Ketoprofen tissue permeation in swine following cathodic iontophoresis.

BACKGROUND AND PURPOSE: Pharmacokinetic assessment of drug tissue permeation following iontophoresis is limited. The depth of ketoprofen tissue permeation following cathodic iontophoresis (4 mA, 40 minutes) and the stereoselectivity of drug delivery were examined in this study. SUBJECTS: Ketoprofen (750 mg) was iontophoresed onto one porcine medial thigh, with passive drug permeation conducted on the other thigh. METHODS: Skin, subcutaneous fascia, and muscle biopsies from the drug delivery sites were harvested and stored separately, and the "R" and "S" ketoprofen enantiomers were determined. Results. Iontophoretic and passive applications yielded equivalent total ketoprofen concentrations in the skin and fascia. In contrast, multivariate analysis demonstrated that the ketoprofen concentration in the first centimeter of muscle following iontophoresis was greater than the drug concentration in the deeper underlying muscle layers and greater than that delivered to any muscle layer following passive delivery. No transcutaneous stereoselective delivery) of ketoprofen was detected. CONCLUSION AND DISCUSSION: Compared with passive delivery, iontophoresis enhances nonstereoselective ketoprofen permeation into the fascia-muscle interface. With delivery to deeper tissue sites, however, there is no apparent enhancement over passive application.

Studies on the hygiene of drinking water for laboratory animals. 1. The effect of various treatments on bacterial contamination.

Daily autoclaving of drinking-water bottles or daily replacement of their contents resulted in drinking water hygienically acceptable for laboratory rats. However, daily autoclaving of the bottles imposes an additional workload which many institutions cannot afford. The daily replacement of the drinking water is not desirable, since with the usual routines it is virtually impossible to guarantee a bottle is returned to the same cage. A reliable method of preventing bacterial growth for more then 1-2 days in the drinking water of conventional laboratory rats is its acidification with hydrochloric acid to pH 2.3-2.5.

Studies on the hygiene of drinking water for laboratory animals. 2. Clinical and biochemical studies in rats and rabbits during long-term provision of acidified drinking water.

The reaction of rats and rabbits to long-term application of acidified drinking water (pH 2.3-2.5) was observed over a 7-months period. The following parameters were studied: growth curves initiated at weaning, haematology, blood glucose, total serum protein, creatinine, inorganic phosphate, sodium, potassium, calcium, alkaline phosphatase, creatinine kinase, serum glutamate oxalacetate transaminase, and serum glutamate pyruvate transaminase in serum as well as the acid-base status in arterial blood; in addition in rabbits gamma-glutamyl transferase and lactate dehydrogenase were examined in the serum. No significant changes were seen in comparison to the control groups.

Tissue extraction and high-performance liquid chromatographic determination of ketoprofen entantiomers.

Local transcutaneous delivery of non-steroidal anti-inflammatory drugs avoids gastrointestinal side effects and concentrates drugs in the intended tissues. An extraction and HPLC method was developed for ketoprofen in skin, fascia and muscle. Tissue samples were homogenized in NaHCO3. After methylene chloride removal of lipids, the aqueous layer was acidified with HCl and back extracted into isooctane/isopropanol. Ketoprofen was derivatized with ethylchloroformate/S-(-)-alpha-phenylethylamine in triethylamine, then detected by HPLC. Ketoprofen recovery was linear (1-33 microg/g) and was detected in these tissues following in vivo cathodic iontophoresis (160 mA*min). This represents the first non-radioactive method for determination of ketoprofen in tissues following transcutaneous iontophoresis.

Cell wall alterations in staphylococci growing in situ in experimental osteomyelitis.

When cells of both Staphylococcus aureus and Staphylococcus epidermidis are grown in batch culture in nutrient-rich media, their cell walls are regular in thickness, their cell size is within the normal range for each species, and their septation patterns are orderly. When cells of each of these species are examined directly in infected tissue in the rabbit tibia model infection, their cell wall thickness is often much increased and very irregular around the circumference of the cell, their cell size is often increased, and their septation patterns are often severely deranged. All of these alterations in cell wall structure occur in the absence of antibiotics, and we suggest that they may be an expression of phenotypic plasticity in response to altered environmental conditions such as specific nutrient limitations, the presence of antibacterial factors, and growth of the cells on hard surfaces such as rabbit bone or plastic catheters. Some of these specific cell wall alterations are also seen when staphylococcal cells are exposed, in vitro or in vivo, to antibiotics such as clindamycin, but we emphasize that growth in tissue alone is sufficient for their induction.

An electron microscopic study of the effect of clindamycin therapy on bacterial adherence and glycocalyx formation in experimental Staphylococcus aureus osteomyelitis.

After induction of experimental osteomyelitis with Staphylococcus aureus in a rabbit tibia model, clindamycin phosphate (280 mg/kg/day) was used to treat the infected animals for 1, 2 and 3 week periods. Scanning electron microscopy of samples of infected bone tissue taken at necropsy revealed masses of coccoid profiles embedded in a matrix of condensed exopolysaccharide material which adhered to the bone in both infected control animals and in infected animals treated for 1 week with clindamycin phosphate. After 2 and 3 weeks of clindamycin phosphate treatment, the infecting bacteria could not be cultured from tissue samples, and scanning electron microscopy of these samples revealed few coccoid profiles adhering to the bone and marrow. Radiological, microbiological, clinical, histological and electron microscopic findings all indicated recovery from the diseased state with increased length of clindamycin phosphate treatment.

Osteomyelitis experimentally induced with Bacteroides thetaiotaomicron and Staphylococcus epidermidis. Influence of a foreign-body implant.

Experimental osteomyelitis was induced in the rabbit tibia with Staphylococcus epidermidis alone, with Bacteroides thetaiotaomicron alone, and with both bacteria as etiologic agents, in the presence or absence of a foreign-body implant. Animals were monitored by clinical observation and roentgenographic, microbiologic, histologic, immunofluorescent microscopic, and electron microscopic methods. Scanning and transmission electron microscopy showed masses of coccoid and rod-shaped bacteria embedded in a matrix of exopolysaccharide and adhered to bone, marrow, and the foreign-body implant (when present). Of the 58 rabbits receiving an implant, osteomyelitis developed in 48 (83%), and bacteria were recovered by culture from 56 (97%). Of the 31 animals without the implant, osteomyelitis developed in 18 (58%), but no bacteria were recovered by culture. Bacterial recovery appeared to be dependent on the presence of the implant. The rate of induction and the severity of osteomyelitis were enhanced by the presence of the foreign-body implant and by the polymicrobic infection.

Effect of ciprofloxacin on experimental osteomyelitis in the rabbit tibia, induced with a mixed infection of Staphylococcus epidermidis and Bacteroides thetaiotaomicron.

After induction of experimental polymicrobic osteomyelitis with Staphylococcus epidermidis and Bacteroides thetaiotaomicron (ciprofloxacin MIC, 0.5 micrograms/ml and 4.0 micrograms/ml, respectively), in the presence of a foreign body implant, in a rabbit tibia model, ciprofloxacin was administered to infected animals for 2- and 4-week periods. At necropsy, rabbits in the 2-weeks-treated group had mean ciprofloxacin levels of 5.94 micrograms/ml in serum, 3.63 micrograms/g in marrow, and 1.88 micrograms/g in bone. Rabbits in the 4-weeks-treated group had mean ciprofloxacin levels of 7.77 micrograms/ml in serum, 5.84 micrograms/g in marrow, and 2.01 micrograms/g in bone. Quantitative bacterial plate counts were conducted on weighed samples of infected bone, marrow, and the catheter implant, taken at necropsy from treated and control rabbits. Variable reduction of bacterial numbers was observed in samples from treated animals, as compared to untreated controls. Samples of infected bone, marrow and catheter, showed comparable evidence of osteomyelitis and bacterial colonization in both treated and control animals. Although relatively high tissue levels of ciprofloxacin were attained, little therapeutic effect was observed.

Effect of ciprofloxacin on subcutaneous abscesses induced with Staphylococcus epidermidis and a foreign body implant in the mouse.

Subcutaneous abscesses were induced in mice with Staphylococcus epidermidis strain G19-85 and a foreign body implant. The MIC of ciprofloxacin for this strain was 0.25 microgram/ml. The ciprofloxacin dosage, 120 mg/kg/day, was divided into three injections, administered to the mice subcutaneously at 8 h intervals. Serum concentration kinetics in normal mice (n = 50) were determined. The peak serum level of ciprofloxacin was 3.18 micrograms/ml at the 15 min sampling time; the trough level was 0.53 micrograms/ml at 8 h. Abscesses were found in 96% (n = 49) of the untreated, infected control mice. Three modes of treatment with ciprofloxacin were tested: (1) four prophylactic injections of ciprofloxacin prior to infection reduced abscess formation to 64% (p less than or equal to 0.0002, n = 50). (2) Eleven therapeutic injections, initiated 4 days after infection, reduced abscess formation to 86% (p less than or equal to 0.17, n = 49). (3) One prophylactic injection prior to surgery and five therapeutic injections after infection reduced abscess formation to 43% (p less than or equal to 0.0001, n = 49). Culture results correlated with the abscess formation rates.

Ciprofloxacin concentrations in serum, bone and bone marrow of rabbits.

Ciprofloxacin concentrations were determined in serum, bone and bone marrow of rabbits. Four experimental groups of animals were examined: group A (n = 6) received a dosage of 60 mg/kg/day intramuscularly for 4 weeks, groups B (n = 6), C (n = 15) and D (n = 15) received dosages of 120 mg/kg/day subcutaneously for 2 days, 2 weeks, and 4 weeks, respectively. In the kinetic portion of the study, peak serum concentrations of ciprofloxacin measured at the 15 min sampling time were: 2.61 +/- 0.27 micrograms/ml in the 60 mg/kg/day group (group A) and 3.24 +/- 0.78 micrograms/ml in the 120 mg/kg/day group (group B). At necropsy, rabbits in group A had mean ciprofloxacin concentrations of 3.60 +/- 2.27 micrograms/ml in serum, 2.24 +/- 1.19 micrograms/g in marrow and 1.19 +/- 0.44 micrograms/g in bone. Rabbits in group B achieved mean levels of 4.02 +/- 1.23 micrograms/ml in serum, 2.48 +/- 0.79 micrograms/g in marrow, and 1.35 +/- 0.40 micrograms/g in bone. Rabbits in group C achieved mean levels of 5.65 +/- 2.16 micrograms/ml in serum, 3.74 +/- 1.33 micrograms/g in marrow and 1.92 +/- 0.94 micrograms/g in bone. Rabbits in group D achieved mean levels of 7.24 +/- 2.50 micrograms/ml in serum, 4.48 +/- 1.68 micrograms/g in marrow, and 1.93 +/- 0.54 micrograms/g in bone. Differences between mean values for the four experimental groups were not statistically significant.

An electron microscopic study of the effect of clindamycin on adherence of Staphylococcus aureus to bone surfaces.

Discs of rabbit tibia, 5 mm thick, were utilized to study the adherence of Staphylococcus aureus to the bone surface in the presence and absence of clindamycin. Bacteria were grown in broth media containing the bone slices and varying concentrations of clindamycin. In the absence of the antibiotic, S. aureus adhered extensively to bone surfaces and formed large microcolonies which were surrounded by an amorphous matrix. In the presence of 0.025 micrograms/ml of clindamycin (0.1 MIC), S. aureus adhered less to bone surfaces, forming smaller and fewer microcolonies. In the presence of 0.0625 micrograms/ml of clindamycin (0.25 MIC), S. aureus adhered to the bone surfaces only sparsely, forming small microcolonies with very little matrix holding them together, and leaving very large areas of the bone surface uncolonized. In the presence of 0.125 micrograms/ml of clindamycin (0.5 MIC), bone surfaces were basically clean, with only one or two cells (no microcolonies) found in crevices and indentations of the bone surface. In the presence of 0.25 micrograms/ml (1 MIC) no bacteria adhered to the bone surfaces.

Foreign-body-associated experimental osteomyelitis induced with Bacteroides fragilis and Staphylococcus epidermidis in rabbits.

Bacteroides fragilis and Staphylococcus epidermidis, alone and in combination, were used to induce foreign-body-associated osteomyelitis in a rabbit model. In this model, a catheter, used as a foreign body, was implanted into the medullary cavity of the tibia. Only two of five animals infected with S. epidermidis alone developed culture-positive osteomyelitis, whereas all three animals infected with B. fragilis alone developed osteomyelitis. All six animals infected with both microorganisms developed culture-positive osteomyelitis. Roentgenographic and histologic evaluations confirmed the diagnosis of osteomyelitis. Transmission and scanning electron microscopy showed that when the two microorganisms are involved in a mixed infection, S. epidermidis predominates on the foreign body and B. fragilis predominates in the infected bone and marrow.

Bacterial adherence and glycocalyx formation in osteomyelitis experimentally induced with Staphylococcus aureus.

A surgical procedure allowed the placement of a silicone rubber catheter in the marrow cavity of the tibia of a rabbit and also allowed the introduction of a sclerosing agent (sodium morrhuate) and cells of Staphylococcus aureus. Osteomyelitis developed in 60% of the animals so treated, and the infecting microorganism was recovered from the infected tibias of the animals that developed this disease. All blood cultures taken 24 h after the infection were negative for S. aureus. Radiological findings consisted of osteolytic changes, the occurrence of sequestration and periosteal reactions, and sclerosis in the infected bones. Sections of bone prepared for histological examination confirmed the diagnosis of osteomyelitis. Transmission and scanning electron microscopy of samples of bone marrow, bone chips, and the catheters taken from the infected tibiae revealed gram-positive cocci embedded in a very extensive matrix of ruthenium red-staining glycocalyx adhering to the bone and the implanted catheter. It is proposed that this extensive glycocalyx served a protective function for the bacteria and was important in bacterial adherence and thus played an important role in bacterial persistence and the development of osteomyelitis in these rabbits.