Biocompatible materials developments for new medical implants.

Recent work on modifying silicone rubber to improve water permeability and biocompatibility is described. In addition, modifications to the interface between an active implanted device and the body are reported, which have led to reduced power consumption and improved device performance.

Fibrin glue in the management of anal fistulae.

OBJECTIVE: Fibrin glue has been used as a sphincter sparing approach for the treatment of anal fistulae for two decades. However, there is uncertainty about its short and long-term efficacy. The objective of this review was to ascertain the role of fibrin glue in the management of anal fistulae, including assessment of recurrence rates, continence disturbance and other complications. METHODS: We searched Medline (January 1966 to February 2004), the Cochrane database, and EMBASE using the terms anal fistulae, fistula-in-ano, and fibrin glue. Relevant papers from the reference lists of these articles and from the authors' personal collections were also reviewed. A systematic review of all articles relating to the use of fibrin glue in the treatment of anal fistulae was performed. This included 19 studies. Reviewers performed data extraction independently. Outcomes evaluated included recurrence rates, continence disturbance, septic complications, adverse drug reactions, and duration of follow-up. Heterogeneity of the clinical trials made direct comparisons difficult and meta-analysis impossible. RESULTS: The success rates reported in published studies range from 0% to 100%. Differences in patient selection (including fistula aetiology and type), treatment protocols, and follow-up duration may contribute to such diverse results. CONCLUSIONS: Fibrin glue is simple to use, has a minimal morbidity and should not affect later treatment options in the event of its failure. It is therefore theoretically attractive as a first line treatment in the management of those types of anal fistula in which it has been shown to work. However, further research into 'biological' glues is merited and these subject to randomised controlled study.

A rapid spectrophotometric assay for tetracycline in gingival crevicular fluid.

AIM: The aim of this study was to investigate a rapid spectrophotometric assay for its potential to measure tetracycline levels in gingival crevicular fluid (GCF). MATERIALS AND METHODS: The technique involves complexation of tetracycline with molybdenum in order to shift the absorbance spectrum away from that region where interference with plasma proteins is a problem. The sensitivity of the assay and reproducibility of elution were examined together with an assessment of the effect of plasma proteins. The assay was also tested in a small pilot clinical project, measuring tetracycline levels in GCF following placement of a test gel formulation in 25 periodontal pockets in 5 patients. RESULTS: The in vitro results showed good sensitivity of the assay over the concentration range tested (0.5-200 microg tetracycline) and with little effect of plasma proteins. Elution from the paper strips was reproducible with a good linear correlation between direct and filter absorbed assays (r=0.9989, p<0.01). The pilot clinical study indicated a mean half-time of tetracycline in GCF of 28 min with confidence intervals of 21 to 34 min, although wide variation between the drug levels of individual periodontal pockets was seen. CONCLUSIONS: The results indicate good sensitivity for this assay to measure tetracycline hydrochloride in vivo. The potential for rapidly processing large numbers of samples contrasts with the assay time and limited sample throughput of other methods such as high pressure liquid chromatography (HPLC) and suggests that the technique may be a useful addition to current techniques for measuring tetracycline hydrochloride in vivo.

Significantly increased lesion size by using the near-infrared photosensitizer 5,10,15,20-tetrakis (m-hydroxyphenyl)bacteriochlorin in interstitial photodynamic therapy of normal rat liver tissue.

BACKGROUND AND OBJECTIVE: Penetration of tissues by activating light ultimately limits the size of the lesions achievable in interstitial photodynamic therapy. Measurements of the wavelength-dependence of tissue optical properties suggest that substantial improvements may be possible, particularly in pigmented organs such as the liver, by using drugs absorbing at near infrared wavelengths. STUDY DESIGN/MATERIALS AND METHODS: In this study, the extent of light induced necrosis with the photosensitive agents Photofrin (activated at 632 nm), meta-tetra(hydroxyphenyl)chlorin (mTHPC) (activated at 652 nm) and 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin (mTHPBC) (activated at 740 nm) are compared in normal rat liver. Interstitial irradiation of mTHPBC-sensitized liver tissue resulted in significantly larger necrotic areas than irradiation of Photofrin and mTHPC-sensitised livers. CONCLUSION: The results illustrate the advantage of near-infrared photosensitizer activation and point to a specific role for mTHPBC in the interstitial treatment of liver tumours.

In vivo photodynamic characteristics of the near-infrared photosensitizer 5,10,15,20-tetrakis(M-hydroxyphenyl) bacteriochlorin.

This paper describes the photodynamic characteristics of the new near-infrared photosensitizer 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin (mTHPBC or SQN400) in normal rat and mouse tissues. A rat liver model of photodynamic tissue necrosis was used to determine the in vivo action spectrum and the dose-response relationships of tissue destruction with drug and light doses. The effect of varying the light irradiance and the time interval between drug administration and light irradiation on the biological response was also measured in the rat liver model. Photobleaching of mTHPBC was measured and compared with that of its chlorine analog (mTHPC) in normal mouse skin and an implanted mouse colorectal tumor. The optimum wavelength for biological activation of mTHPBC in rat liver was 739 nm. mTHPBC was found to have a marked drug-dose threshold of around 0.6 mg kg-1 when liver tissue was irradiated 48 h after drug administration. Below this administered drug dose, irradiation, even at very high light doses, did not cause liver necrosis. At administered doses above the photodynamic threshold the effect of mTHPBC-PDT was directly proportional to the product of the drug and light doses. No difference in the extent of liver necrosis produced by mTHPBC was found on varying the light irradiance from 10 to 100 mW cm-2. The extent of liver necrosis was greatest when tissue was irradiated shortly after mTHPBC administration and necrosis was absent when irradiation was performed 72 h or later after drug administration, suggesting that the drug was rapidly cleared from the liver. In vivo photobleaching experiments in mice showed that the rate of bleaching of mTHPBC was approximately 20 times greater than that of mTHPC. It is argued that this greater rate of bleaching accounts for the higher photodynamic threshold and this could be exploited to enhance selective destruction of tissues which accumulate the photosensitizer.

Biodistribution and bioactivity of tetra-pegylated meta-tetra(hydroxyphenyl)chlorin compared to native meta-tetra(hydroxyphenyl)chlorin in a rat liver tumor model.

It has been proposed that the construction of a photosensitizer-polymer conjugate would lead to an increased selective retention of the drug in tumor tissue resulting in an enhancement of selective tumor destruction by light in photodynamic therapy. In this study the kinetics of a tetra-pegylated derivative of meta-tetra(hydroxyphenyl)chlorin (mTHPC-PEG) were compared with those of native meta-tetra(hydroxyphenyl)chlorin (mTHPC) in a rat liver tumor model. In addition, the time course of bioactivity of both drugs was studied in normal liver tissue. Pegylation of mTHPC resulted in a two-fold increase in the plasma half-life time, a five-fold decrease in liver uptake and an increase in the tumor selectivity at early time intervals after drug administration. However, although mTHPC concentrations in liver decrease rapidly with time, mTHPC-PEG liver concentrations increased as a function of time. This led to a loss of tumor selectivity at all but the earliest time points, whereas with mTHPC tumor selectivity increased with time. For both drugs the time course of bioactivity in the liver parallels drug concentration levels with extensive necrosis after irradiation of mTHPC-PEG-sensitized liver tissue up to drug-light intervals of 120 h. It is concluded that on balance mTHPC-PEG does not appear to show any benefits over native mTHPC for the treatment of liver tumors, as normal liver tissue accumulates the compound. However, pegylation is a potentially promising strategy with an increase in tumor selectivity and reduced liver uptake if accumulation in the liver can be prevented.

mTHPC Polymer Conjugates: The In Vivo Photodynamic Activity of Four Candidate Compounds.

The in vivo photodynamic activities of four poly(ethylene glycol) (PEG) conjugates of the photosensitiser 5,10,15,20-tetrakis-(m-hydroxyphenyl)chlorin (mTHPC, temoporfin, Foscan(®)) were compared with that of mTHPC over a range of drug-light intervals using acute tumour necrosis and skeletal muscles swelling in a mouse model in order to ascertain the influence of linking group stability and PEG chain length on the photodynamic activity. The four compounds examined contained either PEG 2000 or PEG 5000 attached by carbonate or triazine linkages at the phenol hydroxyl groups of the mTHPC.All compounds tested caused tumour necrosis at drug-light intervals of between one and four days. mTHPC produced tumour necrosis of over 5 mm at drug-light intervals of 1 and 2 days with limited muscle damage at early drug-light intervals. The relatively labile carbonate-linked conjugates gave tumour necrosis similar to mTHPC but produced severe muscle and systemic phototoxicity on irradiation at 4-24 h after injection. The more stable triazine-linked conjugates produced no significant muscle damage at any of the drug-light intervals tested, but gave only limited tumour necrosis under the conditions tested. PEG chain length had relatively little effect on the patterns of bioactivity.It is concluded that both classes of mTHPC PEG conjugates may be suitable for photodynamic therapy if the problems of stability and early photosensitivity in the case of the carbonates and reduced potency in the case of the triazines can be overcome through improved formulations and PDT treatment regimens.

Mitochondrial electron transport chain activity, but not ATP synthesis, is required for drug-induced apoptosis in human leukaemic cells: a possible novel mechanism of regulating drug resistance.

There is increasing evidence for an association between mitochondrial function and susceptibility to apoptosis. It has been shown that the vinblastine-resistant leukaemic cell line CEM/VLB100 has a more active mitochondrial electron transport chain (ETC) than the parental CCRF-CEM cell line. Inhibition of mitochondrial DNA replication by ethidium bromide (EB) depleted the activity of the ETC and reduced cellular respiratory rate. Depletion of mitochondrial DNA was associated with increased resistance to vinblastine-induced apoptosis in both cell lines. In contrast, the highly specific inhibitor of the energy producing mitochondrial enzyme F1Fzero-ATPase, oligomycin, rendered CEM/VLB100 cells more sensitive to vinblastine by inhibiting the energy-dependent P-glycoprotein (Pgp) pump, suggesting that the effect of EB is independent of energy generation and ATPase activity. Both mitochondrial ETC depletion and ATPase inhibition decreased vinblastine-induced cell cycle changes in the CCRF-CEM cell line, suggesting that cell cycle changes are dependent on ATP generation. However, EB-induced ETC depletion in CEM/VLB100 cells inhibited apoptosis in response to high concentration of vinblastine, but not G2M arrest. We suggest that: (1) over-expression of Pgp by drug-resistant cells may up-regulate mitochondrial energy production; (2) mitochondrial ETC activity is required for DNA fragmentation in response to vinblastine, but the mechanism is independent of Pgp activity and ATP generation; (3) down-regulation of mitochondrial ETC activity may confer resistance to vinblastine-induced apoptosis; (4) the mitochondrial ETC is involved in vinblastine-induced apoptosis downstream of microtubule disruption and cell cycle changes.

Metabolic adaptation of terminal ileal mucosa after construction of an ileoanal pouch.

BACKGROUND: The major nutrients for the large bowel and small bowel mucosa are, respectively, butyrate and glutamine. The degree of mucosal adaptation that may occur in response to changes in nutrient supply and faecal stasis after the formation of an ileoanal pouch is poorly understood. METHOD: The ability of ileal mucosal biopsies, from nine patients with ulcerative colitis and from 18 with an ileoanal pouch, to oxidize [14C]-glucose, glutamine and butyrate to carbon dioxide was quantified. RESULTS: Glucose, glutamine and butyrate were oxidized respectively at a median of 12.5 (95 per cent confidence interval (4-22), 77 (34-207) and 194 (81-321) pmol microgram-1 h-1 by ileal mucosa and 12.9 (6-21), 35 (11-57) and 194 (73-737) pmol microgram-1 h-1 by pouch mucosa. CONCLUSION: Ileoanal pouch construction and subsequent bacterial colonization and faecal stasis resulted in a significant (P < 0.05) reduction in the mucosal ability to oxidize glutamine whereas there was no difference in the rate of butyrate oxidation.

Characterization of a murine model for the rapid assessment of acute photodynamic response in tumour and muscle.

A murine implanted colorectal tumour model is described in which a measurement of tumour depth of necrosis is combined with a simultaneous measurement of muscle damage. The response of this model to a range of photodynamic therapy parameters was characterized using the chlorin photosensitizer mTHPC (temoporfin, Foscan(R)R). Both tumour depth of necrosis (measured directly and on histology) and muscle swelling correlate with the dose of photosensitizer and light, and are shown to be repeatable and consistent with published values obtained under similar conditions using established models.

Tissue photosensitizer detection by low-power remittance fluorimetry.

A simple adaptation of a commercial spectrofluorimeter which allows the semiquantitative determination of photodynamic therapy photosensitizer fluorescence in accessible tissues is described. Light from a xenon lamp is directed via a monochromator onto the tissue surface by a bifurcated random fibre bundle. Tissue fluorescence is directed to the emission monochromator and photomultiplier of the fluorimeter by the second limb of the fibre bundle. Although relatively simple, this device can be used to carry out a wide range of useful measurements in clinical and experimental photodynamic therapy. The sensitivity and reproducibility of the measurements were determined using mouse tumour and muscle tissue fluorescence measured in vivo compared with photosensitizer content measured by high performance liquid chromatography. As an illustration of the potential applications of such systems, the time courses of fluorescence in the skin of patients treated with the photosensitizers Photofrin(R) and metatetra(hydroxyphenyl)chlorin (mTHPC) (temoporfin) and the photobleaching of 5-aminolaevulinic acid-derived protoporphyrin IX during treatment, are described.

A comparison of novel light sources for photodynamic therapy.

A diode laser, light-emitting diode (LED) array bandwidth 25 nm, full width half maximum (FWHM) and filtered arc lamp (bandwidth 40 nm, FWHM), all with peak emission at about 650 nm, suitable for the photosensitizer tetra(meta-hydroxyphenyl)chlorin (mTHPC), were compared with a copper vapour laser pumped dye laser, using depth of necrosis in normal rat liver as a measure of photodynamic effect.A three-way comparison between a DL10K dye laser, the LED array and the filtered arc lamp resulted in mean depths of necrosis of 4.64, 4.29 and 4.04 mm, respectively, at 20 J cm(-2), the values for the laser and arc lamp being significantly different at the 5% level. A further comparison of a narrower linewidth DL20K dye laser with the LED array, using a light dose of 20 J cm(-2), showed a significant difference between the mean depths of necrosis of 4.97 and 4.05 mm, respectively (p=0.01).A final study, comparing the DL20K dye laser with the diode laser and a light dose of 10 J cm(-2), demonstrated no significant difference in depths of necrosis (3.23 and 3.25 mm, respectively). The results obtained in the three studies are attributed to the relative bandwidths of light emission for the various sources. A simple mathematical model is presented explaining the results in terms of the relative activation of the photosensitizer and the consequent threshold fluence required for the induction of necrosis.It is concluded that, in order to achieve the same depth of effect as a laser when using the broad band sources, the incident fluence would have to be approximately doubled. However, when the low cost and ease of use of the non-laser sources are taken into consideration, these devices are likely to find widespread applications in clinical photodynamic therapy.

Haematoporphyrin derivative--photodynamic therapy of colorectal carcinoma, sensitized using verapamil and adriamycin.

The p-glycoprotein export mechanism may have an effect on the cytotoxicity of chemotherapy or photodynamic therapy (PDT) by reducing cytotoxic drug or photosensitizer concentration within cells. In tissues over-expressing this protein, modulation with verapamil (an antagonist of p-glycoprotein) may be useful in reversing this form of treatment resistance. This study examined the bioactivity of the interaction of photodynamic therapy using Haematoporphyrin derivative (HpD), chemotherapy and the response modifier verapamil. Multicellular spheroids derived from the human colorectal cancer line HRT 18 were used in vitro and bioactivity assessed using growth retardation. Bioactivity was observed to be greatest when all three agents and light irradiation were combined. This application may be clinically useful in the treatment of colorectal carcinoma by improving the efficacy of PDT using HpD.

Distribution and excretion of radiolabeled temoporfin in a murine tumor model.

The biodistribution and excretion of temoporfin (tetra[m-hydroxyphenyl]chlorin, m-THPC), a recently developed photosensitizer, was investigated in BALB/c mice. [14C]temoporfin was administered intravenously (0.73 mumol/kg) to tumor-free mice or to mice implanted with the Colo 26 colorectal carcinoma. Blood, tissue and fecal samples were collected for 35 days and 10 days postdose from tumor-free mice and tumor-bearing mice, respectively. Blood concentrations fell rapidly such that at later time points they were indistinguishable from background counts. Tumor concentrations rose to a peak of 0.34 microgram temoporfin equivalents/mL at 2 days and then declined in parallel (log plot) with the blood concentrations. Tumor: tissue ratios at 2 days for skin, adipose tissue and skeletal muscle underlying the tumor were 1.5, 2.3 and 3.8, respectively. By 4 days the corresponding values were 1.6, 3.4 and 4.0. Nearly 40% of the administered radioactivity was excreted in the feces in the first 24 h and more than 80% had been excreted by 20 days. Less than 0.2% of the dose was recovered from the urine. An elimination half-life of 10-12 days was calculated from the excretion data.

Increased activity and sensitivity of mitochondrial respiratory enzymes to tumor necrosis factor alpha-mediated inhibition is associated with increased cytotoxicity in drug-resistant leukemic cell lines.

The drug-resistant leukemic cell lines, CEM/VLB100 and K/DAU600, are more sensitive to tumor necrosis factor alpha (TNFalpha)-mediated cytotoxicity compared with their parental cell lines, CCRF-CEM and K562 cl.6. Drug-resistant leukemic cell lines have more active mitochondrial function, which is associated with a greater susceptibility to TNFalpha-induced respiratory inhibition. TNFalpha blocked electron transfer at three sites, NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), and cytochrome c oxidase (complex IV). Respiratory rate and electron transport chain enzyme activities were significantly inhibited in the drug-resistant, TNF-sensitive cell lines. Respiratory inhibition preceded cell death by at least 5 to 8 hours. The respiratory failure was not compensated for by appropriate up-regulation of the glycolytic pathway. Increasing mitochondrial respiratory rate and enzyme activities by long-term culture with 2 mmol/L adenosine 5'-diphosphate (ADP) and Pi sensitized both drug-sensitive and drug-resistant cells to TNFalpha-induced cytolysis. Intramitochondrial free radicals generated by paraquat only had a limited and delayed effect on respiratory inhibition and cytolysis in comparison with the effect of TNFalpha. We conclude that TNFalpha-induced cytotoxicity in leukemic cells is, at least in part, mediated by inhibition of mitochondrial respiration. Free radical generation by TNFalpha may not directly lead to the observed inhibition of the mitochondrial electron transport and other mechanisms must be involved.

Butyrate metabolism in the terminal ileal mucosa of patients with ulcerative colitis.

The rate of oxidation of butyrate, glutamine and glucose was investigated in terminal ileal mucosal biopsy samples from nine patients with ulcerative colitis undergoing restorative proctocolectomy and from 12 patients undergoing laparotomy for reasons other than ulcerative colitis. Substrate oxidation was assayed using a radiolabelled isotope technique. Butyrate was the preferred fuel substrate, followed by glutamine and then glucose (median (95 per cent confidence interval) 567 (262-894), 63 (35-123) and 8.1 (5.1-18) pmol micrograms-1 h-1 respectively; P < 0.01, Mann-Whitney U test) in normal terminal ileal mucosa. The patients with ulcerative colitis had a significantly reduced rate of butyrate oxidation compared with the control group (194 (81-321) versus 567 (262-894) pmol micrograms-1 h-1, P < 0.05). Normal terminal ileal mucosa oxidized butyrate in greater quantities than glucose and glutamine. Ulcerative colitic terminal ileal mucosa exhibited an impaired rate of butyrate oxidation.

Adjuvant intraoperative photodynamic therapy for colorectal carcinoma: a clinical study.

The local recurrence rate of colorectal carcinoma after surgery is unacceptable in most series, and adjuvant therapies have made only a small impact on this. There is experimental evidence that adjuvant intraoperative photodynamic therapy (AIOPDT) may be effective. AIOPDT involves systematically photosensitizing the patient preoperatively with a drug (HpD) which relatively localizes to tumour and is activated using visible light. At operation the resected tumour bed is illuminated with a predetermined uniform light energy density to eradicate microscopic tumour deposits left at the lateral resection margin. We have previously investigated technical and biological factors leading to this clinical trial. Seventeen patients have received AIOPDT in a potentially effective dose, and safety and technical matters have been investigated. Cutaneous phototoxicity occurred in 3 patients. Three patients had anastomotic breakdown, none considered attributable to PDT. The intraoperative technique was a practical option. AIOPDT carried a low patient morbidity and should be investigated in prospective clinical trials to determine if local recurrence rates can be decreased.

Butyrate oxidation is impaired in the colonic mucosa of sufferers of quiescent ulcerative colitis.

The short chain fatty acids, acetate, propionate, and butyrate are produced by colonic bacterial fermentation of non-starch polysaccharides. Butyrate is the major fuel source for the colonic epithelium and there is evidence to suggest that its oxidation is impaired in ulcerative colitis. Triplicate biopsy specimens were taken at colonoscopy from five regions of the large bowel in 15 sufferers of ulcerative colitis. These patients all had mild or quiescent colitis as assessed by clinical condition, mucosal endoscopic and histological appearance. The rate of oxidation of glucose, glutamine, and butyrate through to carbon dioxide was compared with that in biopsy specimens from 28 patients who had no mucosal abnormality. Butyrate (272 (199-368)) was the preferred fuel source for the colitic mucosa followed by glutamine (33 (24-62)) then glucose (7.2 (5.3-15)) pmol/micrograms/hour; medians and 95% confidence intervals, p < 0.01. There was no regional difference in the rate of utilisation of these metabolites. In the group with colitis the rate of butyrate oxidation to carbon dioxide was significantly impaired compared with that in normal mucosa decreasing from 472 (351-637) pmol/micrograms/hour to 272 (199-368) pmol/micrograms/hour; median and 95% confidence intervals, p = 0.016. The rate of glucose and glutamine utilisation were not significantly different between normal and colitic mucosa. These data confirm that in quiescent ulcerative colitis there is an impairment of butyrate oxidation.

New technique to measure mucosal metabolism and its use to map substrate utilization in the healthy human large bowel.

A new method of assessing substrate utilization in gastrointestinal mucosal specimens is described. Small human endoscopic biopsy specimens with wet weights ranging between 1.4 and 12.2 mg were used to quantify the oxidation of three metabolic substrates, glucose, glutamine and butyrate, through to carbon dioxide over a 2-h period. The technique proved to be reproducible and capable of distinguishing variations in mucosal metabolism between individuals (P < 0.0001 for each substrate). Results were similar to those obtained previously using human and rat colonocytes. To characterize the metabolism of the healthy large bowel, specimens were obtained from five regions in 15 patients who had a normal colonoscopic examination. The results show that butyrate is the preferred fuel source of large bowel mucosa, followed by glutamine, then glucose (P < 0.01). There was no significant regional variation in utilization of the three substrates between the five regions; with respect to glutamine, this is contrary to previous findings.