Enhancing absorption of fluorescein and LHRH across hindgut epithelia in a marsupial, the common brushtail possum Trichosurus vulpecula.

We have identified differences in transport properties of intestinal epithelia in the marsupial brushtail possum, compared to eutherian mammals. To determine whether differences in its permeability to hydrophilic compounds also occur, the absorption of sodium fluorescein and luteinizing hormone releasing hormone (LHRH) was assessed in vitro and the ability of chemical enhancers and a metabolic inhibitor to promote their absorption investigated. The apparent permeability of colonic and caecal tissues to fluorescein and LHRH and transepithelial resistance (Rt) in the absence or presence of ethylenediamine tetra-acetic acid (EDTA), sodium deoxycholic acid (SDA), dithiothreitol (DTT), polyacrylic acids (PAA), or the inhibitor bacitracin were determined. The effects of SDA and/or DTT on adherent mucus and the release of lactate dehydrogenase (LDH) were also assessed. In the absence of treatment, both tissues had comparable amounts of adherent mucus, Rt and low permeabilities to fluorescein and LHRH. All chemical enhancers increased fluorescein permeability, but SDA at concentrations >0.5 mM also induced LDH release. DTT alone and in combination with SDA reduced the amount of adherent mucus. Bacitracin inhibited LHRH metabolism and increased LHRH permeability. These data indicate that the possum hindgut epithelium represents a significant barrier to the uptake of hydrophilic compounds, similar to that in eutherians.

Polymer microfiber mechanical properties: a system for assessment and investigation of the link with fibrous capsule formation.

A novel microtensile testing instrument was developed to assess the mechanical properties of small-diameter polyethylene, polyurethane, and polyester microfibers. The instrument had a root-mean-square error of 2.96 microN for force measurement and 1.91 microm for displacement measurement. Microfibers ranging in diameter from 1.0 to 10.9 microm were strained at 2 mm/s in the device, and the slopes of their stress-strain curves (material moduli) were determined. Correlations between material modulus and previously published data on fibrous capsule presence and thickness for implanted polyethylene, polyurethane, and polyester microfibers were investigated. Results for the 1.0-5.9-microm microfiber diameter range showed that neither the percentage of unencapsulated fibers nor the capsule thickness correlated well with modulus. Correlation coefficients were 0.04 and 0.09, respectively. However, for the 6.0-10.9 microm diameter range the correlations were strong, 1.00 for both percentage of unencapsulated fibers and capsule thickness. It is suggested that the results reflect the greater attachment and mechanical interaction of cells with microfibers for the 6.0-10.9 microm-diameter range than for the 1.0-5.9 microm-diameter range.

The metabolic barrier of the lower intestinal tract of salmon to the oral delivery of protein and peptide drugs.

Oral delivery of peptide and protein drugs has potential advantages for the aquaculture industry. The bioavailability of proteins and peptides from the intestinal tract is very low. This can be attributed in part to the proteolytic activities of the intestine. Bovine serum albumin (BSA), human (hLHRH) and salmon (sLHRH) luteinizing-hormone releasing hormones were used to evaluate the proteolytic activity of anterior, middle and posterior sections of the Quinnat salmon (Oncorhynchus tshawytscha) intestinal tract. The lumenal proteolytic activities of the posterior intestinal section towards BSA were approximately half that of the anterior and middle sections. The half-lives of the LHRH analogues in the posterior were twofold longer than for the anterior and middle sections. Proteolytic activity of the posterior mucosal homogenates towards BSA was fourfold higher than the middle mucosal homogenates. LHRH analogues were hydrolysed by the posterior mucosal homogenate, whereas in the middle mucosal homogenate they were stable. Soybean trypsin inhibitor was shown to be the most effective inhibitor of lumenal proteolytic activity towards LHRH analogues. Sodium deoxycholate, EDTA and bestatin significantly inhibited the posterior mucosal hydrolytic activity towards the LHRH analogues. The posterior intestine of salmon is the most favourable site for the delivery of BSA and LHRH analogues with respect to the lumen, however the higher proteolytic activity of the posterior mucosa has to be overcome.

Enzymatic degradation of luteinizing hormone releasing hormone (LHRH) by mucosal homogenates from the intestine of the common brushtail possum (Trichosurus vulpecula).

The peptidolytic activity of fresh and frozen mucosal homogenates from five regions (duodenum, jejunum, ileum, caecum and colon) of possum intestine from Trichosurus vulpecula towards human Luteinizing Hormone Releasing Hormone (LHRH) was investigated. The rank of order of specific peptidolytic activity of the mucosal homogenates was jejunum > ileum > caecum> duodenum = colon, with a 3 to 4 fold difference between the least and the most active segment in both frozen and fresh samples. The formation of peptides LHRH (1-3), LHRH (1-4) and LHRH (1-5) suggest endopepetidase-24.18, endopeptidase-24.15 and angiotensin converting enzyme (ACE) might be responsible for the peptide degradation in mucosal homogenates. The inhibition of LHRH degradation by mucosal homogenates was evaluated in four regions (jejunum, ileum, caecum and colon) of possum intestine. Ethylenediaminetetraacetic acid (EDTA, 5 mM), sodium deoxycholate (SDA, 10 mM) and bacitracin (3.5 or 9 mM) inhibited the degradation of LHRH in mucosal homogenates from small intestine and hindgut. However, the serine protease inhibitor, soybean trypsin-chymotrypsin inhibitor (SBTI), did not prevent degradation of LHRH. It is concluded that combining peptides with inhibitors may enhance oral delivery of bioactive peptides or proteins to possums.

Inhibition of proteolysis in luminal extracts from the intestine of the brushtail possum.

The proteolytic activity of luminal extracts from five regions (duodenum, jejunum, ileum, caecum and colon) of the brushtail possum intestine towards bovine serum albumin (BSA) and human luteinizing hormone releasing hormone (LHRH) was investigated. There were no significant differences in degradation rates between fresh and previously frozen extracts from any region of the possum intestine. The inhibition of degradation of BSA by luminal extracts from two regions (jejunum and ileum) and of LHRH from four regions (jejunum, ileum, caecum and colon) was evaluated. Soybean trypsin-chymotrypsin inhibitor (SBTI), sodium deoxycholate, Carbopol 934P, bacitracin and bestatin significantly inhibited the degradation of both LHRH and BSA (P < 0.05). SBTI almost totally inhibited the proteolysis of BSA and the peptidolysis of LHRH in extracts from the small intestine. This finding suggests that serine proteases such as chymotrypsin are responsible for the protein and peptide degradation in luminal extracts. It is concluded that including serine protease inhibitors in a formulation may enhance oral delivery of bioactive peptides and proteins to possums.

Protein and peptide degradation in the intestine of the common brushtail possum (Trichosurus vulpecula).

A protein (bovine serum albumin: BSA) and a peptide (luteinizing hormone releasing hormone: LHRH) were used to evaluate proteolytic activity in the intestine of common brushtail possums (Marsupiala, Trichosurus vulpecula). Luminal and mucosal extracts were isolated from the duodenum, jejunum, ileum, caecum, proximal colon and distal colon, their protein content assessed and specific activities in metabolising LHRH and BSA determined in vitro. The degradation of LHRH by luminal extracts was compared with that by the pancreatic enzymes, chymotrypsin, trypsin, and elastase. The protein concentration (microg x mg-1) of mucosal extract in the duodenum was higher ( P<0.05) than in the proximal colon, but that of luminal extracts did not differ significantly between regions. Proteolytic activity of luminal extracts was greater ( P<0.01) in the jejunum and ileum than in the hindgut. In the small intestine, proteolytic activity of luminal enzymes far exceeded that of mucosal enzymes ( P<0.05). All three pancreatic enzymes hydrolysed LHRH, but chymotrypsin had the greatest activity. This study has demonstrated that, in possums, proteolysis occurs primarily in the small intestine through luminal enzymes, with chymotrypsin playing a major role. The possum hindgut contributes little to the metabolism of peptides and proteins, identifying it as a potential site to target for their absorption following oral delivery.

Comparative efficacy and pharmacokinetics of racemic bupivacaine and S-bupivacaine in third molar surgery.

PURPOSE: To compare the efficacy and pharmacokinetics of racemic bupivacaine (rac-bupivacaine) with S-bupivacaine as primary local anesthetic agent in bilateral impacted third molar extractions. METHOD: A randomised, double blind, two period cross-over design was employed. Six subjects (2 males, 4 females; age 19-25 years; weight 69.2+/-9.4 kg) received bupivacaine hydrochloride injection (6.6 ml) as rac-bupivacaine (0.5% as salt) or S-bupivacaine (0.5% as base) prior to extraction of impacted third molars on one side and three weeks later on the other side. Anesthesia, blood loss associated with surgery and post-operative pain experience were evaluated. Plasma samples were analysed for bupivacaine enantiomers by chiral HPLC. RESULTS: In 7/12 operations, anesthesia adequate for surgery was delayed (>10 min) or unsatisfactory requiring lidocaine rescue medication. Despite this, there were no significant differences in onset and duration of anesthesia, blood loss or post-operative pain experience between the two arms of the study. Pharmacokinetic parameters were not significantly different and there was no evidence of chiral inversion after dosing with S-bupivacaine. CONCLUSIONS: Both study drugs were inadequate as single anesthetic agent for third molar surgery. Any decision to use S-bupivacaine for oral surgery must rest on evidence that it is less toxic than the racemic drug.

Particulate contamination in parenteral nutrition solutions: still a cause for concern?

OBJECTIVES: In consideration of a US Federal Drug Administration recommendation that all parenteral nutrition admixtures should be administered through an in-line filtration device, this observational study examined the number, size distribution, and sources of particulate contamination in parenteral nutrition admixture infusion systems. METHODS: Samples were drawn from the terminal connection of the infusion tubing before connection to the patient. The particles were sized and counted by optical microscopy and further investigated by electron microscopy and energy disperse spectroscopy. RESULTS: Large numbers of particles were found, and information gained about their possible origin. CONCLUSIONS: This study provides further support for the adoption of this Federal Drug Administration recommendation.

The effects of oral selegiline hydrochloride on learning and training in the dog: a psychobiological interpretation.

1. Twenty two healthy, non-problem dogs were assessed for their acquisition of three different learning tasks on consecutive days and on the extinction of the response in the third. In Task 1, dogs were trained to walk in a circle on command. In Task 2, dogs were trained to retreat and sit on a mat. In Task 3, dogs were assessed for the acquisition and extinction of an operant response (pawing a panel). 2. Dogs were orally administered a placebo or selegiline hydrochloride at a dose of 0.5 mg/kg for a period of three weeks prior to testing and during the test period. 3. Dog's treated with selegiline tended to perform better at tasks which were clearly lured with a motivationally significant cue, performing a first correct response sooner and requiring fewer reinforcements to reach the success criterion. They were also significantly more likely to walk over a novel object placed on the floor of the test arena. In the absence of a significant lure, the selegiline treated dogs took significantly longer to reach the required performance criterion for the operant task. These dogs also extinguished their response more rapidly than the control group 4. In the third task, selegiline treated dogs were significantly less likely to look away and throughout all tasks these dogs tended to be less distracted than the placebo group. 5. These findings and other reports associated with the effects of selegiline on learning may be explained by reference to the effects of selegiline on dopaminergic structures associated with positive incentive motivation.

Activity of pancreatic endopeptidases towards luteinizing hormone-releasing hormones.

LHRH and its analogues have low oral bioavailability; this is in part due to their degradation by peptidases present in the intestinal lumen. To determine the appropriate inhibitors to co-administer with LHRH oral formulations, the peptidases involved in their digestion have to be identified. Human (hLHRH) and salmon (sLHRH) LHRH analogues contain a number of potential cleavage sites for the lumenal pancreatic secreted serine endopeptidases: chymotrypsin, trypsin and elastase. The rate of LHRH degradation by equimolar concentrations of chymotrypsin, trypsin and elastase were examined separately in vitro, at pH 8.0, 15 degrees C. At a molar ratio of 1:1000 (enzyme:LHRH), both LHRH analogues were rapidly hydrolysed by alpha-chymotrypsin with half-lives of 2.5+/-0.3 and 2.7+/-0.4 min (mean+/-S.D., n=3), respectively, whereas in the presence of elastase both LHRH analogues were slowly hydrolysed with half-lives of 90+/-15 and 114+/-21 min (mean+/-S.D., n=3), respectively. Trypsin had no activity towards either LHRH analogues after 2 h incubation. The degradation of the LHRH analogues by elastase is likely to be a property of the chymotrypsin impurity. It is concluded that protection of the LHRH analogues from alpha-chymotrypsin is a requirement for the development of oral absorbable product.

Carbomer inhibits tryptic proteolysis of luteinizing hormone-releasing hormone and N-alpha-benzoyl-L-arginine ethyl ester by binding the enzyme.

PURPOSE: To determine the mechanism by which Carbomer inhibits the enzymatic activity of trypsin in hydrolysis of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) and luteinizing hormone-releasing hormone (LHRH). METHODS: Inhibition of enzymatic activity was studied by measuring the formation of metabolites from LHRH and BAEE. Binding of trypsin and substrates to 0.35% (w/v) Carbomer at pH 7.0 was studied by centrifugal filtration. Gel filtration and reverse phase HPLC was used to determine the stability of trypsin. RESULTS: Carbomer reduced the rate of hydrolysis of BAEE and LHRH by trypsin to 34% and 28% of the control activity, respectively. The rate of metabolite formation for both substrates followed pseudo-zero order kinetics in the presence and absence of carbomer. Binding studies showed that 68% of the trypsin protein and 10% of BAEE was bound to carbomer, but no LHRH was bound. No low molecular weight autolysis products of trypsin could be identified by gel filtration. Reverse phase HPLC analysis of the unbound carbomer-treated-trypsin suggests a number of conformational forms of trypsin. The equilibrium binding capacity was 30 microg of trypsin to 1000 microg of carbomer. CONCLUSIONS: Decreased hydrolysis of LHRH and BAEE by trypsin in the presence of carbomer is due to enzyme-polymer interaction.

Controlled release opportunities for oral peptide delivery in aquaculture.

Over the last decade, fish supplies for human consumption have reached over 100 million tons. Due to overfishing, future increases in demand can only be met from the aquaculture industry. This will require increased research in areas such as the control and manipulation of fish reproduction. There is increasing interest in the oral delivery of peptides that control gamete reproduction. However, compared to mammalian species, little is known about the barriers to peptide delivery and methods to improve such delivery. The three major barriers to peptide delivery are the enzymatic barriers sourced from the host luminal and membrane bound peptidases, the immunological cells present within both the enterocytes and underlying connective tissue and the physical barrier of the epithelial cells. Furthermore, the anatomy and physiology of the gastrointestinal tract of these species are markedly different when compared to higher vertebrates and therefore must be considered when designing appropriate delivery systems. Research to date has focused on the oral delivery and subsequent pharmacodynamic responses to the peptides associated with growth and reproduction. However, minimal work has been undertaken to overcome the identified barriers and therefore any future investigations need to attend to these obstacles before the oral delivery of bioactive peptides can become a commercial reality.

A pilot study of the disposition of pilocarpine in plasma, saliva and urine after a single oral dose.

Concentrations of pilocarpine in plasma, saliva and urine from three healthy male volunteers were measured using a fluorescence derivatisation method, following administration of a single 10 mg oral dose. Pharmacokinetic parameter values were estimated from concentration-time profiles. Linear correlations between plasma and saliva pilocarpine concentrations (r2=0.945, n=10, p<0.001; r2=0.954, n=12, p<0.001) and plasma concentrations and salivation rate (r2=0. 863, n=12, p<0.001; r2=0.862, n=15, p<0.001) were established. Pilocarpine and an unidentified metabolite, respectively 20.3% and 34.7% of the oral dose, were excreted into urine.

Stereoselective urinary excretion of bupivacaine and its metabolites during epidural infusion.

A sensitive and efficient chiral assay for bupivacaine and its three principal metabolites desbutylbupivacaine, 4'-hydroxybupivacaine, and 3'-hydroxybupivacaine has been applied to urine from five male patients receiving postoperative epidural infusions of rac-bupivacaine fentanyl over 60-120 hr. The fraction of the dose of bupivacaine (total dose 840-2093 mg) accounted for in urine was 75 +/- 6%. The rate of excretion of bupivacaine enantiomers' approximated a steady state after approximately 30 hr with values of 1.27 +/- 0.26 and 0.76 +/- 0.13 mg hr-1 for (R)- and (S)-enantiomers, respectively. The fraction of the dose of bupivacaine enantiomer excreted unchanged in the urine (fe) varied from 14.3% to 39.1% for (+)-(R)-bupivacaine and 9.2% to 14.0% for (-)-(S)-bupivacaine in the five patients. The rate of excretion of all metabolites also reached a steady state after approximately 30 hr and the relative amounts of metabolites excreted into urine (fm) suggest bupivacaine is subject to regioselective and stereoselective clearance, which may vary from patient to patient.

Simultaneous analysis of lignocaine and bupivacaine enantiomers in plasma by high-performance liquid chromatography.

A sensitive analytical procedure is described for the simultaneous determination of lignocaine and the enantiomers of bupivacaine in biological fluids using diazepam as an internal standard. After solvent extraction into hexane, the local anaesthetics were separated using an alpha1-acid glycoprotein (AGP) column and detected at 214 nm. Calibration curves were linear (r2>0.99) in the concentration range of 5 to 500 ng/ml for the enantiomers of bupivacaine and 12.5 to 1000 ng/ml for lignocaine. The corresponding limits of detection were 4 ng/ml and 10 ng/ml, respectively. The method was applied to the analysis of plasma from a healthy woman undergoing tubal ligation.

Serum pilocarpine esterase activity and response to oral pilocarpine.

Pilocarpine is used orally to treat xerostomia but patients vary widely in their response and ability to tolerate this drug. To elucidate the potential pharmacokinetic contribution of serum to this variability, the enzymatic hydrolysis of pilocarpine in human serum in vitro was investigated using a stability indicating HPLC assay. The reaction at 37 degrees C follows Michaelis-Menten kinetics (K(m) = 2.78 +/- 0.48 mmol/liter, Vmax = 79 +/- 13 nmol min-1 ml 1; n = 5) and produces pilocarpic acid as the only detectable product. The distribution of pilocarpine esterase activity in a group of healthy young adults at age 21 (n = 163; 87 males, 76 females) was examined by incubating serum samples with pilocarpine (10 mmol/ liter) at 37 degrees C for 60 min. The distribution was positively skewed and ranged from 4 to 132 nmol min-1 ml-1 with a mean value of 55 +/- 23 nmol min-1 ml 1. The means for males and females were not significantly different. Similar measurements in xerostomia patients undergoing treatment with oral pilocarpine showed that those with higher serum esterase activity tolerated pilocarpine well and tended to require higher doses for relief of xerostomia, whereas those with low activity were sensitive to the adverse effects of the drug and were adequately treated with a lower dose. The results suggest that at least some of the variability in response to oral pilocarpine is due to differences in serum pharmacokinetics.

Sensitive high-performance liquid chromatographic assay for pilocarpine in biological fluids using fluorescence derivatisation.

A sensitive assay for pilocarpine in biological fluids has been developed involving HPLC of a fluorescent derivative of 4-bromomethyl-7-methoxycoumarin. Pilosine as internal standard was added before the derivatisation step. The fluorescent derivatives were well resolved and separated from excess reagent and endogenous compounds on a cyanopropyl silica column. The detection limit of pilocarpine in biological fluids was 1.0 ng/ml and the assay was linear up to a concentration of 150 ng/ml. The assay was applied to a preliminary study of pilocarpine disposition in man after a single oral dose. This is the first report of pilocarpine excretion into saliva.

Are there any successful men from criminogenic backgrounds?

In the Cambridge Study in Delinquent Development, a prospective longitudinal survey of 411 London males, a vulnerable group of 63 boys from criminogenic backgrounds was defined on the basis of the best nonbehavioral predictors of delinquency at age 8-10 (low family income, large family size, convicted parents, low intelligence, and poor parental child-rearing behavior). These males were followed up to age 32, and the more successful men were defined according to criteria such as the absence of convictions and of other deviant behavior, good relationships with wives and children, and good accommodation and employment histories. Hence, "success" here refers to satisfactory social adjustment. The more successful men were those who had been neurotic at age 10, those who had few or no friends at age 8, those without convicted parents or behavior problem siblings, those with mothers who had a high opinion of their sons, and those who did not spend their leisure time with their fathers. At age 8-10 they were already better behaved and less daring than those later judged as the unsuccessful men. There was some tendency for shyness to act as a protective factor against delinquency for non-aggressive boys but as an aggravating factor for aggressive boys.

Refolding of denatured thioredoxin observed by size-exclusion chromatography.

Molecular sieve chromatography can resolve interactive systems into populations having different effective hydrodynamic volumes. In this report, the advantages of such resolution to protein folding are illustrated by using moderate pressure to decrease analysis time and lowered temperature to slow down the kinetics of conformational change. A 300-mm Bio-Sil TSK-125 size-exclusion column was equilibrated with a series of different concentrations of guanidine hydrochloride at 2 degrees C in 50 mM phosphate buffer, pH 7.0. Samples of native Escherichia coli thioredoxin, denatured thioredoxin, or thioredoxin equilibrated with the column solvent were injected, and the effluent was monitored at 220 nm. Injection of equilibrated protein samples defined three denaturant concentration zones identical with those observed by spectral measurements: the native base-line zone where only compact protein is observed in the effluent profile; the transition zone in which both compact and denatured forms are observed in slow exchange; and the denatured base-line zone in which only denatured protein is observed. Unfolding was observed by injection of native protein into columns having isocratic denaturant concentrations in the transition and denatured base-line zones. Effluent profiles indicated a dynamic conversion of compact to denatured protein with a time constant which appeared to decrease markedly with increasing denaturant concentration. Refolding was observed by injection of denatured protein into columns having isocratic concentrations in the transition and native base-line zones. As the denaturant concentration was decreased, the effluent profiles evidenced a persistent slow conversion of denatured to compact protein which was suddenly accelerated about midway in the native base-line zone.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation and analysis of reactive blue 2 bonded to silica via variable spacer groups.

A variety of aminoalkylated microparticulate porous silicas have been prepared and used to displace the reactive chloride from reactive blue 2 thereby immobilizing the dye in a mild and predictable manner. Methods of analysis of immobilized reactive blue 2 are compared and a rapid new method is recommended which is clean and non-destructive. The biospecific elution of a dehydrogenase from an immobilized dye column is demonstrated.