In vitro and in vivo evaluation of e-PTFE and alkali-cellulose membranes for guided bone regeneration.

Guided bone regeneration (GBR) is employed to encourage the formation of new bone in osseous defects by restricting the infiltration of soft tissues. While a variety of membranes have been evaluated for this surgical procedure, the non-resorbable material of choice is currently expanded polytetrafluoroethylene (e-PTFE). A new alkali-cellulose membrane produced by a biotechnological process has been developed as an alternative to e-PTFE for GBR. In this study, the biocompatibility of this novel alkali-cellulose membrane and e-PTFE was compared using tissue culture and an in vivo GBR model. In vitro both materials supported the attachment, migration and differentiation of osteoblast-like cells in culture for up to 3 weeks. The in vivo model was based upon full-thickness transcortical bone defects in the mandibular rami of Sprague-Dawley rats. The right rami were used as controls, contralateral defects being covered bucally and lingually with either e-PTFE or alkali-cellulose membranes. Pathological and histomorphometric analysis was undertaken at 4 and 10 weeks post-implantation. Bone regeneration associated with alkali-cellulose membranes was predominantly endochondral in type in contrast to e-PTFE which induced direct bone formation (intramembranous ossification). The amount of new bone formed in defects was similar for both types of membrane, but alkali-cellulose membranes induced significantly greater inflammatory response; characterized by lymphocytes, macrophages and multinucleated giant cells. Degradation and possible exposure of individual cellulose fibres may account for the poor performance of alkali-cellulose membranes in vivo. This animal and in vitro study indicates that when choosing a non-resorbable membrane for GBR, e-PTFE membranes are likely to perform better than those produced from alkali-cellulose.

Clinical pharmacokinetics of 2'-deoxy-2'-methylidenecytidine (DMDC), a deoxycytidine analogue antineoplastic agent.

This article reviews the clinical pharmacokinetics of a deoxycytidine analogue of cytarabine, 2'-deoxy-2'-methylidenecytidine (DMDC). DMDC belongs to the antimetabolite class of anticancer drugs and is phosphorylated into its active, triphosphate, form within the tumour cell. Cancer cell death appears to be a result of the impairment of DNA synthesis by the triphosphate form. DMDC undergoes deamination to the inactive 2'-deoxy-2'-methylideneuridine (DMDU), its main plasma metabolite. Following intravenous administration at 30 to 450 mg/m2, DMDC has low systemic clearance (10 to 15 L/h/m2), moderate volume of distribution (nominally similar to total body water) and a short elimination half-life of between 2 and 6 hours. Renal clearance of DMDC accounts for approximately 30 to 50% of total clearance. Following oral administration of DMDC at 12 to 50 mg/m2, mean maximum DMDC plasma concentrations are within the 100 to 400 microg/L range and are generally reached within 2 hours. Oral bioavailability of DMDC is in the order of 40%, largely as a result of first-pass metabolism in the gut and liver. This first-pass effect results in considerable interpatient variability in systemic exposure to DMDC after oral administration. The systemic availability of DMDC is proportional to the administered dose and, although there was evidence that systemic exposure to DMDC decreased on repeated administration, there are no excessive time-dependent changes in systemic exposure to DMDC. Following oral administration, DMDC is metabolised in the gut wall and liver by deamination to DMDU. The kidneys eliminate DMDC and DMDU, with up to 50% of the administered dose recovered in urine, on average, as parent drug and metabolite. Dose escalation to the maximum tolerated dose was facilitated by a pharmacokinetically guided dose escalation strategy. DMDC has shown activity in non-small-cell lung cancer and colorectal cancers following oral administration. Several tumour responses are observed at the highest doses of DMDC, indicating a possible dose-response relationship with this drug. The main clinical adverse event of DMDC therapy is myelotoxicity. The haematological toxicity of DMDC was schedule dependent; twice daily administration was associated with greater toxic effects than a once daily regimen. A pharmacokinetic-pharmacodynamic model characterised the relationship between plasma DMDC concentrations and the time-dissociated toxicity. This model-dependent approach may be used to predict the consequences of as-yet-untested therapy as well as relating acceptable risks of haematological toxicity to target drug exposure.

Models of schedule dependent haematological toxicity of 2'-deoxy-2'-methylidenecytidine (DMDC).

OBJECTIVE: DMDC (2'-deoxy-2'-methylidenecytidine) is-a potential antitumour deoxycytidine analogue of cytosine arabinoside. The major dose-limiting toxicity of DMDC is haematological depression, particularly neutropenia, and therefore quantitative exposure-toxicity relationships for DMDC are warranted. METHODS: Data on the survival fraction at nadir of leukocytes, neutrophils and platelets from 66 patients receiving a once-daily regimen and 85 patients receiving a twice-daily regimen of DMDC were related to DMDC concentration-time profiles using area under the plasma concentration-time curve (AUC), threshold and general models. A semiphysiological model of neutrophils versus time after DMDC administration included transient compartments to imitate the differentiation stages in the bone marrow. RESULTS: The relationship between plasma DMDC concentration-time profiles and the haematological toxicity at nadir was best described using an AUC-dependent model with separate functions for once- and twice-daily dosing, indicating schedule dependence of DMDC effects, even if differences in treatment duration had a similar explanatory value. Twice-daily dosing was associated with greater toxic effects than once-daily dosing. The AUC required for a 70% reduction in the neutrophils was 16 mg.h/l and 4.2 mg.h/l for the once- and twice-daily regimens, respectively. The semiphysiological model included nine proliferating transient compartments that were sensitive to DMDC in a schedule-dependent manner, five non-mitotic, non-sensitive compartments and one compartment for circulating neutrophils. CONCLUSIONS: The haematological toxicity of DMDC is schedule dependent. The survival fractions of leukocytes, neutrophils and platelets are predicted to be lower when given on a twice-daily regimen than on a once-daily regimen. A semiphysiological model with transient compartments successfully described the entire time course of neutropenia after DMDC administration.

Plasma copolymer surfaces of acrylic acid/1,7 octadiene: surface characterisation and the attachment of ROS 17/2.8 osteoblast-like cells.

The purpose of this study was: (a) to examine the effect of plasma-gas composition on plasma polymer oxygen/carbon (O/C) ratio, functional group composition and stability in water, and then (b) to examine cell attachment to surfaces containing different concentrations of O/C and functional groups. Oxygen-functionalised surfaces were deposited by means of the plasma copolymerisation of acrylic acid/1,7-octadiene. The use of a diluent hydrocarbon allowed the deposition of surfaces with a range of O/C concentrations. Plasma copolymer surfaces were characterised by X-ray photoelectron spectroscopy (XPS). Changes in functional group composition with % acrylic acid monomer and the non-dispersive and dispersive parts of the surface energy of these plasma copolymers were measured. The solubility of the plasma copolymers was assessed by means of XPS. The degree of attachment of ROS 17/2.8 osteoblast-like cells to plasma copolymer surfaces deemed to be 'stable' in aqueous medium was measured. Tissue culture polystyrene (TCPS) was included as a control. Attachment was found to be greatest to the plasma copolymer surface with an O/C of 0.11. This surface had a carboxylic acid concentration of ca. 3%. Attachment did not correlate with increased surface wettability (i.e. the non-dispersive component of the surface energy).

Dependence of in vitro biocompatibility of ionomeric cements on ion release.

The in vitro biocompatibility of a group of ionomeric cements (ICs) was evaluated with respect to their ion release properties. These ICs were made from a defined series of glasses with the general formula 1.5SiO2.0.5P2O5.Al2O3.(1.0-Z)CaO.0.75CaF2 where Z was the mole fraction (ranging from 0-0.1) of an alkali metal oxide, either sodium or potassium or a mixture of both. For these alkali metal ICs, the amount of sodium released was directly related to the sodium content of the constituent glass. Similarly, the amount of potassium released was directly related to the potassium content. There was no correlation between the aluminum content of the glass and the aluminum ion release. Increasing the monovalent cation concentration, however, produced ICs with increased fluoride release. The biocompatibility of the ICs, as assessed by in vitro cell growth and viability measurements, was inversely proportional to aluminum ion release. Fluoride ion release, although important in terms of in vitro biocompatibility, would appear to be less important than aluminum ion release in determining the overall biocompatibility of the ICs studied.

Immunochemical detection of parathyroid hormone-related protein in the saccus vasculosus of a teleost fish.

Using antisera to regions of human parathyroid hormone-related protein (PTHrP) the saccus vasculosus (SV) of the sea bream (Sparus aurata) has been shown to contain immunoreactive PTHrP. By immunohistochemistry (IHC) the epithelial coronet cells in fixed and wax-embedded SV tissue reacted with antisera to the prepro region of human PTHrP (-13 to +2), the N-terminus PTHrP (1-16), and the midmolecule PTHrP (50-69). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of saccus extracts and incubation media contained two major proteins of 14.3 and 15 kDa. By Western blotting these two proteins both reacted with the three antisera used for IHC, suggesting that they are immunochemically similar to human PTHrP (1-84). Ultrastructurally the coronet cells of Sparus saccus vasculosus resembled coronet cells described for other teleosts, with an abundant smooth endoplasmic reticulum (SER) which was more highly organized in the coronets. IHC at EM level showed reaction mainly with the membranes of the SER. These results suggest that S. aurata saccus vasculosus may produce a PTHrP-like molecule similar to human PTHrP.

Parathyroid hormone-related protein is a factor in normal fish pituitary.

Using antibodies to the amino-terminal region of human parathyroid hormone-related protein (PTHrP) we have demonstrated PTHrP immunoreactivity in pituitaries and plasma of the sea bream (Sparus aurata). Pituitary cells at two distinct locations contained immunodetectable PTHrP; an anterior group in the rostral pars distalis which also contained immunoreactive thyroid stimulating hormone (TSH), and a posterior group lying at the border of the pars intermedia and proximal pars distalis between cells which stained with antibody to human corticotrophin-like intermediate lobe peptide. By Western blot analysis pituitary extracts contained two immunoreactive isoforms of PTHrP, one of 29 kDa and the other of 26 kDa. Media of pituitaries incubated for up to 14 days in Krebs-Ringer bicarbonate also had several isoforms of immunodetectable PTHrP, two of them corresponding to the 29- and 26-kDa molecular forms but there were in addition both larger and smaller molecules. The concentration of PTHrP in sea bream plasma was comparable with levels observed in human subjects with humoral hypercalcaemia of malignancy. There was no reaction between pituitary cells or pituitary extracts and antibody to human parathyroid hormone. Thus sea bream pituitary contains immunoreactive PTHrP, which appears to be released into medium during in vitro incubation and which may be a significant source of plasma immunoreactive PTHrP in vivo.

Activation of bovine plasma benzylamine oxidase (BzAO) by 1-methyl-4-(2-methylphenyl)-1,2,3,6-tetrahydropyridine.

We have shown previously that certain analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are potent inhibitors of human and bovine plasma benzylamine oxidase (BzAO: EC 1.4.3.6). Inhibition was competitive, reversible and allosteric. Under certain conditions competitive inhibitors of allosteric enzymes can act as allosteric activators. In the present work, 1-methyl-4-(2-methylphenyl)-1,2,3,6-tetrahydropyridine (2'-CH3MPTP) was found to activate bovine plasma BzAO at low substrate and 2'-CH3MPTP concentrations. At higher 2'-CH3MPTP concentrations, the activation was negated.

Inhibition of human benzylamine oxidase (BzAO) by analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

A sensitive assay for human plasma BzAO, involving the conversion of 14C-benzylamine to 14C-benzaldehyde, was developed. MPTP and several of its analogues were found to be competitive inhibitors of the enzyme. Ki values for the MPTP analogues in the presence of human plasma BzAO were determined. The analogues had a different rank order of inhibition of human plasma BzAO compared with the rank order of inhibition of bovine plasma BzAO found previously. MPTP and 1-methyl-4-(2-methylphenyl)-1,2,3,6-tetrahydropyridine (2'-CH3-MPTP), which are potent nigrostriatal toxins, were weak inhibitors of human plasma BzAO.