Robin Ledger

Carbomer Inhibits Tryptic Proteolysis of Luteinizing Hormone-Releasing Hormone and N-α-Benzoyl-L-Arginine Ethyl Ester by Binding the Enzyme

AbstractPurpose. To determine the mechanism by which Carbomer inhibits the enzymatic activity of trypsin in hydrolysis of N-α-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 μg of trypsin to 1000 μg 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.

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 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.

The Rationale and Efficacy of Problem-based Learning and Computer Assisted Learning in Pharmaceutical Education

This paper explores two modes of teaching at schools of pharmacy and the benefits to be gained using such methods:namely, problem-based learning (PBL) and computer assisted learning (CAL). Possible advantages of PBL over traditional approaches include its greater relevance to the ability to the practice of pharmacy, its ability to promote the retention and application of knowledge and its encouragement of self-directed life-long learning. Possible disadvantages include higher costs, both in resources and staff time. The current enthusiasm for PBL seems to justified and its use is likely to increase further. CAL aims to bridge the gaps not fully covered by traditional and PBL methods. Using PBL and CAL as a supplementary resource for conventional teaching and learning is desirable for providing optimal opportunities to learn the processes needed to identify and treat drug related problems confronting todays practising pharmacist.

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.

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.

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.

Isocratic liquid chromatographic assay for monitoring the degradation of luteinizing hormone releasing hormone by extracts from the gastrointestinal tract of possums

A simple HPLC method to separate human luteinizing hormone releasing hormone (LHRH) from its metabolites using an isocratic elution is described. Intact LHRH and five metabolites were separated in 11.4 min. The calibration curve (peak area versus concentration) was linear over the concentration range 1.25-35 microg/ml (r(2)=0.99) with the intercept not significantly different from zero (P>0.05). Intra-day and inter-day variability of the assay was less than 5% for repeat injections of 5, 14.5 and 29 microg/ml. The method was applied to evaluate the susceptibility of LHRH to enzymes present in the lumen and mucosal extracts of the gastrointestinal tract of possums. The major degradation products of LHRH were identified by HPLC separation, amino acid analysis and mass spectrometry as LHRH (1-5), LHRH (1-4), LHRH (1-3) and LHRH (3-4).