Chitosan hydrogels incorporating colloids for sustained drug delivery.

In today's biomedical research, a huge effort is being made towards the development of efficient drug delivery systems, achieving sustainable and controlled delivery of drugs. Chitosan (CS) hydrogels are high water content materials with very relevant biological properties to that purpose. Their use for a local and delayed delivery has already been demonstrated for a wide variety of therapeutic agents. One relatively recent strategy to improve these CS-based systems consists in the insertion of colloids, embedding drugs, within their three-dimensional matrix. This provides a second barrier to the diffusion of drugs through the system, and allows to better control their release. The main objective of this review is to report the many existing complex systems composed of CS hydrogels embedding different types of colloids used as drug delivery devices to delay the release of drugs. The various biomedical applications of such final systems are also detailed in this review.

Chitosan hydrogels for sustained drug delivery.

Sustainable and controlled delivery of drugs is at the centre of a huge amount of undertaken researches. The ability of hydrogels, high water content materials, to achieve a local and delayed-delivery has already been demonstrated for a wide variety of therapeutic agents and various polymer natures. In particular, chitosan, a natural polymer, stands out as a first choice material for hydrogels elaboration in biomedical, cosmetic, and health related applications, owing to its interesting properties (as biocompatibility, biodegradability, antimicrobial capacity, and mucoadhesivity). Moreover, chitosan also allows drugs to go easier through biological barriers. The main objective of this review is to report the various uses of chitosan hydrogels as drug delivery devices to control and/or delay the release of drugs loaded into their three dimensional matrix. A wide spectrum of corresponding biomedical applications of these systems can be encountered in the literature, whatever the physicochemical nature of drugs (hydrophilic, hydrophobic, macromolecular), as detailed in this review.

Embedment of liposomes into chitosan physical hydrogel for the delayed release of antibiotics or anaesthetics, and its first ESEM characterization.

This work describes the characterization of an original liposomes/hydrogel assembly, and its application as a delayed-release system of antibiotics and anaesthetics. This system corresponds to drug-loaded liposomes entrapped within a chitosan (CS) physical hydrogel. To this end, a suspension of pre-formed 1,2-dipalmitoyl-sn-glycero-3-phosphocoline liposomes loaded with an antibiotic (rifampicin, RIF), an anaesthetic (lidocaine, LID), or a model fluorescent molecule (carboxyfluorescein, CF), was added to a CS solution. The CS gelation was subsequently carried out without any trace of chemical cross-linking agent or organic solvent in the final system. Liposomes within the resulting gelled CS matrix were characterized for the first time by environmental scanning electron microscopy. The release of drugs from the assembly was investigated by fluorescence or UV spectroscopy. The cumulative release profiles of RIF and LID (and also CF for comparison) were found to be lower from the "drug-in-liposomes-in-hydrogel" (DLH) assembly in comparison to "drug-in-hydrogel" (DH) system.

Biodistribution and dosimetry of (99m)Tc-RP527, a gastrin-releasing peptide (GRP) agonist for the visualization of GRP receptor-expressing malignancies.

UNLABELLED: The aim of this study was to determine the human biodistribution and radiation dosimetry of (99m)Tc-RP527, a promising radioligand for the visualization of gastrin-releasing peptide (GRP) receptor-expressing human malignancies. METHODS: Whole-body scans were obtained up to 48 h after intravenous injection of 555 MBq (99m)Tc-RP527 in each of 6 subjects. Blood samples were taken at various times up to 48 h after injection. Urine was collected up to 48 h after injection for calculation of renal clearance and whole-body clearance. Time-activity curves were generated for the thyroid, heart, breasts in women, testes in men, and liver by fitting the organ-specific geometric mean counts, obtained from regions of interest, on the respective images as a function of the time after injection. The MIRD formulation was applied to calculate the absorbed radiation dose for various organs. RESULTS: The serial whole-body images showed rapid hepatobiliary excretion, resulting in low background and potentially high-contrast imaging of the thoracic region. Imaging of abdominal tumors may prove problematic, however, because of the extensive bowel activity. (99m)Tc-RP527 was predominantly cleared by the kidneys and to a lesser extent by the gastrointestinal tract. The mean excretion in the urine (+/-SD) at 48 h after injection was 58.3 +/- 5.4 percentage of the injected activity corrected for decay to the time of injection. The highest absorbed doses were received by the excretory organs (i.e., the urinary bladder and gallbladder wall). The average effective dose of (99m)Tc-RP527 was estimated to be 0.0095 mSv/MBq. CONCLUSION: The biodistribution of (99m)Tc-RP527 revealed low lung, myocardial, and liver uptake, which allowed early imaging of the supradiaphragmatic region with a favorable dosimetry (including effective dose) for administered activities required for SPECT imaging.

Is there a role for agonist gastrin-releasing peptide receptor radioligands in tumour imaging?

Gastrin-releasing peptide (GRP) has been shown to be a tumour growth stimulating agent for a number of normal and human cancer cell lines. The tumour growth effect is a direct result of GRP binding to membrane G-protein coupled GRP receptors (GRP-R) on the cell surface. Available data on the role of GRP and GRP-R in human lung, prostate, breast, colorectal and gastric carcinoma are reviewed and it is suggested that radiolabelled agonists are preferable to antagonists for imaging and therapy as they appear to be internalised, yielding a higher target/background ratio. The use of rhenium or indium radiolabels for therapy may provide a new approach to GRP/bombesin expressing tumours.

Evaluation of 99mTc-RP128 as a potential inflammation imaging agent: human dosimetry and first clinical results.

UNLABELLED: 99mTc-RP128 is a bifunctional peptide chelate designed to target the tuftsin receptor, expressed by neutrophils, monocytes, and macrophages. Studies in animal models of both infectious and noninfectious inflammation have shown a positive correlation between accumulation of 99mTc-RP128 and quantitative measures of inflammation. A phase 1 trial was conducted with the objective of determining the safety, biodistribution, and human dosimetry of 99mTc-RP128 in eight healthy volunteers. For evaluation of the potential of 99mTc-RP128 for imaging sites of inflammation, 10 patients with active rheumatoid arthritis were studied. METHODS: Normal biodistribution was determined using the conjugate view method up to 24 h after intravenous injection of 280 MBq 99mTc-RP128. Dosimetry calculations were based on standard MIRD methodology, using the International Commission on Radiological Protection model 30 of the gastrointestinal tract and a voiding bladder model with an interval of 4.8 h. For rheumatoid arthritis patients, whole-body scans and spot views of the hands, knees, and feet were obtained at 1 and 3 h after injection of 475 MBq 99mTc-RP128. RESULTS: 99mTc-RP128 was cleared rapidly from the blood by renal excretion, and no major organs showed significant accumulation. The synovia of the major joints were visualized for all subjects. The effective dose equivalent and the effective dose were calculated to be 0.011 and 0.0094 mSv/MBq, respectively. The highest dose was to the bladder wall, which received 0.076 mGy/MBq. In all rheumatoid arthritis patients, we observed a markedly increased uptake in several affected joints. Painful and swollen joints were detected with a sensitivity of 76% and 69%, respectively. Seventy-three percent of the joints with radiographic signs of erosion were scintigraphically positive. In some patients, lines of increased activity were observed and were considered to correspond to uptake in the synovium lining tendon sheaths in the wrists and hands. CONCLUSION: This study shows that 99mTc-RP128 is safe and can successfully be used to visualize clinically affected joints in patients with long-standing rheumatoid arthritis. A proposed radioactive dose of 450-500 MBq will produce an effective dose well within the range of effective doses for commonly used radiopharmaceuticals.

Technetium-99m RP527, a GRP analogue for visualisation of GRP receptor-expressing malignancies: a feasibility study.

Gastrin-releasing peptide (GRP) receptor scintigraphy could allow prediction of response to GRP receptor-targeted treatment options, early non-invasive diagnosis and in vivo prognostic stratification of GRP receptor-positive tumours. This study reports on the imaging characteristics and efficacy for tumour detection of technetium-99m RP527, a 99mTc chelated targeting peptide derived from bombesin, which binds GRP receptors with high affinity. Ten patients (four men and six women, mean age 56.4 years) either suffering from metastasised prostate (n, number of patients = 4) or breast carcinoma (n=1) or presenting with a clinical diagnosis highly suggestive for breast carcinoma (n=5) were included in the study. In the latter five patients, 99mTc-RP527 scintigraphy was performed prior to diagnostic, e.g. biopsy, and staging examinations. Final diagnosis in these patients was breast carcinoma in all five. In all patients, whole-body planar scans and tomographic images were acquired 1 h and 5-6 h post injection of 555 MBq 99mTc-RP527 and tumour to normal tissue (T/N) ratios determined. 99mTc-RP527 showed specific uptake in four of six breast and one of four prostate carcinomas. T/N ratios derived from planar and tomographic images increased significantly (P<0.01) from 1.65 (SD 1.53) and 3.35 (SD 3.04) to 2.58 (SD 1.26) and 7.23 (SD 8.46), respectively. T/N ratios derived from tomographic images were consistently higher (P<0.01). The data presented suggest that 99mTc-RP527 results in specific tumour localisation and exhibits good imaging characteristics with a good T/N ratio that may be further enhanced by single-photon emission tomography.

The detection and quantitation of inflammation in the central nervous system during experimental allergic encephalomyelitis using the radiopharmaceutical 99mTc-RP128.

RP128 is a novel agent which readily chelates 99mTc to form a radiopharmaceutical which binds in vivo to the tuftsin receptor located specifically on neutrophils and monocyte-macrophages, therefore removing the need for in vitro cell labelling prior to intravenous administration. We have assessed the ability of 99mTc-RP128 to detect central nervous system (CNS) inflammation in experimental allergic encephalomyelitis (EAE), an animal model of the human disease multiple sclerosis. The radiopharmaceutical was recorded at significantly increased levels in all EAE diseased CNS tissues, compared to normal and control samples, at 0.5, 1 and 3 h post-injection using a dual radioisotope technique to correct for non-extravasated tracer (P<0.05). Moreover, extravascular accumulation of the agent could be clearly demonstrated in inflammatory tissues with minimal loss of sensitivity when the secondary isotopic correction for blood volume was omitted. In addition, 99mTc-RP128 successfully monitored glucocorticoid suppression of inflammation (P<0.05), recording a typical dose-response to increasing steroid concentration. Clearly, 99mTc-RP128 can quantitatively detect CNS inflammation and assess responses to therapy indicating potential value as an imaging agent both clinically and as a research aid. Furthermore, the rapid in vivo labelling by 99mTc-RP128 of specific inflammatory cells combined with the ability to monitor the progress of anti-inflammatory therapeutics may recommend the agent for use in a variety of inflammatory conditions.

Endogenous corticosteroids modulate lymphoproliferation and susceptibility to experimental allergic encephalomyelitis in the Brown Norway rat.

Successful induction of the experimental autoimmune disease allergic encephalomyelitis (EAE) depends, in part, upon species susceptibility. The Lewis rat is highly susceptible to EAE whereas the Brown Norway (BN) strain is resistant to induction. Endogenous glucocorticoids influence the manifestation of the disease and recovery from neurological deficits. Moreover, abrogation of the curative steroid-mediated effects converts the condition to a terminal state. In the present study treatment of EAE-inoculated BN rats with the steroid antagonist RU486 (Mifepristone) failed to influence the resistance to symptoms. Similarly, adrenalectomy (ADX) prior to sensitisation did not allow the development of clinical EAE but did facilitate neuroperivascular accumulation of inflammatory-type cells. However, RU486 treatment after ADX induced neurological and histological signs of EAE in the majority of animals. Lymphocyte proliferation studies on cells isolated from BN rats treated with RU486 revealed an enhanced responsiveness to mitogenic and antigenic stimulation. These results strongly implicate endogenous steroids in the expansion of immune cell numbers which would be an absolute requirement for the expression of autoimmune-based neurological disease in otherwise resistant rats.

Serum corticosterone, interleukin-1 and tumour necrosis factor in rat experimental endotoxaemia: comparison between Lewis and Wistar strains.

1. Circulating corticosterone, interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF alpha) activities in serum of Lewis and Wistar rats were measured following injection of lipopolysaccharide (LPS). IL-1 was measured as 'lymphocyte activation factor' (LAF) activity following precipitation of inhibitory activity with polyethylene glycol. TNF alpha activity was measured as cytotoxic activity. 2. Compared to the Wistar, the Lewis rat had higher circulating LAF and TNF activities following LPS, and release of both cytokines was prolonged in this strain. 3. Corticosterone increases in response to LPS were less in the Lewis than in the Wistar rat following the initial peak at 1 h; basal corticosterone was lower in the Lewis rat. 4. Adrenalectomized Lewis rats had even greater amounts of circulating LAF and TNF activities following LPS than did intact animals; the effect of adrenalectomy was not however mimicked by acute treatment with the steroid receptor antagonist, RU486, suggesting that endogenous corticosteroids did not acutely control cytokine release. 5. Although in vivo administration of anti-murine IL-1 alpha antiserum significantly lowered LAF activity of serum, circulating corticosterone in response to LPS was not affected. Similarly, treatment with anti-murine TNF alpha monoclonal antibody (mAb) abrogated TNF activity without affecting corticosterone, suggesting that other mediators may be responsible for corticosterone release following LPS. 6. This 'overproduction' of inflammatory cytokines together with lower circulating corticosterone may contribute to the susceptibility of the Lewis rat to diseases such as adjuvant arthritis or experimental allergic encephalomyelitis.

Development of specific antibody and in vivo response to antigen in different rat strains: effect of dexamethasone and importance of endogenous corticosteroids.

Endogenous glucocorticoids undoubtedly play a role in the control of immune responses: their contribution to inter-strain variation is unknown. The development of specific IgG and IgE was measured following inoculation with ovalbumin in Lewis, Fischer, Wistar and Brown Norway rats. The Lewis gives a smaller IgG and IgE response than the other strains and the response in vivo to antigen injected into the paw correlates with the titre of specific antibody. Treatment with the steroid receptor antagonist RU486 (mifepristone) following inoculation reveals that in the Lewis, and to a lesser extent in the Brown Norway, the development of a specific IgG response is limited by endogenous corticosteroids. The IgG response in different strains is differently sensitive to treatment with the synthetic glucocorticoid dexamethasone, the Lewis being particularly resistant. The importance of control by endogenous corticosteroids should not be overlooked in contributing to strain differences in immune response.

Sex steroids affect glucocorticoid response to chronic inflammation and to interleukin-1.

The influence of gender and sex hormones upon both the hypothalamic-pituitary-adrenal (HPA) axis and the immune and inflammatory responses is well recognized, but it is not clear to what extent the two effects are interdependent. We have investigated this interaction using a chronic inflammation model. Corticosterone levels were measured in mature BALB/c male and female mice, which were intact, sham-operated or gonadectomized. No significant differences were found between groups in baseline corticosterone, but systemic inflammation (cotton-induced granulomas) resulted in stimulation of the HPA axis in a reproducible pattern. Corticosterone levels were higher in sham-operated females than in males, but gonadectomy had opposing effects in the two genders, resulting in reduced levels in females but significantly increased levels in males. A similar pattern emerged after stimulation by ether exposure or injection of interleukin-1 beta. In the chronic inflammatory model, replacement of ovariectomized females with physiological levels of progesterone restored a response similar to that of intact females. Physiological levels of 5 alpha-dihydrotestosterone prevented the increase in corticosterone levels caused by castration in males and also resulted in reduced corticosterone levels in sham-operated females. Oestradiol treatment did not affect corticosterone levels. Release of interleukin-1 by peritoneal macrophages from intact and gonadectomized mice with chronic inflammation followed a similar pattern, females releasing more than males. These data suggest a complex inter-relationship between sex steroids, inflammatory stimuli and the HPA axis, such that females have a greater tendency than males to generate activating signals and in addition have a greater sensitivity to such factors.

The local anti-inflammatory action of dexamethasone in the rat carrageenin oedema model is reversed by an antiserum to lipocortin 1.

1. A local pre-injection of 1 micrograms dexamethasone sodium phosphate strongly inhibited (> 60% inhibition at 3 h; P < 0.001 at all time points) the development of carrageenin-induced paw oedema in the rat induced by a subplantar injection of 0.1 ml, 2% carrageenin. 2. Coinjection of a polyclonal rabbit antiserum raised against human 1-188 recombinant lipocortin 1, which also recognised the rat protein, reversed the inhibitory action of dexamethasone (P < 0.05 at 4 h and 5 h). At the highest volume used (40 microliters) control antisera were without any effect. 3. These data further support the concept that lipocortin 1 is involved in the anti-inflammatory mechanism of action of the glucocorticoids.

Glucocorticoid-and non-glucocorticoid induction of lipocortins (annexins) 1 and 2 in rat peritoneal leucocytes in vivo.

1. We have studied the occurrence, distribution and disposition of lipocortins (annexins) 1, 2 and 5 in mixed peritoneal leucocytes obtained from rats in which glucocorticoid levels were altered by adrenalectomy, administration of the glucocorticoid antagonist, RU486, or by injection of dexamethasone or hydrocortisone, as well as from rats in which the peritoneal cells were elicited by inflammatory stimuli. 2. In cells obtained from untreated rats with an intact adrenal cortex, lipocortins 1, 2 and 5 were readily detectable: the majority of each of the proteins was apparently located intracellularly with much smaller amounts in the membrane. Lipocortin 1 and to a lesser extent lipocortin 5 were also seen in a Ca(2+)-dependent association with the external plasma membrane. Following administration of RU486 (2 x 20 mg kg-1) the amounts of lipocortin 1 and 2 in cells were greatly reduced. Conversely, injection of hydrocortisone (1 mg kg-1) or dexamethasone (0.08 mg kg-1) caused an increase in the amount of lipocortin 1 and 2 in peritoneal cells within 30 min. Lipocortin 5 was unchanged by any manipulation of glucocorticoid levels. 3. Lipocortins 1 and 2 were elevated in both intracellular and membrane-associated fractions of macrophages elicited by intraperitoneal injection in inflammogens. This phenomenon also occurred in adrenalectomized animals. 4. Our data indicate that glucocorticoids control the synthesis of some members of the lipocortin family in rat mixed peritoneal cells but also suggest the existence of a separate system for controlling the generation of this protein. The significance of these observations is considered in relation to the mechanism of glucocorticoid hormone action on eicosanoid production.

Evidence for the presence and location of annexins in human platelets.

In this study the identity of annexins in human platelets has been determined together with their ability to be released by agents which induce platelet degranulation. The presence of proteins cross-reacting to antibodies against annexins I and V was detected in human platelets. However, the study provided evidence that these annexins are not located on the surface of the plasma membrane in a Ca++ dependent manner. Moreover, activation of platelets with several agents which induced platelet degranulation did not cause release of annexins I or V as determined by both immunoblotting and ELISA.

Steroid inhibition of oedema formation in the rat skin.

1. A model has been developed to compare the inhibitory effects of the topical steroid, betamethasone-17-valerate, to those of systemically administered betamethasone upon oedema responses induced by 5-hydroxytryptamine (5-HT), platelet activating factor (PAF) and zymosan-activated serum (ZAS) +/- prostaglandin E1 (PGE1), measured in the rat skin by use of 125I-labelled human serum albumin. 2. Systemic betamethasone had a selective, time- and dose-dependent inhibitory effect upon oedema treatment, with 1 mg kg-1 and a 3 h pretreatment having the greatest effect of the doses and times employed. 3. Topical betamethasone inhibited the oedema responses to all of the stimuli showing no apparent selectivity. 4. Topical betamethasone inhibits inflammatory stimuli in a different manner from systemic betamethasone. The broad spectrum of inhibition suggests that topical betamethasone acts by affecting a fundamental feature of the inflammatory response common to all of the stimuli.

Site of anti-inflammatory action of dexamethasone in rabbit skin.

Systemic pretreatment of rabbits with dexamethasone results in a time-dependent inhibition of oedema responses caused by intradermal injection of both 'direct-acting' and 'neutrophil-dependent' stimuli. Local injection of actinomycin D, an inhibitor of RNA synthesis, inhibits this effect. Our studies suggest that a major action of dexamethasone in this model may be a local inhibition of increased permeability of the vascular endothelium.

Antipyretic actions of human recombinant lipocortin-1.

The effect of human recombinant lipocortin-1 (hrLC-1) on the pyrogenic actions of the synthetic polyribonucleotide polyinosinic:polycytidylic acid (poly I:C) has been studied in conscious rabbits. Poly I:C (2.5 micrograms kg-1) given i.v. produced a biphasic fever with a first peak after 90-105 min and a second peak between 225-240 min. hrLC-1 (50 micrograms kg-1) given i.v. simultaneously with the poly I:C produced a significant reduction in the febrile response but without complete suppression. The thermal response index over 5 h (TRI5) was 4.69 +/- 0.51 for poly I:C given with saline and the TRI5 for poly I:C given with hrLC-1 was 2.66 +/- 0.45 (values are for n = 5 +/- s.e. mean, P less than 0.05). hrLC-1 administered alone had no effect on body temperature and its antipyretic activity was lost on heating. In a separate series of experiments 1 h pretreatment with dexamethasone (1 mg kg-1) given i.v. reduced the pyrogenic response (TRI5) to poly I:C (2.5 micrograms kg-1) from 4.87 +/- 0.54 without dexamethasone to 2.00 +/- 0.25 (n = 5, P less than 0.05) and dexamethasone given alone had no effect on body temperature. These data demonstrate that LC-1 possesses antipyretic actions and raises the possibility that the antipyretic actions of dexamethasone are mediated through the induction of LC-1.

Anti-inflammatory lipocortin 1 production by peripheral blood leucocytes in response to hydrocortisone.

The presence and amount of the anti-inflammatory protein lipocortin 1 was determined in plasma and peripheral blood leucocytes by a highly specific, enzyme-linked immunosorbent assay. Within 120 min of a single intravenous dose of 100 mg hydrocortisone, the intracellular concentrations of lipocortin 1 in peripheral monocytes in 7 of 8 healthy men increased by a median of 225% (range 129-507%) compared with pretreatment levels, and mononuclear cell-surface lipocortin increased by a median of 224% (range 76-483%). Placebo injections had no effect. There was no increase at any time in free plasma or polymorph-associated lipocortin. In 3 of 4 subjects, induction of lipocortin was also observed when whole unseparated blood was incubated in vitro after steroid administration, but cells which had first been isolated and purified were refractory to such induction. Thus rapid changes in the concentration of an active anti-inflammatory protein can occur in man after normal therapeutic doses of hydrocortisone.