Liposomal amphotericin B-the past.

The discovery of amphotericin B, a polyene antifungal compound, in the 1950s, and the formulation of this compound in a liposomal drug delivery system, has resulted in decades of use in systemic fungal infections. The use of liposomal amphotericin B formulation is referenced in many international guidelines for the treatment of fungal infections such as Aspergillus and cryptococcal disease and Candida infections, as well as other less common infections such as visceral leishmaniasis. With the development of liposomal amphotericin B, an improved therapeutic index could be achieved that allowed the attainment of higher drug concentrations in both the plasma and tissue while simultaneously lowering the toxicity compared with amphotericin B deoxycholate. In over 30 years of experience with this drug, a vast amount of information has been collected on preclinical and clinical efficacy against a wide variety of pathogens, as well as evidence on its toxicity. This article explores the history and nature of the liposomal formulation, the key clinical studies that developed the pharmacokinetic, safety and efficacy profile of the liposomal formulation, and the available microbiological data.

Tissue pharmacokinetics and pharmacodynamics of AmBisome® (L-AmBis) in uninfected and infected animals and their effects on dosing regimens.

By selecting a unique combination of lipids and amphotericin B, the liposome composition for AmBisome® (L-AmBis) has been optimized resulting in a formulation that is minimally toxic, targets to fungal cell walls, and distributes into and remains for days to weeks in various host tissues at drug levels above the MIC for many fungi. Procedures have been standardized to ensure that large scale production of the drug retains the drug's low toxicity profile, favorable pharmacokinetics and antifungal efficacy. Tissue accumulation and clearance with single or multiple intravenous administration is similar in uninfected and infected animal species, with tissue accumulation being dose-dependent and the liver and spleen retaining the most drug. The efficacy in animals appears to be correlated with drug tissue levels although the amount needed in a given organ varies depending upon the type of infection. The long-term tissue retention of bioactive L-AmBis in different organs suggests that for some indications, prophylactic and intermittent drug dosing would be efficacious reducing the cost and possible toxic side-effects. In addition, preliminary preclinical studies using non-intravenous routes of delivery, such as aerosolized L-AmBis, catheter lock therapy, and intravitreal administration, suggest that alternative routes could possibly provide additional therapeutic applications for this antifungal drug.

Toxicity and efficacy differences between liposomal amphotericin B formulations in uninfected and Aspergillus fumigatus infected mice.

Because of the reduced toxicity associated with liposomal amphotericin B preparations, different amphotericin B liposome products have been made. In the present study, we compared the amphotericin B liposomal formulations, AmBisome(®) (AmBi) and Lambin(®) (Lbn), in uninfected and Aspergillus fumigatus infected mice, using several in vitro and in vivo toxicity and efficacy assays. The results showed that the formulations were significantly different, with Lbn 1.6-fold larger than AmBi. Lbn was also more toxic than AmBi based on the RBC potassium release assay and intravenous dosing in uninfected mice given a single 50 mg/kg dose (80% mortality for Lbn vs. 0% for AmBi). Renal tubular changes after intravenous daily dosing for 14 days were seen in uninfected mice given 5 mg/kg Lbn but not with AmBi. Survival following A. fumigatus challenge was 30% for 10 mg/kg Lbn and 60% for 10 mg/kg AmBi. When the BAL and lungs were collected 24 h after the second treatment, AmBi at 10 or 15 mg/kg or 15 mg/kg Lbn lowered the BAL fungal burden significantly vs. the controls (P ≤ 0.05), while there was no difference in lung fungal burden amongst the groups. In contrast, lung histopathology at this same early timepoint showed that AmBi was associated with markedly fewer fungal elements and less lung tissue damage than Lbn. In conclusion, given the differences in size, toxicity, and efficacy, AmBi and Lbn were not physically or functionally comparable, and these differences underscore the need for adequate testing when comparing amphotericin B liposome formulations.

Impact of Proximal and Distal Pocket Site-Directed Mutations on the Ferric/Ferrous Heme Redox Potential of Yeast Cytochrome-c-Peroxidase.

Cytochrome-c-peroxidase (CCP) contains a five-coordinate heme active site. The reduction potential for the ferric to ferrous couple in CCP is anomalously low and pH dependent (Eo = ~-180 mV vs. S.H.E. at pH 7). The contribution of the protein environment to the tuning of the redox potential of this couple is evaluated using site directed mutants of several amino acid residues in the environment of the heme. These include proximal pocket mutation to residues Asp-235, Trp-191, Phe-202 and His-175, distal pocket mutation to residues Trp-51, His-52, and Arg-48; and a heme edge mutation to Ala-147. Where unknown, the structural changes resulting from the amino acid substitution have been studied by X-ray crystallography. In most cases, ostensibly polar or charged residues are replaced by large hydrophobic groups or alternatively by Ala or Gly. These latter have been shown to generate large, solvent filled cavities. Reduction potentials are measured as a function of pH by spectroelectrochemistry. Starting with the X-ray derived structures of CCP and the mutants, or with predicted structures generated by Molecular Dynamics (MD), predictions of redox potential changes are modeled using the Protein Dipoles Langevin Dipoles (PDLD) method. These calculations serve to model an electrostatic assessment of the redox potential change with simplified assumptions about heme iron chemistry, with the balance of the experimentally observed shifts in redox potential being thence attributed to changes in the ligand set and heme coordination chemistry, and/or other changes in the structure not directly evident in the X-ray structures (e.g. ionization states, specific roles played by solvent species, or conformationally flexible portions of the protein). Agreement between theory and experiment is good for all mutant proteins with the exception of the mutation Arg 48 to Ala, and His 52 to Ala. In the former case, the influence of phosphate buffer is adduced to account for the discrepancy, and measurements made in a bis-tris propane/2-(N-morpholino)ethanesulfonic acid buffer system agree well with theory. For the latter case, an unknown structural element relevant to His-52, and/or solvent influence in the mutant akin to anion binding in the distal pocket (though lacking proof that it is) manifests in this mutant. The use of exogenous (sixth) ligands in dissecting the contributions to control of redox potential are also explored as a pathway for model building.

Reflections on building community: a different perspective on academic-community partnerships using the integration matrix.

Implementation of interdisciplinary rehabilitation services and clinical training in community settings is a complex process. Documented methods for analyzing, evaluating and monitoring the process within federally funded grant projects are limited. This paper reports on the use of one proposed method, the Integration Matrix of Konrad, as a framework for formative assessment and analysis of project operations. Based upon ongoing experiences implementing a Health Resources and Services Administration (HRSA) funded clinical training program in a community setting, a revision of the Integration Matrix is proposed to better reflect a community-centered, pervasive communication approach to program operations and analysis.

Cunninghamella bertholletiae infection in a bone marrow transplant patient: amphotericin lung penetration, MIC determinations, and review of the literature.

Infections caused by Cunninghamella bertholletiae, an opportunistic fungal organism, have an extremely high mortality rate. A fatal case of C. bertholletiae fungal pneumonia occurred in a man who had received an allogeneic bone marrow transplant. Aggressive debridement and high-dose liposomal amphotericin B failed to eradicate the infection. Right lung tissue samples obtained during lobectomy were assayed for amphotericin B concentrations by high-performance liquid chromatography, and minimum inhibitory concentration (MIC) determinations of amphotericin B against C. bertholletiae were determined by the macrobroth dilution method. The MIC for the isolate of C. bertholletiae was 4 microg/ml. Amphotericin B lung concentrations averaged 9.5 microg/ml (range 3.7-13.8 microg/ml), with a corresponding serum trough concentration of 0.9 microg/ml. To our knowledge, this is the first reported case of amphotericin B concentrations measured at the site of infection in a patient with a pulmonary Cunninghamella infection, together with a corresponding MIC of the organism. The patient's death, which occurred despite aggressive debridement and high amphotericin B lung concentrations, highlights the need for novel strategies to treat infections caused by invasive molds such as C. bertholletiae.

Expert practice in physical therapy.

BACKGROUND AND PURPOSE: The purpose of this qualitative study was to identify the dimensions of clinical expertise in physical therapy practice across 4 clinical specialty areas: geriatrics, neurology, orthopedics, and pediatrics. SUBJECTS: Subjects were 12 peer-designated expert physical therapists nominated by the leaders of the American Physical Therapy Association sections for geriatrics, neurology, orthopedics, and pediatrics. METHODS: Guided by a grounded theory approach, a multiple case study research design was used with each of the 4 investigators studying 3 therapists working in one clinical area. Data were obtained through nonparticipant observation, interviews, review of documents, and analysis of structured tasks. Videotapes made during selected therapist-patient treatment sessions were used as a stimulus for the expert therapist interviews. Data were transcribed, coded, and analyzed through the development of 12 case reports and 4 composite case studies, one for each specialty area. RESULTS: A theoretical model of expert practice in physical therapy was developed that included 4 dimensions: (1) a dynamic, multidimensional knowledge base that is patient-centered and evolves through therapist reflection, (2) a clinical reasoning process that is embedded in a collaborative, problem-solving venture with the patient, (3) a central focus on movement assessment linked to patient function, and (4) consistent virtues seen in caring and commitment to patients. CONCLUSION AND DISCUSSION: These findings build on previous research in physical therapy on expertise. The dimensions of expert practice in physical therapy have implications for physical therapy practice, education, and continued research.

The clinical doctorate: a framework for analysis in physical therapist education.

This article explores major considerations for analysis and discussion of the role of the clinical doctorate as the first professional degree in physical therapist education (DPT). A process for this analysis is posed based on a conceptual framework developed by Stark, Lowther, Hagerty, and Orczyk through grounded theory research on professional education. External influences from society and the profession, institutional and programmatic influences, and articulation of critical dimensions of professional competence and professional attitudes as major categories are discussed in relation to the DPT. A series of questions generated from the application of the model are put forth for continued discussion and deliberation concerning the DPT. We conclude that the DPT provides the best pathway to serve society, the patient, and the profession.

Nature of clinical practice for specialists in orthopaedic physical therapy.

Clinical specialization is part of physical therapy's continued development as a profession. Clinical specialization in physical therapy has evolved with little discussion of how specialization is related to the development of professional expertise. The purposes of this paper were to compare the identified clinical competencies in orthopaedic physical therapy to selected clinical reasoning models and expertise development models in physical therapy and interpret these comparisons in light of current theoretical work in expertise. Descriptive content analysis using results from the 1993 Practice Analysis for Orthopaedic Physical Therapy Certified Specialists was done to link attributes identified in 3 selected theoretical models of clinical decision making and practice. Survey materials were linked to theories by use of a binary index (yes/no) of whether theoretical concepts were present or absent in the survey results. The attributes that characterize an expert physical therapy practitioner involve clinical reasoning, and the ability to teach patients. The skills of a master clinician were based not just on the application of knowledge, but also on thinking and reasoning that occurs with experience. We propose that knowledge is gained through the clinician's thinking and reasoning during practice which results in a transformation or change in the clinician's knowledge base. Describing the clinical specialization process in the context of expert theory provides a strong foundation for the specialization process in physical therapy.

Describing expert practice in physical therapy.

In this article, the authors demonstrate how grounded theory may be used to develop models for understanding clinical practice. Through a series of research studies involving novice, experienced, and expert physical therapy practitioners, conceptual frameworks were continually revised based on data obtained from returning to the field and relevant literature available at the time. As concepts and relationships moved to larger themes, a theoretical framework for expertise in clinical practice was proposed. Current work on verifying the theoretical framework continues. Grounded theory is an excellent research approach to bound and help guide a multistage research program involving multiple researchers working in multiple settings.

Secretory pathways in Salmonella Typhimurium-induced fluid accumulation in the porcine small intestine.

The involvement of 5-hydroxytryptamine (5-HT) and 5-HT3 receptors and prostaglandin E2 (PGE2) in Salmonella Typhimurium-induced fluid accumulation in the porcine small intestine was investigated. Salmonella Typhimurium (10(8) and 10(10) cfu) and cholera toxin (CT; 20 microg) were instilled for 8 and 11 h in ligated loops in the porcine jejunum and ileum. Fluid accumulation and concentrations of Na+, K+, Cl-, 5-HT and PGE2 in the fluid accumulated in the loops were measured. The fluid accumulation was also measured when Salmonella Typhimurium (10(10) cfu) and CT (20 microg) were instilled for 8 h in ligated loops in jejunum and ileum in pigs given subcutaneous injections of saline or the 5-HT3 receptor antagonist ondansetron (200 microg/kg). Salmonella Typhimurium (10(10) cfu) and CT both induced fluid accumulation in jejunum and ileum after 8 and 11 h. Both treatments also induced an increase in luminal release of 5-HT and PGE2. The accumulated fluid was iso-osmotic and hyperosmotic in CT- and Salmonella Typhimurium-treated loops, respectively. Ondansetron reduced the Typhimurium-induced fluid accumulation in both jejunum and ileum by c. 40%, while it failed to reduce the response to CT. These results demonstrate that 5-HT and PGE2 are released and 5-HT3 receptors activated in the secretory pathway of Typhimurium in the porcine small intestine.

Effect of ondansetron on Salmonella typhimurium-induced net fluid accumulation in the pig jejunum in vivo.

Two major pathophysiological mechanisms explaining the diarrhoea induced by Salmonella typhimurium have been suggested to be: (a) invasion of the intestine by the bacteria, and (b) an enterotoxin resembling Vibrio cholerae toxin. Cholera toxin is a potent secretagogue in pig small intestine and induces secretion partly by activating 5-hydroxytryptamine receptors, following release of 5-hydroxytryptamine. Ondansetron is a selective 5-hydroxytryptamine-3 receptor antagonist, which reduces the cholera toxin-evoked fluid accumulation in pig jejunum. The aim of this study was to investigate the effect of ondansetron on Salmonella typhimurium-induced fluid accumulation in ligated loops of pig jejunum in vivo. 10(10) colony-forming units of the bacteria was injected into loops and incubated for 8 hr. 200 mg x kg-1 ondansetron given subcutaneously reduced the Salmonella typhimurium-induced fluid accumulation by about 40%. This results suggests the involvement of 5-hydroxytryptamine and 5-hydroxytryptamine-3 receptors in Salmonella typhimurium-induced diarrhoea.

The effect of high altitude and other risk factors on birthweight: independent or interactive effects?

OBJECTIVES: This study examined whether the decline in birth-weight with increasing altitude is due to an independent effect of altitude or an exacerbation of other risk factors. METHODS: Maternal, paternal, and infant characteristics were obtained from 3836 Colorado birth certificates from 1989 through 1991. Average altitude of residence for each county was determined. RESULTS: None of the characteristics related to birthweight (gestational age, maternal weight gain, parity, smoking, prenatal care visits, hypertension, previous small-for-gestational-age infant, female newborn) interacted with the effect of altitude. Birthweight declined an average of 102 g per 3300 ft (1000 m) elevation when the other characteristics were taken into account, increasing the percentage of low birthweight by 54% from the lowest to the highest elevations in Colorado. CONCLUSIONS: High altitude acts independently from other factors to reduce birthweight and accounts for Colorado's high rate of low birthweight.

Effect of 5-HT2 and 5-HT3 receptor antagonists on cholera toxin-induced fluid hypersecretion in the pig jejunum.

5-Hydroxytryptamine is a mediator in cholera toxin-induced hypersecretion in the small intestine. The aim of this study was to determine the effect of the 5-hydroxytryptamine receptor antagonists ketanserin, granisetron, ondansetron and tropisetron on cholera toxin-induced hypersecretion in the pig jejunum. Hypersecretion was induced by cholera toxin in ligated jejunal loops. The antagonists were administered subcutaneously at a dose of 100 micrograms/kg. Furthermore, the effect of intraluminally instilled ondansetron was studied. None of the antagonists altered basal absorption or caused fluid hypersecretion. Cholera toxin caused a dose-dependent electrolyte and fluid hypersecretion. The apparent maximal effect, 6.8 +/- 0.4 mg fluid x mg dry loop-1, was reduced by ondansetron, granisetron and tropisetron by about 40%, 30%, and 20%, respectively, whereas ketanserin had no effect. Intraluminal ondansetron reduced the effect of cholera toxin by about 50%. These results demonstrate that 5-hydroxytryptamine3 antagonists administered subcutaneously reduce the cholera toxin-induced hypersecretion in the pig jejunum. Finally, the results support species differences with respect to the antagonistic effect of the tested drugs in cholera toxin-induced hypersecretion.

Altering substrate specificity at the heme edge of cytochrome c peroxidase.

Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site. This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function.

Circular dichroism and x-ray spectroscopies of Azotobacter vinelandii nitrogenase iron protein. MgATP and MgADP induced protein conformational changes affecting the [4Fe-4S] cluster and characterization of a [2Fe-2S] form.

Nucleotide interactions with nitrogenase are a central part of the mechanism of nitrogen reduction. Previous studies have suggested that MgATP or MgADP binding to the nitrogenase iron protein (Fe protein) induce protein conformational changes that control component protein docking, interprotein electron transfer, and substrate reduction. In the present study, we have investigated the effects of MgATP or MgADP binding to the Azotobacter vinelandii nitrogenase Fe protein on the properties of the [4Fe-4S] cluster using circular dichroism (CD) and x-ray absorption spectroscopies. Previous CD and magnetic CD studies on nitrogenase Fe protein suggested that binding of either MgATP or MgADP to the Fe protein resulted in identical changes in the CD spectrum arising from transitions of the [4Fe-4S]2+ cluster. We present evidence that MgADP or MgATP binding to the oxidized nitrogenase Fe protein results in distinctly different CD spectra, suggesting distinct changes in the environment of the [4Fc-4S] cluster. The present results are consistent with previous studies such as chelation assays, electron paramagnetic resonance, and NMR, which suggested that MgADP or MgATP binding to the nitrogenase Fe protein induced different conformational changes. The CD spectrum of a [2Fe-2S]2+ form of the nitrogenase Fe protein was also investigated to address the possibility that the MgATP- or MgADP-induced changes in the CD spectrum of the native enzyme were the result of a partial conversion from a [4Fe-4S] cluster to a [2Fe-2S] cluster. No evidence was found for a contribution of a [2Fe-2S]2+ cluster to the CD spectrum of oxidized Fe protein in the absence or presence of nucleotides. A novel two-electron reduction of the [2Fe-2S]2+ cluster in Fe protein was apparent from absorption, CD, and electron paramagnetic resonance data. Fe K-edge x-ray absorption spectra of the oxidized Fe protein revealed no changes in the structure of the [4Fe-4S] cluster upon MgATP binding to the Fe protein. The present results reveal that MgATP or MgADP binding to the oxidized state of the Fe protein result in different conformational changes in the environment around the [4Fe-4S] cluster.

The role of aspartate-235 in the binding of cations to an artificial cavity at the radical site of cytochrome c peroxidase.

The activated state of cytochrome c peroxidase, compound ES, contains a cation radical on the Trp-191 side chain. We recently reported that replacing this tryptophan with glycine creates a buried cavity at the active site that contains ordered solvent and that will specifically bind substituted imidazoles in their protonated cationic forms (Fitzgerald MM, Churchill MJ, McRee DE, Goodin DB, 1994, Biochemistry 33:3807-3818). Proposals that a nearby carboxylate, Asp-235, and competing monovalent cations should modulate the affinity of the W191G cavity for ligand binding are addressed in this study. Competitive binding titrations of the imidazolium ion to W191G as a function of [K+] show that potassium competes weakly with the binding of imidazoles. The dissociation constant observed for potassium binding (18 mM) is more than 3,000-fold higher than that for 1,2-dimethylimidazole (5.5 microM) in the absence of competing cations. Significantly, the W191G-D235N double mutant shows no evidence for binding imidazoles in their cationic or neutral forms, even though the structure of the cavity remains largely unperturbed by replacement of the carboxylate. Refined crystallographic B-values of solvent positions indicate that the weakly bound potassium in W191G is significantly depopulated in the double mutant. These results demonstrate that the buried negative charge of Asp-235 is an essential feature of the cation binding determinant and indicate that this carboxylate plays a critical role in stabilizing the formation of the Trp-191 radical cation.