A placebo-controlled study of liposome-mediated gene transfer to the nasal epithelium of patients with cystic fibrosis.

Cystic fibrosis (CF) is a common, serious, inherited disease. The major cause of mortality in CF is lung disease, due to the failure of airway epithelial cells to express a functional product of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A potential treatment for CF lung disease is the expression of CFTR in the airways following gene transfer. We have undertaken a double-blinded, placebo-controlled, clinical study of the transfer of the CFTR cDNA to the nasal epithelium of 12 CF patients. Cationic liposomes complexed with plasmid containing the human CFTR cDNA were administered to eight patients, whilst four patients received placebo. Biopsies of the nasal epithelium taken 7 days after dosing were normal. No significant changes in clinical parameters were observed. Functional expression of CFTR assessed by in vivo nasal potential difference measurements showed transient correction of the CF chloride transport abnormality in two patients (15 days after dosing in one patient). Fluorescence microscopy demonstrated CFTR function ex vivo. In cells from nasal brushings. In total, evidence of functional CFTR gene transfer was obtained in six out of the eight treated patients. These results provide proof of concept for liposome-mediated CF gene transfer.

In vitro drug metabolism and pharmacokinetics of diazepam in cynomolgus monkey hepatocytes during culture for six days.

Diazepam (DZ), N-desmethyl diazepam (NOR) and temazepam (TEM) were used as substrates in drug metabolism studies to characterize the changes in cytochrome P-450 mono-oxygenase pathways in hepatocytes isolated from cynomolgus monkeys, during culture for 6 days. Hepatocytes were incubated with DZ (20 microM), NOR (6 microM) or TEM (20 microM) for 3 hr at 3, 24, 48, 96 and 144 hr post-isolation in culture, and the profiles of disappearance of DZ, as substrate, and appearance of its metabolites determined. Major metabolites were NOR, TEM and oxazepam (OX). The kinetic profiles for the disappearance of DZ and the accumulation of metabolite were analysed using a four-compartment model and constants for the rates of formation of the metabolites were derived. There were significant changes during the period in culture for the rate constants of DZ demethylation, but no alteration in the 3-hydroxylation activities. Rates of DZ metabolism were unchanged during the initial 2 days in culture and well maintained for the subsequent 4 days, despite a fall in total cytochrome P-450 to 23% of initial values after 6 days. Cynomolgus monkey hepatocytes produce similar metabolite profiles for DZ to those found in man, both in vitro and in vivo, indicating that cynomolgus monkey hepatocytes may represent a relatively stable and valuable model of human drug metabolism.

Comparative drug metabolism of diazepam in hepatocytes isolated from man, rat, monkey and dog.

Diazepam (DZ) was used as a substrate in drug metabolism studies to characterise the differences in metabolite profiles in hepatocytes isolated from four species: Wistar rat, cynomolgus monkey, beagle dog and man. Hepatocytes were incubated with DZ (20 microM) for 180 min at 3 hr post isolation in culture, and the disappearance of parent compound and appearance of its metabolites determined. DZ disappearance was found to be monoexponential in rat, monkey and human cells, but that DZ disappearance in dog hepatocytes was best described by a two compartment process. There were considerable differences in both the rates of formation and the profiles of metabolites produced from DZ in each species. Drug metabolism of DZ was determined in five human hepatocyte preparations. The rates of formation of both the major metabolites, temezepam (TEM) and nordiazepam (NOR) were highly variable between samples, and oxazepam (OX) was detected in only three of the preparations. There was no evidence of further metabolism of these metabolites, and the profiles were comparable with in vivo findings. In a single case, human hepatocytes were cultured for five days, and DZ was used as substrate to characterise the changes in drug metabolising activities. There was a rapid loss in the production of OX in the initial 24 hr, and a complete loss of 3-hydroxylation activities in the succeeding 120 hr. N-demethylation activities, however, were well maintained, and the appearance of NOR declined to 47% of initial rate. The hepatocytes of all species were found to produce NOR and TEM as metabolites; NOR representing the principal metabolite in the dog, monkey and human cells. In the dog, TEM was found only as a minor metabolite. OX was a significant metabolite in the monkey and a minor metabolite in the dog and human hepatocytes, but was not detected in rat cultures. The principal metabolite in rat cells was 4'-hydroxy diazepam, which was rapidly further metabolised to its glucuronide. The drug metabolising activities of the hepatocyte cultures towards DZ were comparable with the drug metabolism of DZ found in vivo in each species. These findings substantiate hepatocytes as an in vitro model of hepatic metabolism.

General practitioners in hospital: a move toward medical workforce integration.

The reintegration of the medical profession's clinical activities is a goal worth pursuing. For the patient it could have considerable advantages with continuity of care, greater coordination of services, quality assurance, and a more cost effective service. For the medical profession there would be a greater cohesion, improved peer review, cross fertilisation of ideas and a more balanced advocacy role.

Diazepam metabolism in cultured hepatocytes from rat, rabbit, dog, guinea pig, and man.

Diazepam metabolism has been investigated in cultured hepatocytes from rat, rabbit, dog, guinea pig, and man. The metabolite profile obtained by HPLC analysis of the culture medium indicated that substantial differences exist corresponding to known species differences in the metabolite profile of diazepam in vivo. These differences were attributed to a combination of the rate at which a metabolite was formed and the rate at which it is removed from the medium by further metabolism. The intrinsic clearance of nordiazepam in hepatocytes from each of the species exhibited the most marked species variation (rat much greater than guinea pig greater than rabbit greater than human greater than dog). Species that exhibited a high intrinsic clearance for nordiazepam were also those species that exhibited significant hydroxylation at the 4'-site of the molecule. The disappearance of diazepam was rapid in rat, dog, and guinea pig hepatocytes, but slow in human hepatocytes. Moreover, rat and human hepatocytes exhibited different saturability of diazepam clearance with respect to diazepam concentration accounting, at least in part, for the different rates of diazepam metabolism in the different species. These results support the value of hepatocytes in drug metabolism studies and especially in studies of species differences in metabolism.

Education--the concern of the profession? Undergraduate medical education.

In this brief review not all aspects could be covered. The main requirements for improvement are: Reorganise the Medical Council - expand the role of its educational committee and establish a curriculum advisory body. Begin a continuous evaluation process of medical education. Colleges and medical associations begin debating their role, involving lay organisations in the process. Curriculum committees and medical schools look at four areas of criticism (begin planning introduction of early clinical contact, problem solving, critical approach, self learning) and contemplate reduction in length of course. The direction of medical education following Flexner was the centres of medical excellence, these became the teaching hospitals and they have become the trap leading to a distorted view of medicine. That they have produced many advances is not disputed. That they will continue to do so is to be expected, although they are now retarding progress in the important and neglected areas that hold the most potential for benefit--prevention and primary care. The new direction of excellence must not be solely into institutions, but must now be into the minds of all the individual members of the profession. The institutions will change, as society does, along with increasing knowledge, but if possession of the habits of continual critical inquiry is equally represented in all members of the profession, then they will adapt more quickly and appropriately to the requirements in the future.

Education--the concern of the profession? Early postgraduate medical education.

Overall what is suggested is a tried and proven apprentice type system, with continuing role models, more stable patient care teams, defined educational objectives, all with some flexibility and a reduction in the tendency to over supply in specific areas. The transition would be difficult, it could not happen without change (already suggested) in the undergraduate curriculum and it would also require changes in other areas, peripheral to the areas of hospital organisation already discussed. The changes necessary for the full effect of these proposals to be felt, will be those occurring in medical society. The changes will be in the methods of practice, in organisation and payment and in the relationship of different parts of the profession (aiming for abolition of the dichotomy existing between primary care and the hospital), all sections taking an active part in planning and delivering medical education. A clear commitment must be made to continually evaluate medical education's performance and relevancy, connecting this to institutions (medical schools, hospitals, colleges etc) geared and prepared to respond. A reconstituted and reorientated Medical Council would be pivotal to this being successful. This two year spell inflicted on the young members just entering our profession is inexcusable in humanitarian terms, wasteful in educational terms, and ineffective in management terms.

Drug metabolising activity of freshly isolated human hepatocytes.

Hepatocytes have been isolated from samples of adult human liver by removal of extracellular calcium followed by perfusion with collagenase. The hepatocytes were isolated with a yield of up to 39 X 10(6) cells/g and with a viability of up to 74%. The cells were active in the oxidation of aldrin and 7-ethoxycoumarin. They also catalysed the conjugation of 7-hydroxycoumarin. Monooxygenase activity of the hepatocytes was linear for at least 60 min. Maintenance of the hepatocytes in suspension at 4 degrees C for 19 h resulted in a 15% loss in viability. This was accompanied by a 50% decrease in monooxygenase activity expressed per viable cell. It is concluded that human hepatocytes can be isolated in sufficient yield and with satisfactory viability for use in a range of studies on drug metabolism and toxicity.