Kazusaburo Kataoka

Inhibition of Experimental Abdominal Aortic Aneurysm in the Rat by Use of Decoy Oligodeoxynucleotides Suppressing Activity of Nuclear Factor κB and ets Transcription Factors

Background—Two phenomena, inflammation and matrix degradation, contribute to the progression of abdominal aortic aneurysm (AAA). Importantly, the inflammation is regulated by the transcription factor nuclear factor (NF)–&kgr;B, whereas the destruction and degradation of elastin fibers by matrix metalloproteinases (MMP) are regulated by ets. Thus, we developed a novel strategy to treat AAA by simultaneous inhibition of both NF-&kgr;B and ets by using chimeric decoy oligodeoxynucleotides (ODN). Methods and Results—AAA was induced in rats by transient aortic perfusion with elastase, whereas transfection of decoy ODN was performed by wrapping a delivery sheet containing decoy ODN around the aorta. Gel-mobility shift assay at 7 days after treatment demonstrated that both NF-&kgr;B and ets binding activity were simultaneously inhibited by chimeric decoy ODN. Transfection of chimeric decoy ODN resulted in significant inhibition of the progression of AAA such as aneurysmal dilation at 4 weeks after treatment as compared with control, accompanied by a reduction of MMP expression. Moreover, the destruction of elastin fibers was inhibited in the aorta transfected with chimeric decoy ODN. Importantly, transfection of chimeric decoy ODN demonstrated potent inhibition of aneurysmal dilatation compared with NF-&kgr;B decoy ODN alone, whereas scrambled decoy ODN had no effects. Interestingly, the migration of macrophages was significantly inhibited by chimeric decoy ODN. Conclusions—We demonstrated that inhibition of the progression of AAA was achieved by a novel strategy with chimeric decoy ODN used against NF-&kgr;B and ets in rat model. NF-&kgr;B and ets are considered to play an important role in the pathogenesis of AAA.

Regression of Abdominal Aortic Aneurysms by Simultaneous Inhibition of Nuclear Factor κB and Ets in a Rabbit Model

Because current therapy to treat abdominal aortic aneurysm (AAA), and particularly to manage small AAA, is limited to elective surgical repair, we explored less invasive molecular therapy by simultaneous inhibition of the transcription factors nuclear factor (NF)&kgr;B and ets using a decoy strategy. Both NF&kgr;B and ets were shown to be markedly activated in human AAA. In addition, NF&kgr;B- and ets-positive cells were increased in the aneurysm wall, and a part of the expression of NF&kgr;B and ets was detected in migrating macrophages. Thus, we used chimeric decoy oligodeoxynucleotides (ODNs) containing consensus sequences of both NF&kgr;B and ets binding sites to treat AAA. Inhibitory effects of chimeric decoy ODNs on matrix metalloproteinase-1 and -9 expression were confirmed by ex vivo experiments using a human aorta organ culture. To examine the regressive effect in a rabbit already-formed AAA model, transfection by wrapping a delivery sheet containing chimeric decoy ODNs around the aneurysm was performed 1 week after incubation with elastase. Importantly, treatment with chimeric decoy ODNs significantly decreased the size of AAA. Interestingly, significant preservation of elastic fibers was observed with chimeric decoy ODN treatment, accompanied by a reduction of matrix metalloproteinase-2 and -9 and induction of macrophage apoptosis. Regression of AAA was also associated with an increase in elastin and collagen type I and III synthesis in the aneurysm wall. Minimally invasive molecular therapy targeted to the inhibition of NF&kgr;B and ets is expected to be useful for AAA through the rebalance of matrix synthesis and degradation.

Hypertension Accelerated Experimental Abdominal Aortic Aneurysm Through Upregulation of Nuclear Factor κB and Ets

In this study, we focused on the effect of hypertension on the transcription factors nuclear factor &kgr;B (NF&kgr;B) and ets in the mechanisms of abdominal aortic aneurysm (AAA), and we investigated how hypertension affects the progression of AAA. AAA was produced by elastase perfusion in hypertensive rats and normotensive rats. The size of AAA rapidly increased in hypertensive rats as compared with normotensive rats. Western blot analysis demonstrated that the expression of matrix metalloproteinase (MMP)-2, -3, -9, and -12, as well as intercellular adhesion molecule, was increased in hypertensive AAA rats, accompanied by upregulation of NF&kgr;B and ets. Moreover, in situ zymography showed that the activity of MMPs was increased in the aorta of a hypertensive AAA model as compared with that in a normotensive AAA model. Interestingly, transfection of chimeric decoy oligodeoxynucleotide (ODN) resulted in significant inhibition of aortic dilatation both in normotensive and hypertensive rats at 4 weeks after transfection. Destruction of elastic fibers was also significantly inhibited by transfection of chimeric decoy ODN in both hypertensive rats and normotensive rats. The expression of MMP-2, -3, -9, and -12, as well as intercellular adhesion molecule, was significantly attenuated by the chimeric decoy ODN, accompanied by inhibition of the migration of macrophages. Also, the effect of chimeric decoy ODN was confirmed in an organ culture. The present study demonstrated that hypertension accelerated the progression of experimental AAA through upregulation of NF&kgr;B and ets. Inhibition of NF&kgr;B and ets could be a novel therapeutic strategy to treat AAA in hypertensive patients.

Vitamin A and E Requirements During Total Parenteral Nutrition

Studies were undertaken to determine rational dosages of vitamin A and E during long-term total parenteral nutrition (TPN). Four kinds of vitamin prescriptions containing different amounts of vitamin A and E were prepared from commercially available products and/or hospital pharmacy products. Patients were divided into four groups according to the vitamin prescription used. Plasma vitamin levels of different patient groups were determined by a modified fluorimetric method and were compared with those of a normal subject group. The stability of vitamin A and E in TPN solution after admixing was determining by measuring the remaining vitamin contents by high pressure liquid chromatography. From the results, it was concluded that 1) about 50% of vitamin A was decomposed by sunlight (about 2000 lux) 3 hr after admixing and an orange-colored vinyl cover could protect its photodecomposition; 2) vitamin E was stable at any condition tested; 3) 2500 IU of vitamin A and 15 IU of vitamin E could meet the daily requirements; 4) the plasma levels of vitamin A and E were correlative (p less than 0.01); and 5) concomitant administration of vitamin E was essential to keep the poorer level of vitamin A in plasma.

Verification of surface contamination of Japanese cyclophosphamide vials and an example of exposure by handling

Purpose: Cyclophosphamide (CP) contamination has been detected in Japanese hospitals. In other countries, the surface contamination of CP vials has been reported; however, the manufacturing process of Japanese CP vials is unknown, so the conditions are not necessarily the same as in other countries. This study aimed to establish whether vial surface contamination also occurs in Japan. Method: Contamination of vial surfaces was examined with a wipe test. Urine samples were taken from a pharmacist, engaged solely in dispensing work, for 29 h. It was also investigated whether CP vials were dispensed during the urine sampling period. In addition, vial surfaces, purposely coated with CP and then washed, were examined using wipe tests. Result: CP was detected at 30–60% in vials, which was 11–62 ng (0.10–0.54 ng/cm2). One of the urine samples was contaminated (CP 13.5 ng); this was taken on Day 2 (11:35 AM). CP was not detected among the washed vials. Discussion: This study shows that the surface of Japanese CP vials was contaminated and that it was probable that healthcare workers were exposed to CP. CP absorption by the pharmacist was probably due to dermal uptake while dispensing. Washing the vial is considered effective to avoid CP exposure. Manufacturers should be more proactive to prevent contamination and healthcare workers should comply with exposure prevention rules. Cytotoxic drugs should be included in institution monitoring lists.

Investigation on residual-related error and the effect of solution properties using protective devices for the reconstitution of cytotoxic agents in actual situations

Purpose. Three products can be used in Japan for the reconstitution of cytotoxic agents: PhaSeal, Chemo CLAVE and Chemo Mini Spike (CMS). The low preparation volume may be affected by residual-related volume in their devices. In this study, the residual-related error in their devices was examined and compared. Method. The blank of each component of these devices was weighed using a precision electric balance. After ejecting distilled water (DW) for injection, each was weighed again with the balance. In addition, for etoposide in the cases of PhaSeal and Chemo CLAVE, the components of the devices were similarly weighed. Result. The weight gains of each device after ejecting DW were as follows: CMS-V (440 mg) greater than the combined components of Chemo CLAVE (128—171 mg)/CMS-MT (123 mg) greater than the combined components of PhaSeal (13—56 mg). For etoposide, the weight gains of PhaSeal (208 mg) and Chemo CLAVE (223 mg) showed no significant difference. The priming volume of each device was calculated from the specific gravity of water. The residual-related volume was ‘CMS-V > Chemo CLAVE, CMS-MT > PhaSeal’, although this was very slight in actual situations. Conclusion. The residual-related volume was marked in its low preparation volume. In water-soluble drugs, the residual volume of PhaSeal was lowest of the devices in this study, but in viscous drugs, such as etoposide, the residual volume of PhaSeal was almost identical to Chemo CLAVE; that is, the residual volume of these devices was affected by the solution property. The residual-related volume in the devices will lead to errors; therefore, residual-related errors need to be considered in the use of these devices.

The Environmental Assessment by the Air Cleanliness of the Drug Preparation Room in the Hospital Pharmacy and the Analysis of Influential Factors to Airborne Particle Number.

In this study, the present state of the air cleanliness in the drug preparation room at a hospital pharmacy was evaluated, and factors affecting airborne particle numbers (APN) such as the number and the movement of workers and the materials on working clothes and cloths were investigated. In addition, the effect of environmental conditions on air cleanliness on a clean bench was compared. APN was measured with an Aerosol Particle Counter.The maximum 0.5μm APN values while working in the aseptic preparation room were 3, 610, 1, 312 (less than 10, 000 in GMP) and the non-aseptic room were 8, 008, 2, 660 (less than 100, 000) respectively. The conditions of all rooms were sufficiently suitable for drug preparation according to the criteria of GMP.Concerning factors affecting APN, the movement of workers increased the APN much more than the number of workers. The degree of dispersing particles differed greatly depending on the materials of the working clothes and cloths. A decrease to less than 1 /100 can be obtained by the selection of suitable materials for working clothes such as Overall made of polypropylene non-woven fabric from which few of fibers disperse. It is remarkable that smaller particles are dispersed from clothes even after passing through an air shower. In addition, it was confirmed that the dispersing of particles from cloths and rags was also a problem.As long as prescribed methods were used for the clean bench, the air cleanliness inside the clean bench was kept sufficient even through the external air conditions or locations were not so clean.

Studies on dissolution behaviors of extended-release articles.

The dissolution behavior of fifteen commercial extended-release articles was examined by the dissolution test in order to characterize their behavior in vitro. The articles were classified into six groups according to the formation types. The paddle method for six hours was used for tests, and the media were the first solution (pH 1.2) and the second solution (pH 6.8) in JP XI.Five articles showed different patterns in the above two media, so that their dissolution seemed to depend on pH. A few articles were also effected by pH on the dissolution although they showed time-dependent behavior. The others showed pH-independent (time-dependent) dissolution. The dissolution curves of time-dependent articles approximated to zero-order or firstorder kinetic model. There was, however, no significant relation between the formation type of articles and their dissolution behavior.In addition, for time-dependent articles, simulation curves of dissolution were calculated by using “Weibull function” with the data obtained from the dissolution test up to six hours. Since most of all curves conformed to the observed curves, the parameters of the simulation curves were re-calculated only with the data up to three hours to obtain the prediction curves. The predicted rate fitted well to the observed rate after six hours in many articles. Therefore the prediction curves could be applicable to shorten time for the dissolution test.

Screening Test of Microbiological Status of Solutions for Internal Use and of Radiopaque Media by the Method Described in PAB Notification No.297

The microbial quality control of 41 solutions or suspensions for internal use and of 8 radiopaque media was investigated by the test method described in PAB Notification No.297 (valid on April 1, 1976). All of the 49 pharmaceutical products tested met the standard requirements provided in the Notification, so that they are considered to be under good microbial quality control. Most of the sample solutions of radiopaque media were turbid and were difficult to be put to the test for microbial count. In this point, the test method for the microbial count is desired to be improved. A test method of estimating the possible antimicrobial activity of products is also desired to be standardized, because the antimicrobial activity may affect the final results of the tests in the Notification.

Evaluation of Microbial Contamination of Pharmaceutical Products

Twenty one pharmaceutical products which had shown a positive reaction of microbial contamination in all of the 5 culture media in the previous sterility test were selected for this test. This paper compares the levels of microbial contamination in the latest test with those in the previous test conducted 3 years before, and also analyzes the tendency of the recent quality control of pharmaceutical products in relation to microbial contamination. In the latest study, total aerobic microbial count of more than 10000/g was observed in 3 of the 21 products, showing the same incidence as in the previous test. Total aerobic microbial count of less than 1, 000/g was observed in 15 products, as compared to 10in the previous test. Coliform organisms were detected in 2 products, compered to 6 in the preceding study.