosing, a mean of 34% was recovered in the urine, and 63% was recovered in the feces. After po dosing, a mean of 29% of the radiocarbon was recovered in the urine, and 63% was recovered in the feces (dose adjusted for 14% lost in vomitus). Acrivastine and three major metabolites were detected in the excreta. The metabolites were identified as a side-chain-reduced analog of acrivastine (metabolite 3, 270C81), a gamma-aminobutyric acid analog of 270C81 (metabolite 2), and a benzoic acid analog of 270C81 (metabolite 1). After iv dosing, 34% of the dose was excreted as parent drug, 21% as metabolite 3, 15% as metabolite 2, and 6% as metabolite 1, while after po dosing, 35% of the dose was excreted as parent drug, 18% as metabolite 3, 11% as metabolite 2, and 7% as metabolite 1. Pharmacokinetic analysis of acrivastine plasma concentration-time curves after both routes of administration indicated a mean total body clearance of 17.3 ml/min/kg, a Vss of 0.93 liter/kg, a terminal half-life of 0.7 hr, and an oral bioavailability of 40%. The apparent plasma half-life of the metabolite, 270C81, was 1.5 hr. Analysis of AUC values indicated that greater amounts of 270C81 than acrivastine circulated in plasma after both iv and po dosing, and that first-pass metabolism of acrivastine to 270C81 occurred. The results indicated that acrivastine was extensively metabolized in the dog to 270C81 and suggested that 270C81 itself underwent further metabolism to metabolites 1 and 2.

Orthopedic history and examination in the etiology of overuse injuries.

Overuse injuries, and stress fractures in particular, afflict many runners and military recruits. This investigation sought to identify pretraining factors which may predispose to overuse injuries. Orthopedic and running history questionnaires and an orthopedic examination were administered to 505 trainees entering an intensive military training school. A novel method for evaluating ankle dorsiflexion was developed, and alignment measures, in units of centimeters rather than degrees, were obtained. Over 10% of the trainees were removed from the school for overuse-related injuries, and over half of these were tibial stress fractures. The incidence of clinically diagnosed stress fractures was 6.3%. No single orthopedic history question or combination of questions could discriminate between trainees who did or did not subsequently incur overuse injuries. Results from the running history indicated that those running 25 or more miles.wk-1 (mpw) had a significantly (P less than 0.027) lower incidence of stress fractures (3.0%) than those running 4 or fewer mpw over the previous year (11.5%). The orthopedic examination did not identify any predisposing alignment characteristics, perhaps due to the low incidence of overuse injuries. Population means are presented for future use in comparative studies.

Compatibility of Metarhizium anisopliae var. anisopliae with chemical pesticides.

The effects of various insecticides on the mycelial growth, sporulation and conidial germination of Metarhizium anisopliae var. anisopliae isolate E9 were studied in the laboratory. Chlorpyrifos was the most toxic organophosphate to mycelial growth and sporulation at all concentrations. Temephos, malathion and leptophos were highly toxic to sporulation while malathion was the most inhibitory to germination. The carbamates, carbofuran, methomyl and oxamyl were moderately toxic to mycelial growth and sporulation while oxamyl had an adverse effect on germination. The pyrethroids (pyrethrin, permethrin and resmethrin) and the insect growth regulators (diflubenzuron and methoprene) were not inhibitory to the various developmental stages of isolate E9. The chlorinated hydrocarbons (chlordane, lindane and toxaphene) were more deleterious than all other insecticide groups tested. Among the fungicides, benomyl and maneb produced the greatest inhibition.

A laboratory study of cyromazine on Aedes aegypti and Culex quinquefasciatus and its activity on selected predators of mosquito larvae.

In a laboratory study, the insect growth regulator, cyromazine, exerted a high level of biological activity on Aedes aegypti and Culex quinquefasciatus treated in the 4th larval instar. At 1.5 and 1.0 ppm this IGR produced 97 and 99% inhibition of emergence in adult Ae. aegypti, respectively. In Cx. quinquefasciatus, there was 99% inhibition at 1 ppm and complete inhibition at 1.5 ppm. The overall pupal mortality was higher than larval or adult stages of both species. This material induced different types of morphogenetic abnormalities in pupae and adults of the 2 species similar to those induced by other IGRs. However, most abnormalities were observed in the pupal stage. Adverse effects were not detected on the 4 mosquito predator species during the acute or posttreatment tests.

Anti-estrogenic action of chlordecone in rat pituitary gonadotrophs in vitro.

Estradiol-17 beta (E2) enhances the response of rat pituitary gonadotrophs to gonadotrophin-releasing hormone (GnRH) in vitro. This effect of E2 on rat gonadotrophs in vitro was applied as a model to study the estrogenic or anti-estrogenic activity of chlordecone in gonadotrophs. Rat pituitary cell cultures were treated with E2 (10(-10) M), chlordecone (10(-7) to 10(-5) M), or both before a 6-hr challenge of D-Lys6-GnRH (GnRH-A). Secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) during the 6-hr of GnRH-A treatment was measured. Pretreatment of cells with E2 for 2 days increased the GnRH-A-stimulated LH and FSH secretion approximately twofold. This effect of E2 was antagonized by the coexistence of chlordecone in a dose-dependent manner. The effect of E2 (10(-10) M) could be totally abolished by 10(-6) M chlordecone. Further, chlordecone alone had little or no effect on basal secretion of LH and FSH, but it significantly suppressed the response of gonadotrophs to GnRH-A. [Mirex, a compound with a similar chemical structure to chlordecone, did not suppress GnRH-A-stimulated secretion of LH or FSH under any condition tested.] The present study demonstrated that chlordecone had two specific effects on rat pituitary gonadotrophs: (1) to antagonize the effects of estrogen, and (2) to reduce the response of gonadotrophs to GnRH-A. Thus, chlordecone is not estrogenic in rat pituitary cells in vitro.

alpha-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, inhibits tumor promoter-induced polyamine accumulation and carcinogenesis in mouse skin.

The role of ornithine decarboxylase (OrnDCase, EC 4.1.1.17) and of the polyamines [putrescine (Put), spermidine (Spd), and spermine (Spm)] in mouse skin tumor promotion was investigated by the use of alpha-difluoromethylornithine (CHF2-Orn), an enzyme-activated irreversible inhibitor of OrnDCase. 12-O-Tetradecanoylphorbol 13-acetate (TPA), mezerein, and ethyl phenylpropiolate (EPP) were employed as complete, stage II specific, and nonpromoting agents, respectively. TPA and mezerein, but not EPP, provided for a dose-dependent increase in tissue Put accumulation. The Put level in papillomas developed by TPA (2 micrograms) treatment was approximately equal to 15-fold higher than that of the surrounding skin tissue; Spd accumulation was 2- to 3-fold greater in the papillomas. Put administered (intraperitoneally) with TPA greatly enhanced papilloma yield. CHF2-Orn, given orally or intraperitoneally, abolished the TPA-induced OrnDCase activity and Put accumulation in mouse epidermis. The reduction of polyamine accumulation by CHF2-Orn was directly proportional to reduction of tumor size. CHF2-Orn administered in a two-stage (TPA-mezerein) promotion protocol [Slaga, T. J., Fischer, S. M., Nelson, K. G. & Gleason, G. L. (1980) Proc. Natl. Acad. Sci. USA 77, 3659-3663; Slaga, T. J., Klein-Szanto, A. J. P., Fischer, S. M., Weeks, C. E., Nelson, K. & Major, S. (1980) Proc. Natl. Acad. Sci. USA 77, 2251-2254] reduced tumor size, inhibited by 65-70% the number of papillomas per mouse, and decreased by 40% the percentage of mice with tumors when given with the stage II agent mezerein. CHF2-Orn provided considerably less effect on tumorigenesis when administered with the TPA portion of the protocol, and CHF2-Orn did not inhibit the induction of dark basal keratinocytes by TPA. Based on our results with CHF2-Orn, we suggest that regulation of polyamine biosynthesis, particularly Put, is a critical factor in stage II promotion.