Modified recombinant adenoviruses increase porcine circovirus 2 capsid protein expression and induce enhanced immune responses in mice.

Porcine circovirus type 2 (PCV2) is the primary viral pathogen of porcine circovirus associated disease (PCVAD) and vaccination is an important method to prevent and control the disease. The expression of PCV2 capsid protein (Cap) in adenovirus vector system has been investigated, but the poor immune responses limit its application. In this study, transcriptional enhancer element largest intron of the human cytomegalovirus (Intron A) and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) were applied to increase the immunogenicity of PCV2 Cap adenovirus-based vaccine. Western blot and indirect immunofluorescence assay (IFA) analysis showed that modified adenoviruses with Intron A and WPRE alone or both could significantly increase the expression of Cap compared to the unmodified adenoviruses. Furthermore, the humoral and cellular immune responses of the constructed recombinant adenoviruses were evaluated in mice. Indirect ELISA, virus neutralizing test and western blot showed that modified adenoviruses elicited higher humoral immune responses than unmodified adenovirus, and Intron A-WPRE-modified virus immunized group had better immune response than the others. Besides, the results of lymphocyte proliferation response and cytokines release assay showed that enhanced cellular immune responses were induced by modified adenoviruses. These results demonstrated that Intron A and WPRE significantly improved the expression of the Cap protein in adenovirus vector system and enhanced the immune responses in mice, making the adenovirus vector system more applicable against PCV2.

Association between polymorphisms in the csrRS two-component regulatory system and invasive group A streptococcal infection.

The csrRS two-component regulatory system is an important element in the pathogenesis of group A Streptococcus (GAS). The main goal of this study is to understand the association between csrRS polymorphisms and GAS infection. We sequenced the csrRS genes from 172 clinical isolates, including 81 invasive and 91 noninvasive isolates, and then employed phylogenetic analyses to determine the consequences of the csrRS polymorphisms. In total, 13 and 26 polymorphic loci were detected in the csrR and csrS genes, respectively. These polymorphisms constituted 14 csrR and 25 csrS alleles, producing two CsrR and seven CsrS variants, respectively. Three invasive isolates contained an indel in csrS, but no indel was identified in csrR. The frequency and distribution of polymorphisms in csrR and csrS was significantly different between the invasive and noninvasive infection isolates (p < 0.001). For CsrR, only one noninvasive isolate was identified to have a V29I mutation. The amino acid substitutions in CsrS included S32P (0.6 %), E265G (0.6 %), E265K (0.6 %), I332V (1.7 %), and N498K (82.6 %). Isolates with an N498K single mutation were more likely to be associated with invasive infections (p < 0.001). The dN/dS ratio indicated that both csrR and csrS were under purifying selection. The fixation index suggested a moderate evolutionary differentiation of the csrR and csrS alleles between invasive and noninvasive isolates. The identification of these genetic differences within the csrRS loci will provide a better understanding of the pathogenesis of GAS.

Effect of free-range days on a local chicken breed: growth performance, carcass yield, meat quality, and lymphoid organ index.

An experiment was conducted to evaluate the effect of free-range days on growth performance, carcass yield, meat quality, and lymphoid organ index of a local chicken breed. In total, 1,000 one-day-old male Suqin yellow chickens were raised for 21 d. On d 21, 720 birds with similar BW (536 ± 36 g) were selected and randomly assigned to free-range treatment at 21, 28, 35, and 42 d of age (assigned to free-range treatment for 21, 14, 7, and 0 d, respectively). Each treatment was represented by 5 replicates (pens) containing 36 birds (180 birds per treatment). All the birds were raised in indoor floor pens measuring 1.42 × 1.42 m (2 m(2), 18 birds/m(2)) in conventional poultry research houses before free-range treatment. In the free-range treatment, the chickens were raised in indoor floor houses measuring 3 × 5 m (15 m(2), 2.4 birds/m(2)). In addition, they also had an outdoor free-range paddock measuring 3 × 8 m (24 m(2), 1.5 birds/m(2)). The BW of birds after being assigned to free-range treatment for 7 d decreased significantly compared with that in the conventional treatment (P < 0.05). However, there was no effect of the free-range days on the BW at 42 d of age (P > 0.05). The daily weight gain, feed per gain, daily feed intake, and mortality from 21 to 42 d of age were unaffected by free-range days (P > 0.05). At 42 d of age, the breast yield increased linearly with increasing free-range days (P < 0.05), whereas the thigh, leg, thigh bone, and foot yields decreased linearly (P < 0.05). The lung yield showed a significant increasing and then decreasing quadratic response to increasing free-range days (P < 0.05). The water-holding capacity of the thigh muscle decreased linearly with increasing free-range days (P < 0.05), whereas there was no significant difference in the meat color, shear force, and muscle pH (P > 0.05). The absolute thymus weight and thymus:BW ratio showed a significant increasing and then decreasing quadratic response to increasing free-range days (P < 0.05). The findings of this study suggest that increasing free-range days advantageously affects breast yield, but decreases thigh, leg, thigh bone, and foot yields as well as the water-holding capacity of thigh. No evidence was found that increasing free-range days caused changes in growth performance, meat quality, and lymphoid organs except for changes in water-holding capacity and thymus.

Safety evaluation of phytosterols in laying hens: effects on laying performance, clinical blood parameters, and organ development.

Phytosterols are intended for use as a novel food ingredient with plasma cholesterol-lowering activity. Although phytosterols are naturally present in the normal diet, daily consumption is insufficient to ensure plasma cholesterol-lowering levels. Therefore, phytosterols may be added to the diets to achieve the desired cholesterol-lowering activity. A subchronic laying hen safety study was conducted to examine if high-dose phytosterols could affect the safety of hens. Three hundred sixty 21-wk-old Hy-Line Brown laying hens were randomly assigned to 5 groups with 6 replicates of 12 birds each; after 3 wk, birds were fed diets supplemented with 0, 20, 80, 400, and 800 mg/kg of phytosterols for 12 wk. Throughout the study, clinical observations and laying performance were measured. At the end of the study, birds were subjected to a full postmortem examination: blood samples were taken for clinical pathology, selected organs were weighed, and specified tissues were taken for subsequent histological examination. No treatment-related changes that were considered to be of toxicological significance were observed. Therefore, a nominal phytosterol concentration of 800 mg/kg was considered to be the no-observed-adverse-effect level.

Association of SCD1 and DGAT1 SNPs with the intramuscular fat traits in Chinese Simmental cattle and their distribution in eight Chinese cattle breeds.

Intramuscular fat (IMF) is a key parameter for evaluation of nutritional quality of beef, with its endogenous synthesis regulated by stearoyl CoA desaturase (SCD1) and diacylglycerol-acyl transferase 1 (DGAT1) genes in cattle. The object of this research was to evaluate the effect of SCD1 and DGAT1 polymorphisms on IMF trait in beef cattle and to estimate the frequency distribution of SNPs in the two genes in Chinese cattle populations. The SCD1 and DGAT1 polymorphisms were detected by PCR-single strand conformation polymorphism (PCR-SSCP) method in Chinese Simmental cattle and their associations with IMF traits were analyzed using the general linear model (GLM). The frequency distribution of SNPs in SCD1 and DGAT1 genes were detected by PCR-SSCP method and analyzed in seven other cattle populations. The results showed significant associations of SNPs SCD1-878, SCD1-762, and DGAT1 10433 and 10434 with IMF (%) and shearing force values (SFV; kg) in Chinese Simmental cattle. A haplotype combining SCD1-878C, SCD1-762T, and DGAT1 10433 and 10434-GC had the highest IMF, marbling score and shearing force. The polymorphic investigation indicated that the frequency of SCD1-878C or SCD1-762T was significantly higher in Chinese southern cattle (Leiqiong, Yunnan High pump, BMY or Minnan Cattle) than in Chinese northern cattle (Chinese Simmental, Luxi Cattle, Bohai Black or Chinese Holstein), while the frequency of DGAT1 10433 and 10434-GC in Chinese indigenous breed (Leiqiong, Yunnan High pump, BMY, Luxi Cattle, Bohai Black, or Minnan Cattle) was significantly lower than breeds with imported blood (Chinese Simmental or Chinese Holstein). These findings demonstrated that both the SCD1 and DGAT1 SNPs were prospect genetic markers for IMF traits, and the SCD1 SNPs could be used as a genetic marker for southern or northern blood in Chinese cattle.

Suppressor of cytokine signaling 1 gene expression and polymorphisms in systemic lupus erythematosus.

With the aim of investigating the role of suppressor of cytokine signaling 1 (SOCS1) in the pathogenesis of systemic lupus erythematosus, 107 patients with systemic lupus erythematosus, 101 healthy controls, and 151 patients with ankylosing spondylitis were enrolled in this study. SOCS1 mRNA level was measured by the method of quantitative real-time polymerase chain reaction. SOCS1 polymorphisms were detected by the polymerase chain reaction/restriction fragment length polymorphisms method. Systemic lupus erythematosus disease activity was evaluated with the SLEDAI. This study showed that the SOCS1 mRNA expression was significantly higher in the patients with systemic lupus erythematosus than in the healthy controls (p = 0.0014). Patients with active systemic lupus erythematosus had a higher expression of SOCS1 mRNA than the patients with inactive systemic lupus erythematosus (p = 0.035). There was no significant difference in the frequencies of the SOCS1-1478CA/del polymorphisms among the patients with systemic lupus erythematosus, healthy controls, and patients with ankylosing spondylitis. The genotype frequency of the SOCS1-1478 polymorphisms in the dominant model (CA/del+del/del versus CA/CA) was significantly decreased in the patients with thrombocytopenia compared with those without thrombocytopenia (p(c) = 0.035). Moreover, the allele frequency of SOCS1-1478del was also significantly lower in the patients with thrombocytopenia than in those without thrombocytopenia (p( c) = 0.02). In conclusion, this study demonstrated that the expression of SOCS1 mRNA was significantly increased in patients with systemic lupus erythematosus. Moreover, SOCS1 mRNA levels in patients with active systemic lupus erythematosus were significantly higher than those in the inactive patients. We also found that the systemic lupus erythematosus patients with thrombocytopenia have a lower frequency of SOCS1-1478del compared with patients without thrombocytopenia.

Amoxicillin resistance with beta-lactamase production in Helicobacter pylori.

BACKGROUND: Amoxicillin-resistant Helicobacter pylori with minimal inhibitory concentration (MIC) >or= 256 mg L(-1) was isolated from a gastritis patient. The aims were to investigate the mechanism of high-level amoxicillin resistance in H. pylori. MATERIALS AND METHODS: The beta-lactamase production was determined by means of nitrocefin sticks and the presence of gene encoding the beta-lactam antibiotic resistance enzyme TEM beta-lactamase was analysed by polymerase chain reaction (PCR), sequencing and dot-blot hybridization. Sequencing analysis of pbp1A gene was performed and amoxicillin-susceptible isolate was transformed with pbp1A PCR products from the resistant isolate. The expression of hefC efflux system was analysed using real-time quantitative PCR. RESULTS: Activity of beta-lactamase was detected. Sequence analysis showed that the PCR product derived from H. pylori 3778 was identical to the bla(TEM-1) (GenBank accession EU726527). Dot-blot hybridization confirmed the presence of beta-lactamase gene bla(TEM-1.) By transformation of PCR product of mutated pbp1A gene from H. pylori 3778 into amoxicillin-susceptible strain showed that substitutions in Thr(556)-->Ser, Lys(648)-->Gln, Arg(649)-->Lys and Arg(656)-->Pro contribute to low-level amoxicillin resistance. The MIC of amoxicillin for the transformants was 0.75 mg L(-1). Over-expression of hefC was not found. CONCLUSIONS: High-level amoxicillin resistance is associated with beta-lactamase production in H. pylori. Low-level amoxicillin resistance is linked to a point mutation on pbp1A. Because H. pylori can exchange DNA through natural transformation, spreading of bla(TEM-1) amoxicillin resistance gene among H. pylori is a potential threat when treating H. pylori infection.

Thyrotropin and cyclic nucleotide effects on prostaglandin levels in isolated thyroid cells.

Thyrotropin increases prostaglandin levels in isolated thyroid cells. Since comparable results were obtained with butyrated cyclic adenosine monophosphate derivatives as well as with the phosphodiesterase inhibitors quazodine and theophylline, it appears that cyclic adenosine monophosphate mediates this effect of thyrotropin. These observations suggest that intracellular prostaglandins play a role in modulating thyrotropin action on thyroid.

Omega-3 polyunsaturated fatty acid inhibits the malignant progression of hepatocarcinoma by inhibiting the Wnt/ß-catenin pathway.

OBJECTIVE: Omega-3 polyunsaturated fatty acid (ω-3 PUFA) has been found to possess anti-cancer potential in previous studies. However, the underlying mechanism of ω-3 PUFA in protecting hepatocarcinoma has not been fully elucidated. This study aims to explore the function of ω-3 PUFA in the development of hepatocarcinoma and its potential mechanism. PATIENTS AND METHODS: In this study, human hepatocarcinoma cell line Hep G2 was treated with ω-3 PUFA. Cell counting kit-8 (CCK-8) and cell cloning assay were applied to detect the proliferation of Hep G2 cells. In addition, flow cytometry was performed to analyze the cell cycle and apoptosis rate. At the same time, the effect of ω-3 PUFA on invasion and metastasis of hepatocarcinoma cells were analyzed by transwell assay. Moreover, protein levels of key factors in Wnt/ß-catenin pathway were detected by Western blot. RESULTS: Cell proliferation of Hep G2 cells was decreased after ω-3 PUFA treatment in a time- and dose-dependent manner. CCK-8 assay showed that the IC50 value was 12.8 ± 0.67 µmol/L, 8.8 ± 0.43 µmol/L and 4.6 ± 0.42 µmol/L after ω-3 PUFA treatment for 24 h, 48 h and 72 h, respectively. Besides, ratio of Hep G2 cells blocked at G2/M phase after ω-3 PUFA treatment (5 µmol/L, 10 µmol/L and 20 µmol/L) was increased in a dose-dependent manner (p<0.05). Meanwhile, ω-3 PUFA could increase cell apoptosis (p<0.05) and inhibit cell proliferation. In addition, ω-3 PUFA reduced protein expressions of total, cytoplasmic and nuclear ß-catenin in Hep G2 cells, indicating that the Wnt/ß-catenin pathway is inhibited. Decreased expression levels of Dvl-2, Dvl-3, GSK-3ß (p-ser9), c-myc and survivin, and increased expression levels of GSK-3 (p-tyr216) and Axin-2 were observed in Hep G2 cells treated with ω-3 PUFA, but no significant alteration in total GSK-3ß protein level was observed (p>0.05). CONCLUSIONS: Omega-3 PUFA regulates the malignant progression of hepatocarcinoma by inhibiting proliferation and promoting apoptosis of hepatocarcinoma cells via Wnt/ß-catenin signaling pathway.

Triazolinones as nonpeptide angiotensin II antagonists. 1. Synthesis and evaluation of potent 2,4,5-trisubstituted triazolinones.

A series of 2,4-dihydro-2,4,5-trisubstituted-3H-1,2,4-triazol-3-ones was prepared via several synthetic routes and evaluated as AII receptor antagonists in vitro and in vivo. The preferred compounds contained a [2'-(5-tetrazolyl)biphenyl-4-yl]methyl side chain at N4 and an n-butyl group at C5. A number of these bearing an alkyl or aralkyl substituent at N2 showed in vitro potency in the nanomolar range (rabbit aorta membrane receptor), and several of these, e.g., the 2,2-dimethyl-1-propyl analogue (54, IC50 = 2.1 nM), effectively blocked the AII pressor response in conscious rats with significant duration (2.5 h at 1 mg/kg orally for 54). Among analogues possessing aryl substituents at N2, ortho substitution on the phenyl moiety resulted in several derivatives with in vitro potency in the low nanomolar range. One of these, featuring a 2-(trifluoromethyl)phenyl substituent at N2 (25, IC50 = 1.2 nM), was effective at 1 mg/kg orally in the rat model, with a duration of > 6 h. Implications for hydrophobic and hydrogen-bonding interactions with the AT1 receptor are discussed.

Triazolinone biphenylsulfonamides as angiotensin II receptor antagonists with high affinity for both the AT1 and AT2 subtypes.

Angiotensin II (AII), the endogenous peptide ligand of the AII receptor, has equivalent high affinity for both the AT1 and AT2 receptor subtypes while most of the reported nonpeptide AII antagonists are AT1-selective. In an effort to identify dual AT1/AT2 nonpeptide AII antagonists, we have pursued modifications of previously prepared trisubstituted 1,2,4-triazolinone biphenylsulfonamides which exhibited subnanomolar in vitro AT1 (rabbit aorta) AII antagonism and AT2 (rat midbrain) IC50 values of < 40 nM. Present results show that a suitable amide (or reversed amide) side chain appropriately positioned on the N2-aryl group of these compounds gave > 15-fold enhancement in AT2 binding affinity without sacrificing nanomolar AT1 potency (IC50). This added amide, combined with an appropriate choice of the N-substituent on the sulfonamide and the ortho substituent on the N2-aryl group, led to an analogue (46, L-163,-007) which exhibited subnanomolar AT1 binding affinity and an AT2/AT1 IC50 ratio of 3. This compound showed excellent iv activity at 1 mg/kg and oral efficacy at 3 mg/kg with > 6 h duration in a conscious rat model. Available data suggest that the newly introduced amide side chain, mandatory for low nanomolar binding affinity at the AT2 receptor, is well-tolerated by the AT1 receptor and has minimal effect on the in vivo properties of these molecules.

Triazolinone biphenylsulfonamide derivatives as orally active angiotensin II antagonists with potent AT1 receptor affinity and enhanced AT2 affinity.

Several series of 2,4-dihydro-2,4,5-trisubstituted-3H-1,2,4-triazol-3-ones with acidic sulfonamide replacements of tetrazole at the 2'-position of the biphenyl-4-ylmethyl side chain at N4 were prepared and tested as angiotensin II (AII) antagonists. Preferred substituents on the triazolinone ring were n-butyl at C5 and 2-(trifluoromethyl)phenyl at N2. Subnanomolar IC50 values at the AT1 receptor subtype were observed for a variety of acylsulfonamides, including aroyl, heteroaroyl, and cycloalkylcarbonyl derivatives. Certain other acidic sulfonamides, such as sulfonylcarbamates and disulfimides also displayed high affinity for the AT1 receptor. In addition, AT2 binding for some of these compounds was increased by as much as 1000-fold over the corresponding tetrazole (e.g., AT2 IC50 17 nM for the tert-butyl sulfonylcarbamate 92). When evaluated for inhibition of the AII pressor response, the benchmark benzoylsulfonamide 9 (L-159,913) was efficacious in several species and was superior to losartan (1a) in conscious rhesus monkeys. Several subsequent analogues, including the 2-chlorobenzoyl (18), (3-chlorothiophene-2-yl)carbonyl (51), ((S)-2,2-dimethylcyclopropyl)carbonyl (80), and tert-butoxycarbonyl (92) derivatives, were highly effective in rats, surpassing 9 and losartan in duration of action and/or potency. Compound 18 (L-162,223) displayed very prolonged AII antagonism in the rat model (> 24 h at 1 mg/kg iv). At 1 mg/kg po in rats, 18 and 92 (L-162,234) produced 85-87% peak inhibition of the AII pressor response with duration exceeding 6 h. The identification of triazolinone-based sulfonamide derivatives combining high AT1 affinity, considerably enhanced AT2 potency, and favorable in vivo properties provides insights relevant to the design of dual AT1/AT2 receptor antagonists.

Potent and orally active angiotensin II receptor antagonists with equal affinity for human AT1 and AT2 subtypes.

In order to block the effects induced by the interactions between angiotensin II (AII) and both AT1 and AT2 receptors, we have pursued the discovery of orally active non-peptide AII antagonists that exhibit potent and equal affinity for human AT1 and AT2 receptor subtypes. A series of previously prepared nanomolar (IC50) trisubstituted 1,2,4-triazolinone biphenyl-sulfonamide dual-acting AII antagonists has been modified at five different positions in order to increase AT2 binding affinity, maintain AT1 activity, and reduce the human adrenal AT2/AT1 potency ratio (IC50 ratio) from > or = 10. The targeted human adrenal potency ratio of < or = 1 was achieved with a number of compounds possessing an ethyl group at C5 of the triazolinone and a 3-fluoro substituent at the N4-biarylmethyl moiety. The most favored of these was compound 44 which exhibited subnanomolar potency at both the AT1 (rabbit aorta) and AT2 (rat midbrain) receptors, with a slight preference for the latter, and had a human adrenal AT2/AT1 IC50 ratio of 1. This tert-butyl sulfonylcarbamate with an N2-[2-bromo-5-(valerylamino)phenyl] substituent had excellent iv activity at 1 mg/kg (100% peak inhibition, > or = 4 h duration of action) and is orally active at 3 mg/kg with > 6 h duration of action in a conscious rat model. The present study shows that the NH of the amide on the N2-aryl moiety is not required for subnanomolar binding affinity to either receptor subtype, although a keto functionality at this position is essential for acceptable AT2 binding. Receptor-ligand binding interactions derived from the structure-activity relationships are discussed with respect to both receptor subtypes.

Nonpeptide angiotensin II antagonists derived from 4H-1,2,4-triazoles and 3H-imidazo[1,2-b][1,2,4]triazoles.

By a variety of synthetic routes, we have synthesized a series of 3,4,5-trisubstituted 4H-1,2,4-triazoles and a related series of 3H-imidazo[1,2-b][1,2,4]triazoles and evaluated them in vitro and in vivo as angiotensin II (AII) antagonists. Principal efforts focused on triazoles bearing an n-alkyl substitutent at C3 and a 4-[(2-carboxybenzoyl)amino]benzyl, (2'-carboxybiphenyl-4-yl)methyl, or [2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl side chain at N4. Among numerous variations at C5, benzylthio groups gave the best potency. Particularly noteworthy was 3-n-butyl-5-[(2-carboxybenzyl)thio]-4-[[2'-(1H-tetrazol-5-yl )biphenyl-4 - yl]methyl]-4H-1,2,4-triazole (71, IC50 1.4 nM), which blocked the AII pressor response in conscious rats at 0.3 mg/kg iv with a duration of action of approximately 6 h, similar to that of DuP 753. Although 71 was active orally only at a 10-fold higher dose level, good oral bioavailability was demonstrated for a monoacidic analogue 62. Most potent among the bicyclic derivatives was 2-n-butyl-5,6-dimethyl-3-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]meth yl]- 3H-imidazo[1,2-b][1,2,4]triazole (93, IC50 7.8 nM). The effects of hydrophobic, hydrogen-bonding, and ionic interactions with the AT1 receptor are considered.

Targeting of peptide and protein drugs to specific sites in the oral route.

Although the distal small intestine has less lumenal and apical proteolytic activities, it has high activities of some apical peptidases. Colonic proteolytic activities are substantial, but their nature is less understood. The small intestine has di- and tripeptide transporter, facilitating absorption, and P-glycoprotein, an efflux pump suggested to limit absorption of small peptides. Several peptide and nonpeptide drugs have higher absorption in the ileum; however, enhancement on their absorption by enhancers varies from site to site. Specific delivery systems can target drugs to the distal intestine utilizing distinct regional pHs and specific microbial enzymes, but the key is how to achieve a reliable release.

Study of high density Escherichia coli fermentation for production of porcine somatotropin protein.

Recombinant E. coli strains and culture conditions were studied for the fermentation expression of porcine somatotropin (PST) inclusion bodies under the control of a pL promoter. Our objective was to achieve high cell density together with a high level of recombinant protein expression. Improved fermentation conditions included oxygen enrichment, yeast extract (YE) effect, optimal specific growth to switch on gene expression, and feeding strategies. To maintain a low residual glucose concentration, a medium feed rate was controlled on a real-time basis by using cell density information estimated from on-line carbon dioxide monitoring of a fermentor's exhaust gas. The optimal specific growth rate required to initiate a temperature shift in our system was found to be around 0.2 hr-1. The cell density and PST expression level could reach 55 OD600 and 35%, respectively, after 16 hours of cultivation under optimal conditions by applying computer-controlled nutrient feed. In our recombinant host/vector system, the location of cl gene appears to affect gene expression under YE-supplemented and/or a high cell density culture condition. With cl gene placed on plasmid, our E. coli host no longer showed sensitivity toward YE in PST gene expression.

Effects of polyacrylic polymers on the lumenal proteolysis of peptide drugs in the colon.

The in-vitro effectiveness of polyacrylic acid polymers in inhibiting degradation of insulin, calcitonin, and insulin-like growth factor-I by colonic lumenal contents was determined. Further, the effect of Carbopol 974P, a polyacrylic acid polymer, on colonic absorption of insulin in rats was studied. The results revealed that Carbopol 934P, 971P, and 974P all strongly inhibited microbial proteolytic activities against insulin, calcitonin, and insulin-like growth factor-I. Inhibition by Carbopol polymers was complete or almost complete when the concentration of each polymer in saline or in 50 or 100 mM Tris buffer was 0.4%, where the pH of the medium was lower than 5. The extensive inhibition by these polyacrylic acid polymers seems to correlate with their ability to acidify the incubation medium. Further, in-situ absorption studies showed that Carbopol 974P increased the pharmacological availability of colonic insulin. In summary, Carbopol polymers are useful in minimizing colonic proteolysis of peptide drugs.

Immunohistochemical localization of insulin-degrading enzyme along the rat intestine, in the human colon adenocarcinoma cell line (Caco-2), and in human ileum.

Insulin-degrading enzyme (IDE) has been implicated in the intracellular degradation of insulin in insulin target cells. Knowledge of the existence of this enzyme in the intestine will be beneficial to the achievement of clinical oral efficacy of insulin. A comparative study was conducted with rat intestine, human colon adenocarcinoma (Caco-2) cells, and human ileum. Confocal microscopy analysis using the anti-IDE antibody showed that IDE was localized in the mucosal cells of rat and human intestines, as well as in Caco-2 cells. Immunostaining of this enzyme was homogeneous throughout the cell excluding nucleus, indicating a typical cytosolic distribution in rat and human enterocytes and in Caco-2 cells.

Comparison of site-dependent degradation of peptide drugs within the gut of rats and rabbits.

Intestinal luminal degradation of neurotensin and acetylneurotensin-(8-13) within the gut of rats and rabbits was compared using brush-border membranes. Patterns of differential proteolysis of these two peptides within the intestine were similar within the same species and between the species. In both rats and rabbits, jejunal brush-border membranes had the highest proteolytic activities degrading neurotensin and acetylneurotensin-(8-13), and caecal or ileocaecal brush-border membranes had the lowest activities. In both species, patterns of site-dependent degradation of neurotensin and acetylneurotensin-(8-13) agreed with the distribution profiles of endopeptidase-24.11 and angiotensin-converting enzyme within the gut. The distal intestine of rats and rabbits has the lowest activities degrading these two compounds. The results demonstrate that distribution of peptidases within the gut will affect site-dependent degradation and absorption of peptide drugs.

The involvement of dipeptidyl peptidase IV in brush-border degradation of GRF(1-29)NH2 by intestinal mucosal cells.

GRF(1-29)NH2 is degraded mainly by dipeptidyl peptidase IV (DPP IV) in plasma, resulting in inactivated GRF(3-29)NH2. To understand whether improving stability of GRF(1-29)NH2 in the plasma will result in enhanced stability in intestinal mucosal cells, stability of GRF(1-29)NH2 and [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)NH2 in rat intestine brush-border membrane and homogenate was examined. [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)NH2, resistant to plasma DPP IV, was much more stable than GRF(1-29)NH2 in enterocytes. Gradient HPLC analysis, mass balance analysis and studies of inhibitor effects revealed that GRF(3-29)NH2 was the major metabolite of GRF(1-29)NH2 due to the action of DPP IV during incubation with brush-border membranes. It is concluded that the design of peptide analogues to resist plasma enzymes dramatically increases stability in intestinal epithelium.