Viability and functionality of bovine chromaffin cells encapsulated into alginate-PLL microcapsules with a liquefied inner core.

Implantation of adrenal medullary bovine chromaffin cells (BCC), which synthesize and secrete a combination of pain-reducing neuroactive compounds including catecholamines and opioid peptides, has been proposed for the treatment of intractable cancer pain. Macro- or microencapsulation of such cells within semipermeable membranes is expected to protect the transplant from the host's immune system. In the present study, we report the viability and functionality of BCC encapsulated into microcapsules of alginate-poly-L-lysine (PLL) with a liquefied inner core. The experiment was carried out during 44 days. Empty microcapsules were characterized in terms of morphology, permeability, and mechanical resistance. At the same time, the viability and functionality of both encapsulated and nonencapsulated BCC were evaluated in vitro. We obtained viable BCC with excellent functionality: immunocytochemical analysis revealed robust survival of chromaffin cells 30 days after isolation and microencapsulation. HPLC assay showed that encapsulated BCC released catecholamines basally during the time course study. Taken together, these results demonstrate that viable BCC can be successfully encapsulated into alginate-PLL microcapsules with a liquefied inner core.

Intrathecal grafting of porcine chromaffin cells reduces formalin-evoked c-Fos expression in the rat spinal cord.

Chromaffin cells from the adrenal gland secrete a combination of neuroactive compounds including catecholamines, opioid peptides, and growth factors that have strong analgesic effects, especially when administered intrathecally. Preclinical studies of intrathecal implantation with xenogeneic bovine chromaffin cells in rats have provided conflicting data with regard to analgesic effects, and recent concern over risk of prion transmission has precluded their use in human clinical trials. We previously developed a new, safer source of adult adrenal chromaffin cells of porcine origin and demonstrated an in vivo antinociceptive effect in the formalin test, a rodent model of tonic pain. The goal of the present study was to confirm porcine chromaffin cell analgesic effects at the molecular level by evaluating neural activity as reflected by spinal cord c-Fos protein expression. To this end, the expression of c-Fos in response to intraplantar formalin injection was evaluated in animals following intrathecal grafting of 10(6) porcine or bovine chromaffin cells. For the two species, adrenal chromaffin cells significantly reduced the tonic phases of the formalin response. Similarly, c-Fos-like immunoreactive neurons were markedly reduced in the dorsal horns of animals that had received injections of xenogeneic chromaffin cells. This reduction was observed in both the superficial (I-II) and deep (V-VI) lamina of the dorsal horn. The present study demonstrates that both xenogeneic porcine and bovine chromaffin cells transplanted into the spinal subarachnoid space of the rat can suppress formalin-evoked c-Fos expression equally, in parallel with suppression of nociceptive behaviors in the tonic phase of the test. These findings confirm previous reports that adrenal chromaffin cells may produce antinociception by inhibiting activation of nociceptive neurons in the spinal dorsal horn. Taken together these results support the concept that porcine chromaffin cells may offer an alternative xenogeneic cell source for transplants delivering pain-reducing neuroactive substances.

A new method for encapsulation of living cells: preliminary results with PC12 cell line.

A new method is described for encapsulation of living cells. PC12 rat adrenal pheochromocytoma cells, which have been shown to synthesize, store and release dopamine were employed. The particles are made first and the cells then incorporated in a gentle mechanical procedure. The morphology (by light and electron microscopic observation), stability, rheology, texture and permeability of these microcapsules provided by Kappa Biotech were investigated. Membrane permeability studies demonstrated exclusion of 69,000 Da human serum albumin, but equilibrium of D-glucose and inulin was within 24h, indicating a molecular weight cut-off in the 5000-70,000 Da range. The viability and the function of the encapsulated cells were evaluated by measuring the spontaneous release of dopamine by high performance liquid chromatography with electrochemical detection. The results show that dopamine-secreting cells can be sequestered in a semi-permeable capsule and still display good viability and proliferation for at least 1 month.

[Management of intractable cancer pain: from intrathecal morphine to cell allograft]. / Traitement des douleurs chroniques rebelles d'origine cancéreuse: de la morphinothérapie intrathécale à la thérapie cellulaire.

The durable effectiveness of intrathecal morphine administration is well established for the management of intractable cancer pain, after failure of systemic opioids, secondary to the persistence of non-reversible undesirable side effects. Many patients are referred to late in the disease course. This conservative method to control pain of malignant origin must not be reserved for last resort treatment for terminal patients. Intra-cerebro-ventricular morphine administration is a very effective and generally safe method for controlling intractable cancer pain. Because of the chronic implantation of an intra-ventricular catheter this method is somewhat invasive. Its indications remain a simple and effective alternative when the topography of nociceptive pain is diffuse or cephalic. In clinical practice, intrathecal and/or intra-cerebro-ventricular administration of opioids is limited by cost, the need for specialized maintenance and mechanical malfunctions if implantable drug delivery systems, or by the risk of bacterial contamination and ambulatory constraints when repeated daily injections via an intrathecal access port are used. To answer these limitations, cell therapy using intrathecal chromaffin cell allograft is a promising approach for the management of cancer pain refractory to traditional drug therapy and pain lesion surgery. The basic rationale and preclinical studies on experimental pain models have enabled starting prospective clinical trials. Prior to transplantation, handling and preparation of the chromaffin tissue is critical for allograft viability. The initial results of clinical trials with human chromaffin cell grafts from intractable cancer pain have reported long-lasting pain relief, in correlation with met-enkephalin release into the CSF. Convincing evidence will require controlled studies. The limitations of this innovative cell therapy and especially the lack of human adrenal gland availability point to the need for new sources of cells. Perspectives include xenogenic or engineered cell lines.

[Harvesting of an internal saphenous graft for aorto-coronary bypass using video-surgery]. / Prélèvement du greffon saphène interne pour pontage aorto-coronaire par méthode vidéo-chirurgie.

Classical excision of saphenous vein grafts requires a continuous incision on the leg or the thigh or both. To minimise the trauma due to this method, an endoscopic method has been recently developed. The aim of this paper was to assess the benefits of this new method compared with the classical technique. One hundred and twenty patients requiring aorto-coronary grafts were included in this prospective study and divided into two groups according to the method of saphenous vein harvesting. Group A comprised 60 patients who underwent the classical method os saphenous vein harvesting and Group B 60 patients who benefited from the endoscopic method. No difference was observed between the two groups with respect to mean age, sex ratio, history of diabetes and obliterative arterial disease of the lower limbs. Parsonnet index number of bypass grafts and length of vein excised. The length of the skin wound in group A was 30.8 +/- 8.5 cm compared with only 4.1 +/- 1 cm in Group B (p = 0.006) but the harvesting time was longer by endoscopy (55.7 +/- 23.7 minutes: 72.5 +/- 22.6 minutes for the first 10 patients, 48.5 +/- 24.7 minutes for the last 50 patients) compared with the classical technique (39.8 +/- 6.6 minutes: p = 0.001). Moreover, patients who underwent videosurgery had less operative pain (8% versus 15%) (p = 0.001). The number of infectious complications was slightly lower in Group B (3.3%, 2/60, versus 10%, 6/60), (NS). The authors conclude that harvesting of the saphenous vein by videosurgery reduces postoperative pain and gives a more aesthetic result but with a slightly longer operative time at the beginning of the experience.

One-year chromaffin cell allograft survival in cancer patients with chronic pain: morphological and functional evidence.

The control of chronic pain through transplantation of chromaffin cells has been reported over the past few years. Analgesic effects are principally due to the production of opioid peptides and catecholamines by chromaffin cells. Clinical trials have been reported with allografts consisting of whole-tissue fragments implanted into the subarachnoid space of the lumbar spinal cord (14,19,36). In the present study, allogeneic grafts were successfully used to control chronic pain in two patients over a period of 1 yr based on patient reported pain scores, morphine intake, and CSF levels of Met-enkephalin. Macroscopic examination at autopsy located the transplanted tissue fragments in the form of multilobulated nodules at the level of the spinal axis and cauda equina. Immunocytochemical microscopy showed neuroendocrine cells are positive for chromagranin A (CGA), and enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbetaH). The results suggest that there is a relationship between analgesic effect, Met-enkephalin levels in CSF, and the presence of chromaffin cells surviving in spinal subarachnoid space.

Does intrathecal morphine in the treatment of cancer pain induce the development of tolerance?

OBJECTIVE: This retrospective study was designed to investigate whether chronic lumbar intrathecal administration of morphine leads to the development of opioid tolerance in patients suffering from intractable cancer pain. METHODS: Between 1978 and 1995, 159 patients with refractory cancer pain were treated with intrathecal morphine in our Multidisciplinary Pain Center. The treatment consisted of preservative-free morphine administered through an access port as a single bolus. In this series of patients (n = 159), the daily doses of intrathecal morphine were determined as a function of duration of follow-up. RESULTS: The mean follow-up period was 95 days (range, 5-909 d), the mean starting daily dose of intrathecal morphine was 2.69 mg (range, 1-7.5 mg), and the mean terminal dose was 7.82 mg (range, 1-80 mg). The results demonstrated that only a moderate increase in daily dose of intrathecal morphine was required during the course of treatment (a two- to threefold increase for a 3-mo period). Furthermore, the dose increment was similar for patients followed up for more or less than 60 days. This increase did not result in any central opioid-related side effects, and the pain was managed satisfactorily. CONCLUSION: The requirement for a moderate increase in intrathecal opioid doses reflects the development of tolerance but did not limit the patients' ability to obtain adequate analgesia during the course of their painful disease.

[Centralized preparation in the pharmacy of dilute solutions of ganciclovir: (Cymevan): work load change and economic potential]. / Préparation centralisée en pharmacie des solutions diluées de ganciclovir (Cymévan): prévision de la charge de travail et de l'économie potentielle.

The production of ganciclovir doses by a hospital pharmacy having the necessary facilities to reconstitute anticancer drugs would be of the utmost relevance regarding both protection of the nursing staff and therapeutic safety. As this activity is also economically worthwhile a large number of our colleagues would like to implement it. The aim of this work has was to take advantage of a 40 weeks production of ganciclovir dose by the pharmacy at Rangueil hospital in Toulouse so as to answer some of the questions raised by such a project. The savings achieved by the team at Brabois hospital in Vandoeuvre les Nancy could therefore be validated. They are expressed in fraction of flask per dose produced (0,265). Applied to the number of doses that we delivered over a 40 week period, this coefficient enabled us to estimate from the only datum which is initially available to us, namely the number of flasks used, the extra work load and the savings that could be made: given steady activity, the number of doses the pharmacy will have to prepare is equal to the number of flasks multiplied by 1.418 and, provided one opts for a production process mostly in batches, the savings will be equivalent to 37.5% of the flasks.

Effect of intraperitoneal insulin delivery on growth hormone binding protein, insulin-like growth factor (IGF)-I, and IGF-binding protein-3 in IDDM.

Low plasma insulin-like growth factor (IGF)-I despite high circulating growth hormone (GH) in insulin-dependent diabetes mellitus (IDDM) indicate a hepatic GH resistance. This state may be reflected by the reduction of the circulating GH binding protein (GHBP), corresponding to the extracellular domain of the GH receptor, and the reduction of insulin-like growth factor binding protein (IGFBP)-3, major IGF-I binding protein, upregulated by GH. We carried out two studies. In the first, plasma GHBP activity was compared in patients with IDDM on continuous subcutaneous insulin infusion (CSII) or on conventional therapy and in healthy subjects. In the second study, the 18 patients on CSII at baseline were then treated by continuous intraperitoneal insulin infusion with an implantable pump (CPII) and prospectively studied for GH-IGF-I axis. Although HbA1c was lower in patients on CSII than in those on conventional therapy, GHBP was similarly reduced in both when compared to control subjects (10.2 +/- 0.8 and 11.6 +/- 0.9% vs 21.0 +/- 1.3, p < 0.01). CPII for 12 months resulted in: a slight and transient improvement in HbA1c (Time (T)0: 7.6 +/- 0.2%, T3: 7.1 +/- 0.2%, T12: 7.5 +/- 0.2%, p < 0.02), improvement in GHBP (T0: 10.2 +/- 0.8%, T12: 15.5 +/- 1.5, p < 0.0001), near-normalization of IGF-I (T0: 89.4 +/- 8.8 ng/ml, T12: 146.9 +/- 15.6, p < 0.002) and normalization of IGFBP-3 (T0: 1974 +/- 121 ng/ml, T12: 3534 +/- 305, p < 0.0001). The hepatic GH resistance profile in IDDM does not seem to be related to glycaemic control, but partly to insufficient portal insulinization. Intraperitoneal insulin delivery, allowing primary portal venous absorption, may influence GH sensitivity, and improve hepatic IGF-I and IGFBP-3 generation.

Insulin therapy and GH-IGF-I axis disorders in diabetes: impact of glycaemic control and hepatic insulinization.

In Type 1 diabetes, high circulating growth hormone (GH) in conjunction with low plasma insulin-like growth factor-I (IGF-I) is indicative of a hepatic GH-resistance profile since the liver is the main source of circulating IGF-I. The reduction in specific growth hormone binding protein (GHBP), corresponding to the extracellular domain of the GH receptor, provides an indirect indication of the hepatic density of GH receptors, as does the reduction in IGFBP-3, the major IGF binding protein, which is GH-dependent. Type 1 diabetes is also associated with high levels of IGFBP-1, a binding protein down-regulated by insulin. Although most of these abnormalities have been described in situations of poor glycaemic control, hyperglycaemia does not seem to be the predominant factor in their pathogenesis. Even intensified subcutaneous insulin therapy does not normalize GH, IGF-I, GHBP and IGFBP-3 plasma levels. Some indirect evidence suggests that portal insulinopenia plays a role in the hepatic GH-resistance profile of Type 1 diabetes, i.e. discrepancies between the abnormalities reported in Type 1 and Type 2 diabetes, and the inverse relationship between residual insulin secretion in Type 1 diabetes and some of these abnormalities. Intraperitoneal insulin therapy administered to Type 1 diabetic patients by implantable pumps (without modification of glycaemic control) can improve GHBP activity, practically normalize plasma IGF-I and normalize IGFBP-3. The improvement in GH-IGF-I axis disorders obtained with intraperitoneal insulin therapy (which allows primary portal insulin absorption) provides direct evidence of the central role of portal insulin in the regulation of this system.

Differential regulation of serum growth hormone (GH)-binding protein during continuous infusion versus daily injection of recombinant human GH in GH-deficient children.

Serum GH-binding protein (GHBP) was evaluated in 2 randomly divided groups of prepubertal children presenting with idiopathic GH deficiency and receiving recombinant human GH, either continuously by sc infusion (group 1) or as 1 daily sc injection (group 2). After the first 6 months, group 1 switched from continuous infusion to daily injections for the following 6 months. There was no significant difference in clinical data, GH values, or GHBP levels between the 2 groups before treatment. During the first 6 months, GHBP levels increased in all except 1 of the 8 children in group 1 from 8.6% to 16.9% after 3 months and 22.5% after 6 months. The increment factor ranged from 1.1-7.9, with wide individual variations. In group 2, the mean variation in GHBP was from 8.3-8.2% after 3 months and 10.7% after 6 months. Only 2 of the 10 children in this group showed a significant increase in GHBP levels. During the second period, group 1 maintained their GHBP levels, whereas the 2 children in group 2 tended to a continued increase in their GHBP levels. There was no correlation with the increase in growth velocity, as children in both groups grew equally well, but higher insulin-like growth factor-I levels were found in group 1, although the difference between the two groups was not significant. These data show that GH can increase GHBP levels and that there is a differential effect depending on the mode of GH administration, although the reason for and the role of such regulation remains to be explained.

[Nosocomial infections in an urological department. Incidence and etiological factors]. / Infections nosocomiales dans un service d'urologie. Incidence et facteurs étiologiques.

The authors report a prospective study with active request of data for nosocomial infections (NI) in an urological department during six months. From 453 patients, 43 developed an NI (incidence = 9.5%): urinary tract infections (53.5%), bloodstream infections (16.3%), lower respiratory tract infections (7%), surgical wound infections (2.3%) and sepsis syndrome (20.9%). For microorganisms, most often Pseudomonas aeruginosa (22.5%) and E. coli (20%) were encountered. Other microorganisms were the next-ones: Staphylococcus aureus (15%), Staphylococcus epidermidis (7.5%), Acinetobacter baumanii (7.5%), Streptococcus group D (7.5%), Klebsiella pneumoniae (7.5%), Candida albicans (2.5%) and others (10%). Urological interventions with a high risk of NI were cystectomy with intestinal urinary diversion (68.7%), pyeloplasty for ureteropelvic junction obstruction (40%) and percutaneous nephrolitothomy (30%). Even if the last two interventions classically do not have a high risk of NI, we think that their antibioprophylaxis is recommended.

CSF baclofen levels after intrathecal administration in severe spasticity.

The pharmacokinetic parameters in the CSF of baclofen given to 4 patients as an intrathecal bolus are reported. Considerable inter-individual variability in the parameters was observed. The elimination half-life ranged from 0.9 to 5 h and the clearance from 0.013 to 0.08 l.h-1. In order to optimize treatment, it is suggested that CSF baclofen levels be matched to changes in Hoffman's monosynaptic reflex (H reflex).

Chronic intrathecal baclofen administration for control of severe spasticity.

Baclofen, the most effective drug for treating spasticity, is a specific agonist of gamma-aminobutyric acid-B receptors, and is very abundant in the superficial layers of the spinal cord. Given orally, baclofen does not easily penetrate the blood-brain barrier, and is distributed equally to the brain and spinal cord. Direct intrathecal administration was given in order to change the distribution of the drug by preferentially perfusing the spinal cord. Eighteen patients presenting a severe spastic syndrome were treated with chronic intrathecal infusion of baclofen in the lumbar cerebrospinal fluid. After clinical preselection, 38 patients were implanted with a lumbar access port allowing long-term trials in order to determine the efficacy of baclofen therapy and the effective 12-hour dose. The 18 patients selected for chronic administration were implanted with a programmable pump. The pathology in these cases was: multiple sclerosis (6 cases), posttrauma spastic syndrome (eight cases), and (one case each) cerebral palsy, ischemic cerebral lesion, spinal ischemia, and transverse myelitis. The mean follow-up period was 18 months (range 4 to 43 months). The clinical results were evaluated according to muscular hypertony on Ashworth's scale (changed for occurrence of painful spasms) and functional improvement. Results were better for spastic syndrome secondary to traumatic medullary lesion than for demyelinating disease. Hypertonia was improved in all cases as confirmed by the registration of the Hoffman (H) reflex. Painful muscular spasms disappeared in 14 of the 16 affected patients. Significant functional improvement was noted in nine patients and was considerable in three. The risk of side effects secondary to overdose (such as excessive hypotonia or central depression) and the absence of a specific baclofen antagonist stresses the necessity for accurate determination of the efficient dose. After an initial titration period and adjustment of the therapeutic dose, the individual doses were from 21 to 500 micrograms/24 hrs (mean 160 micrograms/24 hrs). This new conservative method is very effective, perfectly reversible, and safe when administered in conditions favorable to its use.

The stability of morphine in isobaric and hyperbaric solutions in a drug delivery system.

Intrathecal preparations of morphine for use in intractable pain must contain no preservatives. They are generally formulated in saline (isobaric) or dextrose (hyperbaric) which raises questions of stability. The behaviour of morphine in hyperbaric and isobaric solutions stored in a reservoir for implantation has been examined and the effect of temperature and the time of contact of morphine with the different components of the reservoir as well as the sterilization procedure have been investigated. The best stability was observed with a hyperbaric solution in which there was 15 to 20 times less pseudomorphine than in the isobaric solution, which was found to contain 1% pseudomorphine after 1 month of storage at 37 degrees C in the reservoir. Similar solutions stored in ampoules did not degrade.

CSF morphine levels after lumbar intrathecal administration of isobaric and hyperbaric solutions for cancer pain.

The objectives of this study were to compare the pharmacokinetic properties and the duration of analgesia following intrathecal administration (L5-S1) of 2 mg morphine in 2 forms: (1) an isobaric (NaCl 0.9%) and (2) a hyperbaric solution (7% dextrose). The study was carried out on 5 cancer patients with severe, intractable pain in the lower half of the body. Samples of CSF were collected at the level of the 10th thoracic vertebra at regular intervals for 15 h after administration. Morphine concentrations were determined by HPLC. The pharmacokinetic properties of the solutions (I and II) were quite different. Peak levels (I) were reached in 5-15 min (30 and 60 micrograms/ml); they then fell rapidly during the 1st hour (7 and 11 micrograms/ml) with an elimination half-life of 10 and 15 min, followed by a change in slope (elimination half-life of 108 and 140 min). Peak levels (II) were reached in 4-5 h (0.8-3.3 micrograms/ml); they then fell progressively according to a single exponential function (elimination half-life: 144-246 min). The duration of analgesia for a dose of 2 mg was 30 h for solution 2 and 24 h for solution 1. The hyperbaric solution, which produced the same degree of analgesia as the isobaric solution, limited the cephalad diffusion of morphine and reduced or abolished the central depressant effects of the drug.

Spinal versus intraventricular chronic opiate administration with implantable drug delivery devices for cancer pain.

Early publications have separately reported the efficacy, specificity and conservative character of direct spinal and intraventricular morphine analgesia in the treatment of intractable cancer pain. The objectives of this study are to compare efficacy and safety of these sites of local administration in order to determine the indication for each, the clinical effects of different opiates and the choice of various drug administration devices.