Rationale for the administration of systemic 5-FU in combination with heated intraperitonal oxaliplatin.

BACKGROUND: Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) with oxaliplatin (OX) is the standard of care for selected patients with peritoneal carcinomatosis of colorectal origin. Because 5-FU is mandatory to improve efficacy of OX when used by systemic route, several teams now empirically combine intravenous (IV) 5-FU with HIPEC OX, but this practice has yet to be supported by preclinical data. Using a murine model, we studied the impact of IV 5-FU on peritoneal absorption of HIPEC OX. METHODS: Under general anesthesia, 24 Sprague-Dawley rats were submitted to 4 different doses of IV 5-FU (0, 100, 400 and 800 mg/m2) and a fixed dose of HIPEC OX (460 mg/m2) perfused at 40 °C during 25 min. At 25 min, samples in different compartments were harvested (peritoneum, portal vein and systemic blood) and the concentrations of 5-FU and OX were measured by high performance liquid chromatography. RESULTS: Peritoneal absorption of OX was significantly higher (17.0, 20.1, 34.9 and 38.1 nmol/g, p < 0.0001) with increasing doses of 5-FU (0, 100, 400 and 800 mg/m2, respectively). Peritoneal absorption of OX reached a plateau between 400 and 800 mg/m2 of IV 5-FU. CONCLUSION: IV 5-FU enhances peritoneal absorption of HIPEC OX. The most efficient dose of IV 5-FU to be used in combination with HIPEC OX seems to be 400 mg/m2.

Emerging evidence of the impact of kidney disease on drug metabolism and transport.

Several lines of emerging evidence indicate that kidney disease differentially affects uptake and efflux transporters and metabolic enzymes in the liver and gastrointestinal (GI) tract, and uremic toxins have been implicated as the cause. In patients with kidney disease, even drugs that are eliminated by nonrenal transport and metabolism could lead to important unintended consequences if they are administered without dose adjustment for reduced renal function. This is particularly so in the case of drugs with narrow therapeutic windows and may translate into clinically significant variations in exposure and response.

Decreased in vivo metabolism of drugs in chronic renal failure.

Chronic renal failure (CRF) is associated with a decrease in renal excretion of drugs, but its effects on the liver metabolism of xenobiotics are poorly defined. The objectives of this study were to determine the effects of CRF on hepatic cytochrome P450 (CYP450) and its repercussions on in vivo hepatic metabolism of drugs. Two groups of rats were studied: control paired-fed and CRF. CRF was induced by subtotal nephrectomy. Total CYP450 activity and protein expression of several CYP450 isoforms (CYP1A2, CYP2C11, CYP3A1, CYP3A2) were assessed in liver microsomes. In vivo cytochrome P450 activity was evaluated with breath tests using substrates for different isoenzymes: caffeine (CYP1A2), aminopyrine (CYP2C11), and erythromycin (CYP3A2). Creatinine clearance was reduced by 60% (P <. 01) in rats with CRF. Compared with control paired-fed rats, total CYP450 activity was reduced by 40% in rats with CRF. Protein expression of CYP2C11, CYP3A1, and CYP3A2 was considerably reduced (more than 45%, P <.001) in rats with CRF, whereas the levels of CYP1A2 were unchanged. In rats with CRF, there was a 35% reduction in the aminopyrine (CYP2C11) and the erythromycin (CYP3A2) breath tests compared with control animals (P <.001). The caffeine (CYP1A2) breath tests remained comparable to controls. Creatinine clearance correlated with the aminopyrine and erythromycin breath tests (r(2) = 0.73 and r(2) = 0.81, respectively, P <.001). In conclusion, CRF is associated with a decrease in total liver CYP450 activity in rats (mainly in CYP2C11, CYP3A1, and CYP3A2), which leads to a significant decrease in the metabolism of drugs.

Studies on small (<350 microm) alginate-poly-L-lysine microcapsules. V. Determination of carbohydrate and protein permeation through microcapsules by reverse-size exclusion chromatography.

Membrane molecular weight (MW) cut-off is a critical factor for immunoprotection of transplanted microencapsulated cells as well as for graft survival. Our goal was to study dextran and protein permeation through small (<350 microm in diameter) alginate-poly-L-lysine microcapsules made with an electrostatic system. Microcapsules were packed into a column, and gel-sieving chromatography was performed with proteins and dextrans of known MW. The objectives of this study were (1) to validate this approach for the assessment of the MW cut-off of <350 microm-in-diameter microcapsules and (2) to evaluate the effect on MW cut-off of changes in experimental conditions. Elution profiles of proteins suggest that the MW cut-off of our small microcapsules lies between 14,500 and 44,000 Da whereas dextrans > or =19,000 Da were excluded. The increase in poly-L-lysine (PLL) concentration from 0.02 to 0.08% reduced the MW cut-off. Increasing the PLL MW from 11.6 to 69.6 kDa induced no change in the MW cut-off. The results also show that the method can be used to discriminate between adsorption and absorption and that insulin diffuses freely across the microcapsule membrane. This method will be useful in establishing the ideal MW cut-off, in optimizing microcapsule characteristics, and in performing routine quality controls.

Studies on smaller (approximately 315 microM) microcapsules: IV. Feasibility and safety of intrahepatic implantations of small alginate poly-L-lysine microcapsules.

UNLABELLED: The most successful transplantation site of nonencapsulated islets of Langerhans is the liver. Because usual alginate poly-L-lysine microcapsules were too large (700-1200 microm diameter) for intravascular implantations and were almost exclusively implanted intraperitoneally, the question of the preferred implantation site of microencapsulated islets has received little attention. The feasibility of implanting smaller (approximately 315 microm) alginate poly-L-lysine microcapsules into the liver and the effect of such implantations on portal pressure and liver histology was evaluated in Wistar rats. A bolus of 10,000 microcapsules of 315 microm diameter was injected intraportally (group 1; n = 22). The portal pressure increased from 6.4 +/- 1.8 mmHg to a maximum of 19 mmHg, returned to basal levels within 2 h, and remained normal after 2 months. In group 2 (n = 3), following the injection of 10,000 larger microcapsules (420 microm), the portal pressure increased to > 60 mmHg and two out of the three rats died within 3 h. When 5,000 microcapsules of 420-microm diameter were injected (group 3; n = 5), the portal pressure peaked to 30 +/- 8 mmHg and remained elevated after 4 h (12 +/- 3 mmHg), but returned to normal (8 +/- 1 mmHg) after 2 weeks. Histological studies showed normal hepatic architecture without collagen deposition into portal tracts occupied by microcapsules. CONCLUSION: intrahepatic implantations of approximately 315-microm alginate poly-L-lysine microcapsules are feasible and safe. These results justify further investigation of this potential implantation site for microencapsulated islets.

Alginate-poly-L-lysine microcapsule biocompatibility: a novel RT-PCR method for cytokine gene expression analysis in pericapsular infiltrates.

Transplantation of microencapsulated islets of Langerhans is impaired by a pericapsular host reaction that eventually induces graft failure. We are studying the role of cytokines in the pathogenesis of this reaction, using the model of alginate-polylysine microcapsule implantation in rat epididymal fat pads. The objectives were: (1) to develop a method to measure, by semiquantitative PCR, TGF-beta1 gene expression in fat pad pericapsular infiltrates, and (2) to use this method to evaluate TGF-beta1 gene expression 14 days after microcapsule implantation. TGF-beta1 mRNA level was significantly higher in pericapsular infiltrate cells than in nonimplanted tissue cells and saline-injected tissue cells (p < 0.0001 and p < 0.01, respectively). There was no significant difference between the TGF-beta1 mRNA levels of the two types of controls (p = 0.0945). These results suggest that TGF-beta1 plays a role in the pathogenesis of the pericapsular reaction. The method developed can be used to study the role of other fibrogenic cytokines potentially involved. This will shed light on the mechanisms underlying the pericapsular reaction and will serve as a basis for the development of strategies to control this reaction.

Studies on small (<350 microm) alginate-poly-L-lysine microcapsules. III. Biocompatibility Of smaller versus standard microcapsules.

Microencapsulation of islets of Langerhans has been proposed as a means of preventing their immune destruction following transplantation. Microcapsules of diameters <350 microm made with an electrostatic pulse system present many advantages relative to standard microcapsules (700-1500 microm), including smaller total implant volume, better insulin kinetics, better cell oxygenation, and accessibility to new implantation sites. To evaluate their biocompatibility, 200, 1000, 1120, 1340, or 3000 of these smaller microcapsules (<350 microm) or 20 standard microcapsules (1247+/-120 microm) were implanted into rat epididymal fat pads, retrieved after 2 weeks, and evaluated histologically. The average pericapsular reaction increased with the number of small microcapsules implanted (p<0.05; 3000 vs. 200, 3000 vs. 1000, and 1000 vs. 200 microcapsules). At equal volume and alginate content, standard microcapsules caused a more intense fibrosis reaction than smaller microcapsules (p<0.05). In addition, 20 standard microcapsules elicited a stronger pericapsular reaction than 200 and 1000 smaller microcapsules (p<0.05) although the latter represented a 3.4-fold larger total implant surface exposed. We conclude that microcapsules of diameters <350 microm made with an electrostatic pulse system are more biocompatible than standard microcapsules.

Peroxynitrite production by human neutrophils, monocytes and lymphocytes challenged with lipopolysaccharide.

To assess peroxynitrite formation in lipopolysaccharide (LPS)-stimulated human blood, we have measured nitric oxide (NO)-dependent intracellular oxidation of dihydrorhodamine 123 (DHR 123) to rhodamine. LPS increased DHR 123 oxidation in neutrophil granulocytes, monocytes and lymphocytes in a time-dependent fashion. Greater extent of DHR 123 oxidation was detected in neutrophils and monocytes than in lymphocytes. These changes were accompanied by accumulation of rhodamine in the plasma. While intracellular DHR 123 oxidation and rhodamine accumulation in the plasma were not affected by inhibition of constitutive NO synthase at 30 and 60 min after addition of LPS, they were markedly attenuated by inhibition of inducible NO synthase at 4, 8, 16 and 24 h after addition of LPS. These results demonstrate that human leukocytes can produce high amounts of peroxynitrite in response to LPS, and may contribute to the elevated plasma peroxynitrite levels observed during endotoxic shock.

Quantitative method for the evaluation of biomicrocapsule resistance to mechanical stress.

A quantitative method has been developed for the evaluation of biomicrocapsule resistance to mechanical stress. Fluorescein isothiocyanate-labelled dextran (M.W. 2 x 10(6)) was microencapsulated in alginate-poly-L-lysine membranes. Microcapsules of 302.0 +/- 3.2 microns were mixed with 3 mm glass beads and continuously agitated for 0 to 144 h. The percentage of broken capsules was calculated by measuring the fluorescence in the supernatant and in the residual intact capsules after the latter were dissolved. The fluorescence method was validated by comparison with a manual method (handpicking under a stereomicroscope). The highest percentage of broken capsules was obtained with a ratio of 225 +/- 25 glass beads per 1000 microcapsules. The percentage of broken capsules increased linearly from 7.3% at 12 h to 48.3% at 72 h of continuous agitation. The applicability of the method was evaluated by studying microcapsules of potentially different levels of resistance. The results confirmed that capsule resistance is improved by increasing poly-L-lysine concentrations and incubation times. Microcapsules made with guluronic acid-rich alginate were stronger than those made with mannuronic acid-rich alginate. In conclusion, this is a simple, precise and sensitive method for the quantification of biomicrocapsule resistance to mechanical stress.

The rat epididymal fat pad as an implantation site for the study of microcapsule biocompatibility: validation of the method.

The study of microcapsule biocompatibility is hindered by their uneven distribution and low recovery when implanted into the peritoneum. We evaluated the use of the rat epididymal fat pad as a microcapsule implantation site for biocompatibility studies. The recovery rate of microcapsules containing 85Sr-labeled microspheres was 99.6 +/- 0.75%. Microcapsules made from the same batch of nonpurified alginate, were injected into both fat pads of male Lewis rats (n = 18) and retrieved 14 days later. A semiquantitative fibrosis score scaled from 0 to 3.0 showed that the pericapsular reaction was uniform throughout a fat pad, and that the results of the two fat pads were equivalent because the null hypothesis of inequivalence was rejected (P < .001). Thus, this method can be used to compare the biocompatibility of microcapsule of differing compositions.

Studies on small (< 300 microns) microcapsules: II--Parameters governing the production of alginate beads by high voltage electrostatic pulses.

The size of microcapsules is a critical parameter in the immunoisolation of islets of Langerhans by microencapsulation. The use of smaller capsules decreases the total implant volume and improves insulin kinetics and oxygen supply. A high voltage electrostatic pulse system was used for the production of small (< 300 microns) alginate beads, the first step of the encapsulation technique. However, islets often protruded from capsules that were too small, further emphasizing the need for a method to control bead size. A study of 7 parameters [electrostatic pulse amplitude (A), duration (D) and wavelength (lambda), pump flow rate (P), needle gauge, alginate viscosity and distance between electrodes] showed that P (r = 0.981, p = 0.003) and lambda (r = 0.988, p = 0.0002) were the principal determinants of bead size. To detect potential interactions between parameters, 270 combinations of different levels of A, D, lambda, and P were studied. A multivariate regression analysis of these data confirmed that P and lambda are the prime determinants of bead size, and showed that a 2-parameter (P, lambda) model could be used to precisely predict bead size (R2 = 0.84), while keeping the application simple. The precision of the predictive model is only slightly improved by the use of additional parameters. The reliability of the data used to elaborate this model was demonstrated (p = 0.6226) by comparing them with a second data set obtained under the same conditions. A third set of experiments confirmed the applicability of the model. This work has major implications on the preclinical application of microencapsulation since it showed that it is possible to predetermine the bead size.

Method for the quantification of alginate in microcapsules.

Alginate is a key reagent in the preparation of microcapsules for cell transplantation. To address the question of the intracapsular alginate concentration, a sensitive assay has been developed to quantify the alginate content of microcapsules. The method is based on the metachromatic change induced by alginate binding to the dye, 1,9-dimethyl methylene blue (DMMB). The assay has a high sensitivity and precision. It covers a wide concentration range enabling the measurement of alginate in dilute supernatants as well as in microcapsules. For the latter, the membrane is initially dissolved by incubating the microcapsules in an alkaline medium. The effect of potentially interfering substances (poly-L-lysine (PLL), citrate, chloride, sodium) and of pH has been studied. Poly-L-lysine interfered with the assay at pH 6.5 but not at pH 13. Interference by sodium augmented with increasing sodium concentration and reached a plateau at 200 mM. This problem was overcome by routinely adjusting all samples to 500 mM sodium. The other substances tested had a negligible effect on the assay. The reliable measurement of alginate with this new assay will allow the optimization of the intracapsular alginate concentration.

Reconstitution in vitro of the pH-dependent aggregation of pancreatic zymogens en route to the secretory granule: implication of GP-2.

Regulated secretory proteins are thought to be sorted in the trans-Golgi network (TGN) via selective aggregation. To elucidate the biogenesis of the secretory granule in the exocrine pancreas, we reconstituted in vitro the conditions of pH and ions believed to exist in the TGN using the end product of this sorting process, the zymogen granule contents. Protein aggregation was dependent on pH (acidic) and on the presence of cations (10 mM Ca2+, 150 mM K+) to reproduce the pattern of proteins found in the granule. The constitutive secretory protein IgG was excluded from these aggregates. Zymogen aggregation correlated with the relative proportion of the major granule membrane protein GP-2 in the assay. These results show that the glycosylphosphatidylinositol-anchored protein GP-2 co-aggregates with zymogens in the acidic environment believed to exist in the pancreatic TGN, and thus suggest that GP-2 would function as a membrane anchor for zymogen aggregates, facilitating their entrapment in budding vesicles directed towards the regulated secretory pathway.

Protection of islets of Langerhans from antibodies by microencapsulation with alginate-poly-L-lysine membranes.

Microencapsulation of islets has been proposed to prevent their immune destruction following transplantation. An indirect immunofluorescence technique has been developed and used to study the permeability of the alginate-poly-L-lysine microcapsules to antibodies. Wistar rat islets were incubated with the R2D6 monoclonal mouse IgM antibody against rat islets, microencapsulated, and incubated with fluorescein-labeled goat IgG antibodies against mouse IgG and IgM. For the negative controls, the first antibody was omitted or both antibodies were omitted. The positive controls included islets incubated with both antibodies before they were encapsulated. Our study demonstrated that the alginate-poly-L-lysine membranes are not permeable to IgG when poly-L-lysine of molecular weights ranging from 21,000 to 390,000 are used. This simple immunofluorescence technique demonstrated the nonpermeability of the microcapsules to IgG, and could be useful for the initial evaluation of new types of membranes.

In resting conditions, the pancreatic granule membrane protein GP-2 is secreted by cleavage of its glycosylphosphatidylinositol anchor.

GP-2 is the major membrane protein of the exocrine pancreatic secretory granule. It is an integral protein which is anchored by a phosphatidylinositolglycan. In addition to being present in the soluble contents of the granule, GP-2 is also actively secreted by the pancreas. Although 93% of the GP-2 in the resting secretions of anaesthetized rats could be pelleted, Triton X-114 phase extraction showed that 70% of this GP-2 had lost its hydrophobic properties. Proteases have been postulated to release GP-2 from the membrane, but phospholipases also have the capacity to release the protein from the membrane by hydrolysis of its peculiar glycosylphosphatidylinositol membrane anchor. These studies show the presence of inositol 1,2-(cyclic)monophosphate on the secreted hydrophilic GP-2, confirming the involvement of an endogenous phospholipase C in the solubilization of GP-2 by the exocrine pancreas. It is therefore concluded that most of the GP-2 secreted by the pancreas of anaesthetized rats under resting conditions is released from the membrane by a phospholipase C which hydrolyses the phosphodiester bond linking GP-2 to its diradylglycerol anchor.

A competition enzyme-linked immunosorbent assay (ELISA) for the measurement of pancreatic GP-2 glycoprotein.

A competition enzyme-linked immunosorbent assay has been developed for the quantitative detection of soluble and membrane-bound GP-2, a glycoprotein which is confined to the exocrine pancreas. Zymogen granule membranes fixed to microtiter plates with poly-L-lysine were used as the source of antigen. Detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS] was added to the assay in order to reveal all the antigens, more particularly in membranous samples. Presence of detergent at concentrations as high as 0.5% did not interfere with any particular steps of the ELISA. This competition ELISA can detect 10 ng of GP-2 and will be useful for measuring soluble as well as membrane GP-2 in order to elucidate its role in the secretory process of the pancreas as well as in certain pathologies such as cystic fibrosis.

Alterations of pancreatic growth and of GP-2 content in the reserpinized rat model of cystic fibrosis.

The chronically reserpinized rat is an experimental model for cystic fibrosis. In this study, we report the effects of two doses of reserpine (0.5 and 1.0 mg.kg-1.d-1) on the growth of the pancreas and on its content of the glycoprotein GP-2, a characteristic protein of the zymogen granule. An assessment of the effects of secondary malnutrition induced by the drug was also performed by adding a group of pair-fed animals. During the 7 d of treatment, body wt and food intake were monitored. These two parameters were significantly affected from the 4th d on. Pancreatic wt, DNA, protein, and activity of amylase and chymotrypsinogen were measured after 4 and 7 d of treatment; lipase activity and GP-2 content, after 7 d. Although the DNA content never did change, total protein diminished by 27% at the higher dose of reserpine. Pancreatic wt, amylase activity and GP-2 content were reduced by the treatment, while chymotrypsinogen and lipase activities were increased. Effects on pancreatic wt, amylase, chymotrypsinogen, and GP-2 were dose-dependent. Malnutrition had effects similar to reserpine on body wt, protein, amylase, and chymotrypsinogen. Pancreatic wt, lipase, and GP-2, however, were specifically altered by the chronic reserpine treatment. It is concluded from these results that reserpine induces, in the pancreas, specific alterations that are distinguishable from the accompanying malnutrition. These findings support the use of pancreatic wt, lipase, and GP-2 as specific markers of the effects of the drug on the pancreatic tissue in the chronically reserpinized rat model for cystic fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Light and electron microscopy of the exocrine pancreas in the chronically reserpinized rat.

The effects of reserpine injections were studied on the morphology of the pancreas in an experimental model for cystic fibrosis, the chronically reserpinized rat. A detailed examination of the tissue was carried out at the light and electron microscopic levels. The nonspecific effects of secondary malnutrition induced by the drug were assessed with a group of animals pair fed with the treated animals. In a companion paper, we show that pancreatic wt, lipase, and GP-2 contents also are affected by reserpine treatment. In this study, we report that no morphologic differences were observed between the exocrine pancreatic tissue of control and pair-fed animals. By contrast, reserpine induced an accumulation of zymogen granules in 60% of the treated animals and a concomitant decrease of the area occupied by the rough endoplasmic reticulum in the same cells. Finally, in all treated animals, at the light and electron microscopic levels, it was observed that some particular regions of the pancreatic tissue were strongly affected. In these regions, numerous autophagic bodies and lysosomes were observed. Cisternae of the Golgi complex were also more distended. Some acinar cells were in the process of lysis. Several vacuolar inclusions were present in some intralobular duct cells. Cellular material was seen in acinar and intralobular duct lumina. In these same regions, distended intralobular ducts and acinar lumina were observed. These last two features have never been reported in the reserpinized rat but are important manifestations of the pathology in cystic fibrosis patients where obstructions of ducts are believed to trigger focal destruction of the pancreatic tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Reducing conditions induce a total degradation of the major zymogen granule membrane protein in both its membranous and its soluble form. Immunochemical quantitation of the two forms.

The major protein of the pig pancreatic zymogen granule membrane is an integral glycoprotein of 92 X 10(3) daltons (Da) which amounts to 25% of the total proteins of this membrane. When zymogen granule membranes were prepared in presence of 5 mM dithiothreitol (DTT), this glycoprotein specifically vanished from the membrane preparation. During membrane purification two other fractions were produced out of the purified granules: a soluble fraction of zymogens referred to as granule content and a dense pellet. The possibility that DTT could release the 92-kDa protein from the membrane to these other fractions has been rejected. Altogether, addition of DTT during the lysis of the granules induced a total degradation of the 92-kDa protein. This hydrolysis could be inhibited by phenylmethylsulfonyl fluoride but not by N-alpha-p-tosyl-L-lysine chloromethyl ketone or L-1-tosylamide-2-phenylethylchloromethyl ketone. In the course of these experiments, using gel filtration of the granule content, it was found that the 92-kDa protein was also present in the granule content in the form of an aggregate of 300 kDa. A protease was present in this aggregate and could hydrolyse the 92-kDa protein upon addition of DTT. From immunoblotting studies and rocket immunoelectrophoresis, it was found that the soluble 92-kDa protein was antigenically similar to the membrane protein and that 44% of the immunoreactive glycoprotein of the granule was soluble in the content. A cross-reacting fragment of 65 kDa has been observed in all the fractions, yet at different levels.(ABSTRACT TRUNCATED AT 250 WORDS)