Prevalence, conservation and functional analysis of Yersinia and Escherichia CRISPR regions in clinical Pseudomonas aeruginosa isolates.

Here, we report the characterization of 122 Pseudomonas aeruginosa clinical isolates from three distinct geographical locations: Dartmouth Hitchcock Medical Center in New Hampshire, USA, the Charles T. Campbell Eye Microbiology Lab at the University of Pittsburgh Medical Center, USA, and the Aravind Eye Hospital in Madurai, India. We identified and located clustered regularly interspaced short palindromic repeats (CRISPR) in 45/122 clinical isolates and sequenced these CRISPR, finding that Yersinia subtype CRISPR regions (33 %) were more prevalent than the Escherichia CRISPR region subtype (6 %) in these P. aeruginosa clinical isolates. Further, we observed 132 unique spacers from these 45 CRISPR that are 100 % identical to prophages or sequenced temperate bacteriophage capable of becoming prophages. Most intriguingly, all of these 132 viral spacers matched to temperate bacteriophage/prophages capable of inserting into the host chromosome, but not to extrachromosomally replicating lytic P. aeruginosa bacteriophage. We next assessed the ability of the more prevalent Yersinia subtype CRISPR regions to mediate resistance to bacteriophage infection or lysogeny by deleting the entire CRISPR region from sequenced strain UCBPP-PA14 and six clinical isolates. We found no change in CRISPR-mediated resistance to bacteriophage infection or lysogeny rate even for CRISPR with spacers 100 % identical to a region of the infecting bacteriophage. Lastly, to show these CRISPR and cas genes were expressed and functional, we demonstrated production of small CRISPR RNAs. This work provides both the first examination to our knowledge of CRISPR regions within clinical P. aeruginosa isolates and a collection of defined CRISPR-positive and -negative strains for further CRISPR and cas gene studies.

Prevalence, conservation and functional analysis of Yersinia and Escherichia CRISPR regions in clinical Pseudomonas aeruginosa isolates.

Here, we report the characterization of 122 Pseudomonas aeruginosa clinical isolates from three distinct geographical locations: Dartmouth Hitchcock Medical Center in New Hampshire, USA, the Charles T. Campbell Eye Microbiology Lab at the University of Pittsburgh Medical Center, USA, and the Aravind Eye Hospital in Madurai, India. We identified and located clustered regularly interspaced short palindromic repeats (CRISPR) in 45/122 clinical isolates and sequenced these CRISPR, finding that Yersinia subtype CRISPR regions (33 %) were more prevalent than the Escherichia CRISPR region subtype (6 %) in these P. aeruginosa clinical isolates. Further, we observed 132 unique spacers from these 45 CRISPR that are 100 % identical to prophages or sequenced temperate bacteriophage capable of becoming prophages. Most intriguingly, all of these 132 viral spacers matched to temperate bacteriophage/prophages capable of inserting into the host chromosome, but not to extrachromosomally replicating lytic P. aeruginosa bacteriophage. We next assessed the ability of the more prevalent Yersinia subtype CRISPR regions to mediate resistance to bacteriophage infection or lysogeny by deleting the entire CRISPR region from sequenced strain UCBPP-PA14 and six clinical isolates. We found no change in CRISPR-mediated resistance to bacteriophage infection or lysogeny rate even for CRISPR with spacers 100 % identical to a region of the infecting bacteriophage. Lastly, to show these CRISPR and cas genes were expressed and functional, we demonstrated production of small CRISPR RNAs. This work provides both the first examination to our knowledge of CRISPR regions within clinical P. aeruginosa isolates and a collection of defined CRISPR-positive and -negative strains for further CRISPR and cas gene studies.

Pharmacokinetic evaluation of guar gum-based three-layer matrix tablets for oral controlled delivery of highly soluble metoprolol tartrate as a model drug.

The objective of the present study is to carry out pharmacokinetic evaluation of oral controlled release formulation (guar gum-based three-layer matrix tablets) containing highly soluble metoprolol tartrate as a model drug. Six healthy volunteers participated in the study, and a two-way crossover design was followed. The plasma concentration of metoprolol tartrate was estimated by reverse-phase HPLC. The pharmacokinetic parameters were calculated from the plasma concentration of metoprolol tartrate versus time data. The delayed T(max) lower C(max) decreased K(a) unaltered bioavailability and prolonged t(1/2) indicated a slow and prolonged release of metoprolol tartrate from guar gum three-layer matrix tablets in comparison with the immediate release tablet dosage form. The results of the study indicated that guar gum three-layer matrix tablets were able to provide oral controlled delivery of highly water-soluble drug such as metoprolol tartrate in humans.

In vivo pharmacokinetics in human volunteers: oral administered guar gum-based colon-targeted 5-fluorouracil tablets.

The objective of the present study is to compare the guar gum-based colon-targeted tablets of 5-fluorouracil against an immediate release tablet by in vitro dissolution and in vivo pharmacokinetic studies in human volunteers. Twelve healthy volunteers participated in the study. 5-Fluorouracil was administered at a dose of 50 mg both in immediate release tablet and colon-targeted tablet. On oral administration of colon-targeted tablets, 5-fluorouracil started appearing in the plasma at 6 h, and reached the peak concentration (C(max) of 216+/-15 ng/ml) at 7.6+/-0.1 h (T(max)), whereas the immediate release tablets produced peak plasma concentration (C(max) of 278+/-21 ng/ml) at 0.6+/-0.01 h (T(max)). The AUC(0- infinity ) for 5-fluorouracil from colon-targeted tablet and immediate release tablet were found to be 617+/-39 and 205+/-21 ng/ml/h, respectively. Colon-targeted tablets showed delayed t(max), delayed absorption time (t(a)), decreased C(max) and decreased absorption rate constant when compared to the immediate release tablets. The results of the study indicated that the guar gum-based colon-targeted formulation did not release the drug in stomach and small intestine, but delivered it to the colon resulting in a slow absorption of the drug and making it available for local action in colon.

Pharmacokinetic evaluation of guar gum-based colon-targeted drug delivery systems of mebendazole in healthy volunteers.

The pharmacokinetic evaluation of guar gum-based colon-targeted tablets of mebendazole against an immediate release tablet was carried out in human volunteers. Six healthy volunteers participated in the study and a crossover design was followed. Mebendazole was administered at a dose of 50 mg both in immediate release tablet and colon-targeted tablets. On oral administration of colon-targeted tablets, mebendazole started appearing in the plasma at 5 h, and reached the peak concentration (C(max) of 25.7+/-2.6 ng/ml) at 9.4+/-1.7 h (T(max)) whereas the immediate release tablets produced peak plasma concentration (C(max) of 37.2+/-6.8 ng/ml) at 3.4+/-0.9 h (T(max)). Colon-targeted tablets showed delayed t(max) and absorption time, and decreased C(max) and absorption rate constant when compared to the immediate release tablets. The results of the study indicated that the guar gum-based colon-targeted tablets of mebendazole did not release the drug in stomach and small intestine, but delivered the drug to the colon resulting in a slow absorption of the drug and making the drug available for local action in the colon.

Bioavailability studies on guar gum-based three-layer matrix tablets of trimetazidine dihydrochloride in human volunteers.

Guar gum-based three-layer matrix tablets of a highly water-soluble drug, trimetazidine dihydrochloride, were evaluated for their in vivo release in healthy volunteers in comparison with commercially available conventional immediate release tablets. Six healthy volunteers participated in the study and a two-way crossover design was followed. The plasma concentration of trimetazidine dihydrochloride was estimated by reverse-phase HPLC. The pharmacokinetic parameters were calculated from the plasma concentration of trimetazidine dihydrochloride versus time data. The delayed T(max), decreased C(max) and K(a), unaltered bioavailability, and prolonged t(1/2) and MRT indicated a slow and prolonged release of trimetazidine dihydrochloride from guar gum three-layer matrix tablets in comparison with the immediate release tablet dosage form. The guar gum three-layer matrix tablets of trimetazidine dihydrochloride may be useful in providing constant drug delivery with minimum fluctuations.

Effect of the solvent system on the in vitro permeability of nicardipine hydrochloride through excised rat epidermis.

PURPOSE: The present investigation was carried out to study the effect of the solvent system on the permeation of nicardipine hydrochloride across excised rat epidermis in order to select a suitable solvent system for use in the development of a transdermal therapeutic system. METHODS: The solubility of nicardipine hydrochloride in pure and mixed solvent systems was determined. The solvents used were water, propylene glycol, ethanol or various proportions of ethanol and water. The effect of these pure or mixed solvent systems on the skin permeation of nicardipine hydrochloride was also studied using in vitro permeation studies through excised rat epidermis mounted in modified Keshary-Chien diffusion cells. Fourier Transform Infrared Spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC) studies were carried out to study the effect of these solvents on the biophysical properties of rat' stratum corneum. RESULTS: Although the solubility of nicardipine hydrochloride in propylene glycol (51.23 mg/mL) was higher than that in water (7.90 mg/mL) and ethanol (20.01 mg/mL), the flux of the drug from propylene glycol was as low (7.25+/-0.13 microg/cm(2)/h) as that from water (7.05+/-0.15 microg/cm(2)/h) and lower than that from ethanol (21.51+/-0.81 microg/cm(2)/h). The solubility of nicardipine hydrochloride in binary ethanol-water solvent systems in various proportions was higher than in pure solvents. The highest permeability was observed from an ethanol-water (70:30 v/v) solvent system (56.10+/-1.23 micro g/cm(2)/h) which showed the highest solubility (224.21 mg/mL). The DSC and FT-IR data indicated that the binary solvent system containing ethanol-water in the ratio of 70:30 v/v increased the drug permeability through the skin by disrupting the highly ordered intercellular lipid structure of the stratum corneum in comparison with the untreated stratum corneum. CONCLUSIONS: The results of the study indicate that the use of a binary solvent system, ethanol and water in the ratio of 70:30 v/v, is an effective vehicle for the development of a transdermal therapeutic system for nicardipine hydrochloride.

Penetration enhancing effect of menthol on the percutaneous flux of nicardipine hydrochloride through excised rat epidermis from hydroxypropyl cellulose gels.

The aim of the present investigation is to study the penetration enhancing effect of menthol on the percutaneous flux of nicardipine hydrochloride through the excised rat epidermis from 2% w/w hydroxypropyl cellulose (HPC) gel system. The HPC gel formulations containing nicardipine hydrochloride and selected concentrations of menthol (0-12% w/w) were prepared, and evaluated for in vitro permeation of the drug through excised rat abdominal epidermis. The percutaneous flux of nicardipine hydrochloride across rat epidermis was enhanced markedly by the addition of menthol to the HPC gels. A maximum flux of nicardipine hydrochloride (227.70 +/- 1.30 micrograms cm-2 hr-1) was observed with an enhancement ratio of 7.12 when menthol was incorporated at a concentration of 8% w/w in a reservoir HPC system. The differential scanning calorimetry and Fourier transform-infrared spectroscopy data indicated that menthol increased the percutaneous flux of nicardipine hydrochloride through the rat skin by partial extraction of lipids in the stratum corneum. The results suggest that menthol may be useful for increasing the skin permeability of nicardipine hydrochloride from transdermal therapeutic system containing HPC gel as a reservoir.

A three-layer guar gum matrix tablet for oral controlled delivery of highly soluble metoprolol tartrate.

The objective of the study is to design oral controlled drug delivery systems for highly water-soluble drugs using guar gum as a carrier in the form of a three-layer matrix tablet. Metoprolol tartrate was chosen as a model drug because of its high water solubility. Matrix tablets containing either 30 (M1), 40 (M2) or 50% (M3) of guar gum were prepared by wet granulation technique using starch paste as a binder. Three-layer matrix tablets of metoprolol tartrate were prepared by compressing on both sides of guar gum matrix tablet granules of metoprolol tartrate M1, M2 or M3 with either 50 (TL1M1, TL1M2 or TL1M3) or 75 mg (TL2M1, TL2M2 or TL2M3) of guar gum granules as release retardant layers. Both the matrix and three-layer matrix tablets were evaluated for hardness, thickness, drug content uniformity, and subjected to in vitro drug release studies. The amount of metoprolol tartrate released from the matrix and three-layer matrix tablets at different time intervals was estimated by using a HPLC method. Matrix tablets of metoprolol tartrate were unable to provide the required drug release rate. However, the three-layer guar gum matrix tablets (TL2M3) provided the required release rate on par with the theoretical release rate for metoprolol tartrate formulations meant for twice daily administration. The three-layer guar gum matrix tablet (TL2M3) showed no change either in physical appearance, drug content or in dissolution pattern after storage at 40 degrees C/75% RH for 6 months. The FT-IR study did not show any possibility of metoprolol tartrate/guar gum interaction with the formulation excipients used in the study. The results indicated that guar gum, in the form of three-layer matrix tablets, is a potential carrier in the design of oral controlled drug delivery systems for highly water-soluble drugs such as metoprolol tartrate.

In vitro drug release studies on guar gum-based colon targeted oral drug delivery systems of 5-fluorouracil.

Intravenous administration of 5-fluorouracil for colon cancer therapy produces severe systemic side-effects due to its cytotoxic effect on normal cells. The broad objective of the present study was to develop novel tablet formulations for site-specific delivery of 5-fluorouracil to the colon without the drug being released in the stomach or small intestine using guar gum as a carrier. Fast-disintegrating 5-fluorouracil core tablets were compression coated with 60% (FHV-60), 70% (FHV-70) and 80% (FHV-80) of guar gum, and were subjected to in vitro drug release studies. The amount of 5-fluorouracil released from the compression-coated tablets in the dissolution medium at different time intervals was estimated by a HPLC method. Guar gum compression-coated tablets released only 2.5-4% of the 5-fluorouracil in simulated GI fluids. When the dissolution study was continued in simulated colonic fluids (4% w/v rat caecal content medium) the compression-coated FHV-60, FHV-70 and FHV-80 tablets released another 70, 55 and 41% of the 5-fluorouracil respectively. The results of the study show that compression-coated tablets containing 80% (FHV-80) of guar gum are most likely to provide targeting of 5-fluorouracil for local action in the colon, since they released only 2.38% of the drug in the physiological environment of the stomach and small intestine. The FHV-80 formulation showed no change either in physical appearance, drug content or dissolution pattern after storage at 40 degrees C/RH 75% for 6 months. The differential scanning calorimetric study showed that 5-fluorouracil did not interact with the formulation excipients used in the study.

Studies on the development of colon-targeted delivery systems for celecoxib in the prevention of colorectal cancer.

The nonsteroidal anti-inflammatory drugs (NSAIDs) are found to be potential chemopreventive agents of colorectal cancer. Celecoxib, an NSAID with selective cyclooxygenase-2 inhibition, was proved to be effective for the prevention of colon cancer in patients with familial adenomatous polyposis (FAP) and sporadic polyps. In the light of this information, the present study was carried out to develop oral colon-targeting drug delivery systems for celecoxib using guar gum as a carrier. Matrix tablets containing various proportions of guar gum were prepared by wet granulation technique using starch paste as a binder. The tablets were evaluated for hardness, drug content and were subjected to in vitro drug release studies. The amount of celecoxib released from the matrix tablets at different time intervals was estimated by a HPLC method. Guar gum matrix tablets released only 2-4% of celecoxib in the physiological environment of stomach and small intestine depending on the proportion of guar gum used in the formulation. When the dissolution study was continued in simulated colonic fluids (rat caecal content medium), the matrix tablets containing 20% of guar gum released another 37% of celecoxib after degradation by the colonic bacterial action. The matrix tablets containing 30% of guar gum released about 24% of celecoxib in simulated colonic fluids indicating the susceptibility of the guar gum formulations to the rat caecal contents. The results of the study show that the matrix tablets containing either 20 or 30% of guar gum are most likely to target celecoxib for local action in the colon. The guar gum matrix tablets of celecoxib showed no change either in physical appearance, drug content or in dissolution pattern after storage at 40 degrees C/RH 75% for 6 months. Differential scanning calorimetry (DSC) studies indicated no possibility of interaction between celecoxib and guar gum/other formulation excipients.

Three-layer guar gum matrix tablet formulations for oral controlled delivery of highly soluble trimetazidine dihydrochloride.

The present study is carried out to design oral controlled drug delivery systems for highly water-soluble drugs using guar gum as a carrier in the form of three-layer matrix tablets. Trimetazidine dihydrochloride was chosen as a model drug because of its high water solubility. Matrix tablet granules containing 30% (M1), 40% (M2) or 50% (M3) of guar gum were prepared by the wet granulation technique using starch paste as a binder. Three-layer matrix tablets of trimetazidine dihydrochloride were prepared by compressing on either side of guar gum matrix tablet granules of trimetazidine dihydrochloride M1, M2 or M3 with 200 mg of guar gum granules containing either 65% of guar gum (T1M1, T1M2 or T1M3), 75% of guar gum (T2M1, T2M2 or T2M3) or 85% of guar gum (T3M1, T3M2 or T3M3) as release retardant layers. The three-layer matrix tablets were evaluated for hardness, thickness, drug content uniformity, and were subjected to in vitro drug release studies. The amount of trimetazidine dihydrochloride released from the matrix and three-layer matrix tablets at different time intervals was estimated using a HPLC method. The three-layer guar gum matrix tablet (T3M3) provided the required release rate on par with the theoretical release rate for guar gum formulations meant for twice daily administration. The three-layer guar gum matrix tablet (T3M3) showed no change either in physical appearance, drug content or in dissolution pattern after storage at 40 degrees C/RH 75% for 6 months. The DSC study did not show any possibility of interaction between trimetazidine dihydrochloride and guar gum/other formulation excipients used in the study. The results indicated that guar gum, in the form of three-layer matrix tablets, is a potential carrier in the design of oral controlled drug delivery systems for highly water-soluble drugs such as trimetazidine dihydrochloride.

Studies on the development of oral colon targeted drug delivery systems for metronidazole in the treatment of amoebiasis.

The aim of the present study is to develop colon targeted drug delivery systems for metronidazole using guar gum as a carrier. Matrix, multilayer and compression coated tablets of metronidazole containing various proportions of guar gum were prepared. All the formulations were evaluated for the hardness, drug content uniformity, and were subjected to in vitro drug release studies. The amount of metronidazole released from tablets at different time intervals was estimated by high performance liquid chromatography method. Matrix tablets and multilayer tablets of metronidazole released 43-52% and 25-44% of the metronidazole, respectively, in the physiological environment of stomach and small intestine depending on the proportion of guar gum used in the formulation. Both the formulations failed to control the drug release within 5 h of the dissolution study in the physiological environment of stomach and small intestine. The compression coated formulations released less than 1% of metronidazole in the physiological environment of stomach and small intestine. When the dissolution study was continued in simulated colonic fluids, the compression coated tablet with 275 mg of guar gum coat released another 61% of metronidazole after degradation by colonic bacteria at the end of 24 h of the dissolution study. The compression coated tablets with 350 and 435 mg of guar gum coat released about 45 and 20% of metronidazole, respectively, in simulated colonic fluids indicating the susceptibility of the guar gum formulations to the rat caecal contents. The results of the study show that compression coated metronidazole tablets with either 275 or 350 mg of guar gum coat is most likely to provide targeting of metronidazole for local action in the colon owing to its minimal release of the drug in the first 5 h. The metronidazole compression coated tablets showed no change either in physical appearance, drug content or in dissolution pattern after storage at 40 degrees C/75% RH for 6 months.

In vivo evaluation of guargum-based colon-targeted oral drug delivery systems of celecoxib in human volunteers.

The present study involved the in vivo evaluation of orally administered guar gum-based colon-targeted tablet formulations of celecoxib (colon-targeted tablet-20 or colon-targeted tablet-30) as compared with an immediate release capsule in 15 human volunteers. Blood samples were obtained at different time intervals and the plasma concentration of celecoxib was estimated by reversed phase HPLC. The immediate release capsules of celecoxib might have disintegrated very fast in GI tract and absorbed quickly from stomach and small intestine thereby producing peak plasma concentration (Cmax of 478 +/- 57 ng/ml) within 3.8 +/- 0.1 h (Tmax). Though celecoxib could be seen in plasma after oral administration of colon-targeted tablet-20 or colon-targeted tablet-30 between 1 and 2 h, low levels of drug were observed upto 8 h resulting in peak concentration (Cmax) of 78 +/- 6 ng/ml or 88 +/- 15 ng/ml at 10.5 +/- 1.9 h or 13.5 +/- 1.4 h (Tmax) respectively, whereas the immediate release capsules produced peak plasma concentration (Cmax) of 478 +/- 57 ng/ml at 3.8 +/- 0.1 h (Tmax). Colon-targeted tablets showed decreased AUC(0-infinity), Cmax and absorption rate constant, prolonged absorption time (ta), and increased t1/2 in comparison with the immediate release capsules. The results of the study indicated that the guar gum-based colon-targeted tablets of celecoxib did not release the drug significantly in stomach and small intestine, but delivered to the colon resulting in a slow absorption of the drug and making it available for local action in the colon.

Guar gum as a carrier for colon specific delivery; influence of metronidazole and tinidazole on in vitro release of albendazole from guar gum matrix tablets.

PURPOSE: The present investigation is to study the influence of metronidazole and tinidazole on the usefulness of guar gum, a colon-specific drug carrier based on the metabolic activity of colonic bacteria, using matrix tablets of albendazole (containing 20% of guar gum) as a model formulation. METHODS: The matrix tablets of albendazole were subjected to in vitro drug release studies in simulated colonic fluids (4%w/v of rat caecal contents) obtained after oral treatment of rats for 7 days either with varying doses of metronidazole/ tinidazole and 1 mL of 2%w/v of guar gum or with 1 mL of 2%w/v of guar gum alone (control study) after completing the dissolution study in 0.1 M HCl (2 h) and pH 7.4 Sorensen's phosphate buffer (3 h). RESULTS: The guar gum matrix tablets of albendazole were found degraded by colonic bacteria of rat caecal contents and released about 44% of albendazole in simulated colonic fluids (control study) at the end of 24 h indicating the susceptibility of the guar gum formulations to the rat caecal contents. However, the release of albendazole decreased when the drug release studies were carried out in caecal contents of rats treated for 7 days with either metronidazole (10-50 mg/ kg once daily) or tinidazole (10-30 mg/ kg once daily), and the release of albendazole from the matrix tablets was found to be dose dependent. The release of the drug from guar gum formulations was found to increase with a decrease in the dose of metronidazole/tinidazole administered. The antimicrobial activity of metronidazole/ tinidazole against the anaerobic bacteria of the rat"s GI flora might have been inhibited to a varying degree depending on the dose of metronidazole/tinidazole administered. CONCLUSIONS: The results of the study showed that concomitant administration of either metronidazole or tinidazole with guar gum based colon-specific drug delivery systems may interfere with the targeting of drugs to colon.