Cellular immune reconstitution after subcutaneous alemtuzumab (anti-CD52 monoclonal antibody, CAMPATH-1H) treatment as first-line therapy for B-cell chronic lymphocytic leukaemia.

Little information is available on long-term immune reconstitution after therapy with alemtuzumab in B-CLL patients. We present long-term follow-up data for blood lymphocyte subsets analysed by flow cytometry in previously untreated B-CLL patients who received alemtuzumab subcutaneously as first-line therapy. All lymphoid subsets were significantly (P<0.001) and profoundly reduced; the median end-of-treatment counts for CD4(+), CD8(+), CD3(-)56(+) (natural killer (NK)), CD3(+)56(+) (NK-T) and CD19(+)5(-) (normal B) cells were 43, 20, 4, 1 and 8 cells/microl, respectively. The median cell count of all subsets remained at <25% of the baseline values for >9 months post-treatment. CD4(+) and CD8(+) levels in blood had reached >100 cells/microl in >50% of the patients at 4 months after the end of treatment. One patient had a cytomegalovirus reactivation and one patient developed Pneumocystis carinii pneumonia during therapy. No opportunistic or other major infections were recorded during unmaintained, long-term follow-up. There was no correlation between the cumulative dose of alemtuzumab and the severity or length of immunosuppression. CD52(-) T-cell subsets occurred during the treatment and comprised >80% of all CD4(+) and CD8(+) cells in the blood at the end of therapy. These subpopulations declined gradually during unmaintained follow-up. The relationship between these observations and the safety/antitumour effects of alemtuzumab is discussed.

Starch microspheres induce pulsatile delivery of drugs and peptides across the epithelial barrier by reversible separation of the tight junctions.

Non-parenteral administration of peptide drugs is prevented by the limited permeability of the epithelia lining the mucosal tissues. As a new approach to non-parenteral delivery, degradable starch microspheres (dsm) were coated with insulin and administered to the mucosal side of monolayers of human intestinal epithelial (Caco-2) cells in vitro. The microspheres induced a pulsed delivery of insulin across the epithelium that lasted for 1-2 h. The pulsed delivery correlated with a reversible appearance of focal dilatations in the tight junctions between the epithelial cells, indicating that dsm enhance the delivery of insulin by the paracellular route. These results provide an explanation for the previously observed absorption enhancing properties of dsm.

Influence of degradable starch microspheres on the human nasal mucosa.

The effect of the nasal administration of degradable starch microspheres (DSM) on the mucociliary system and the geometry of the nasal cavities were evaluated in 15 healthy volunteers. The baseline values for mucociliary clearance of the right nasal cavity were determined on two separate days for each subject using the saccharin-dyes test. Acoustic rhinometry was performed before and during the saccharin-dyes test. The patients then started the treatment period and inhaled 10 mg of DSM intranasally once daily in each nostril for 8 days. The saccharin-dyes test was performed 5 min after the deposition of the DSM on day 1 and day 8. The geometry of the nasal cavities was determined before, 7 min after deposition, and after the end of the saccharin test. Both tests were also performed two days after the end of the treatment period. Each subject was examined by means of rhinoscopy on every visit during the investigation. No changes in mucociliary clearance or in the geometry of the nasal cavities were found after repeated administration of starch microspheres. Thus, intranasally-administered degradable starch microspheres did not have an adverse effect on human nasal mucociliary clearance, and the DSM did not cause any congestion or decongestion of the mucosa.

Pharmaceutical formulations--suspensions and solutions.

Solutions and suspensions of drugs are used widely in the pharmaceutical industry for production of dosage forms for different routes of administration; for example, oral, parenteral and inhalation. Pharmaceutical solutions and suspensions might appear to be simple formulations but they can present many technical problems both for the manufacturing industry and for the individual pharmacist. Substances can be chemically unstable, insoluble in water, distasteful etc. Suspensions are often used as a dosage form when the drug is insoluble in water and when use of solubilizing agents is not possible. Based on method of preparation, suspensions can be divided into two categories, flocculated and deflocculated systems.

Effect of particle size on ocular permeability of prednisolone acetate in rabbits.

The ocular permeability of two sieved fractions of prednisolone acetate with a mean particle size of less than 5 microns and 5-10 microns was studied in rabbits. The results show that there was not significant difference between the corneal uptake of the two fractions of prednisolone acetate. Prednisolone acetate was rapidly taken up by the cornea where it is hydrolyzed rapidly to prednisolone. Only prednisolone was detected in the aqueous humor showing that prednisolone acetate is completely hydrolyzed in the cornea. Both fractions produced similar concentrations of prednisolone in the aqueous humor. For prednisolone acetate, the permeability rate rather than the dissolution rate seems to be the rate-limiting step for corneal transport.

Precorneal residence time in humans of sodium hyaluronate as measured by gamma scintigraphy.

The aim of the present study was to quantify in man the distribution and clearance of two aqueous sodium hyaluronate (SH) solutions of 0.125% and 0.250% after the administration of 25 microliters onto the cornea. Isotonic phosphate buffer (PB) was used as a reference instillation. No systemic or local medication was given to the seven 18- to 30-year-old, healthy male volunteers. A detailed evaluation of the anterior segment of the eye, as well as a Schirmer test and a break-up time measurement, yielded results within the normal range. The clearance of 0.125% and 0.250% SH solutions radiolabelled with sodium pertechnetate Tc-99m was measured by gamma scintigraphy and compared with that of a PB solution tagged with the same radiolabel. There was no statistically significant difference between the quantities of 0.125% SH and PB solutions remaining in the precorneal space at 20 min (paired t-test, P = 0.78, n = 7). However, in comparing the 0.250% SH with the PB solution, we observed a statistically significant difference (P = 0.01, n = 7) in the amount remaining in the precorneal space after the same interval. Actually, 53% of the radiolabelled 0.250% SH solution remained on the cornea as compared with 30% for the 0.125% SH solution and 18.3% for the PB solution. These results suggest that an SH solution of 0.250% might have a prolonged residence time on the precorneal surface, and that SH could therefore be used as an additive in various drug-release systems for the eye.

Sodium hyaluronate as an ophthalmic vehicle: some factors governing its effect on the ocular absorption of pilocarpine.

Sodium hyaluronate, as an additive to aqueous ophthalmic formulations, has been claimed to increase the ocular contact time and, thereby, the drug bioavailability. In the present study, the effect of sodium hyaluronate on corneal residence time and drug absorption in rabbits was investigated. Addition of sodium hyaluronate (0.125%) to a 3H-pilocarpine HCl solution resulted in increased retention of radioactivity in tear fluid and a 2-fold increase in drug concentration in the cornea and aqueous humor. Further, the effects of concentration and molecular weight of sodium hyaluronate on the miosis induced by pilocarpine in rabbits were studied. A significant increase of miotic response was seen at concentrations just less than 0.1% sodium hyaluronate. Pilocarpine solutions prepared from high mol.wt. sodium hyaluronate exhibited a greater miotic response than those prepared from lower mol.wt. samples. This might indicate that other physicochemical properties of sodium hyaluronate influence drug bioavailability.

Coprecipitation of enzymes with water soluble starch--an alternative to freeze-drying.

Krill proteases were prepared in solid form from a partially purified extract by coprecipitation of the enzymes with water-soluble starch in an organic solvent at 22 degrees C. The precipitation did not affect the activity of the enzymes. The recovery of proteolytic activity was 100%. The thermostability of the krill proteases increased when incorporated in the starch precipitate. No reduction in enzymatic activity could be seen after storage at +50 degrees C for 99 days. After milling the coprecipitate could be dispensed. The enzyme preparation consisted of irregular needle-shaped particles. This simple precipitation technique offers an alternative to freeze-drying or spray-drying.

Biodegradable microspheres. VII: Alterations in mouse liver morphology after intravenous administration of polyacryl starch microparticles with different biodegradability.

Semisynthetic polyacryl starch microparticles are being investigated as drug carriers. In the present paper the possible adverse effects, reflected as morphological alterations, of iv administration of polyacryl starch microparticles were studied in mice. The spleen, lungs, and kidneys displayed a normal morphology after microparticle administration, while dose-dependent reversible alterations of the liver morphology were observed. The alterations initially consisted of vacuolization of the hepatocytes along the sinusoids, followed by unicellular hepatocyte necrosis and formation of granulomas. Later, an increased number of mitotic cells reflected tissue generation and, after two weeks, the tissue morphology was essentially normalized, with the exception of an increased number of binucleated hepatocytes. After repeated administration of the particles in low doses, the same types of alterations were observed but the kinetics of tissue repair was slower. Possible mechanisms inducing these alterations are discussed and comparisons are made with the effects of synthetic polyacrylamide microparticles.

Influence of sodium hyaluronate on the meiotic effect of pilocarpine in rabbits.

Sodium hyaluronate is a potential vehicle for drugs given topically to the eye. In the present study, the effect of sodium hyaluronate on the miosis induced by pilocarpine in rabbits was investigated. Addition of 0.2 and 0.75% sodium hyaluronate to 1% pilocarpine hydrochloride in buffer resulted in an increased effect measured as area under the miosis-time curve (AUC) and duration of miosis. A comparison between a commercially available 1% pilocarpine preparation and the preparations containing sodium hyaluronate showed that sodium hyaluronate improves the effect parameters. No adverse effects with sodium hyaluronate were observed. Possible mechanisms inducing these improvements are discussed.

Macrophage stimulation with some structurally related polysaccharides.

The macrophage-stimulating properties of some structurally related polysaccharides were studied in vitro. When the polysaccharides were presented to the macrophages in a sterically fixed form, i.e. as microparticles, they induced the release of interleukin 1 (IL-1) from the macrophages. Microparticulate 1.3-beta-glucan (curdlan) induced nonspecific macrophage mediated tumour cell killing while 1.4-alpha-glucan (starch), 1.6-alpha-glucan (dextran), and 1.6-alpha-mannan were without effect. The corresponding soluble polysaccharides did not stimulate the macrophages. Kinetic studies showed that although IL-1 was released immediately after stimulation, the macrophages needed a time lag of several days to develop tumour cytotoxicity. The development of cytotoxicity paralleled binding of tumour cells to the macrophages. Resident and inflammatory peritoneal macrophages showed differences in their responses to the polysaccharides. Stationary, resident peritoneal macrophages stimulated by macroparticles secreted high levels of IL-1 but expressed a low cytotoxic activity, while newly recruited inflammatory macrophages released lower levels of IL-1 but readily killed the tumour cells. The influence of cyclo-oxygenase products on the IL-1 release and macrophage cytotoxicity was also investigated. When cyclo-oxygenase was blocked with indomethacin, a significantly higher release of IL-1, and then an increased cytotoxicity, were obtained with 1.3-beta-glucan stimulated macrophages. The results suggest that microparticulate polysaccharides may be useful for studies on the induction of macrophage differentiation and also for studies on nonspecific cellular immune responses in vitro and in vivo.

Biodegradable microspheres. V: Stimulation of macrophages with microparticles made of various polysaccharides.

The interaction between four different microparticulate drug carriers and macrophages was investigated in vitro. The microparticles, consisting of crosslinked starch (1,4-alpha-D-glucan with 1,6-alpha-branches), dextran (1,6-alpha-D-glucan with 1,3-alpha-branches), lichenan (1,3-beta-D-glucan), or mannan (1,6-alpha-D-mannan with 1,2-alpha- and 1,3-alpha-branches), were investigated for their macrophage stimulatory properties. Macrophage stimulation was assayed by the uptake of [14C]glucosamine and stimulatory indices were calculated. Microparticles made of crosslinked lichenan were most stimulatory, followed by the biologically inert mannan and dextran microparticles. Biodegradable starch microparticles were less stimulatory to the macrophages than the other microparticles. All microparticles were phagocytosed to the same extent and stimulated the macrophages to release oxygen radicals. Lichenan, mannan, and dextran microparticles induced morphological changes in the macrophages when given in nontoxic doses. No morphological changes were observed when the macrophages were exposed to starch microparticles or soluble polysaccharides.

Studies on microbial contamination of reused disposable plastic insulin syringes.

The microbial contamination of reused disposable insulin syringes and bottles was studied. Fourteen patients, aged 16-64 years, took part in the study. 50 syringes and 52 bottles were examined for sterility at different stages of use. Twelve (24%) syringes and 4 (8%) bottles were found to be contaminated by micro-organisms, although only in low numbers. The micro-organisms recovered belonged to the normal skin flora of man.

Acrylic microspheres in vivo. X. Elimination of circulating cells by active targeting using specific monoclonal antibodies bound to microparticles.

The elimination from the blood of 51Cr-labelled mouse erythrocytes modified with trinitrophenyl (TNP) groups was followed in mice. After 24 hours, when a stable concentration of the labelled erythrocytes has been attained, monoclonal anti-TNP-antibodies were given intravenously, either in free, soluble form, or bound to microparticles containing immobilized protein A. The anti-TNP-antibodies induced a rapid elimination of the TNP- and 51Cr-labelled erythrocytes. Over the 8-hours time period studied, the elimination rate was significantly faster when the antibodies were administered bound to the particles. After the elimination of the target cells, the radioactivity was found in the liver, spleen and bone marrow. These results and relevant control experiments indicate that a solid carrier 1. can be directed to a specific target cell with a specific antibody and 2. can induce a rapid elimination of the target cell from the circulation.

Biodegradable microspheres II: Immune response to a heterologous and an autologous protein entrapped in polyacryl starch microparticles.

Human serum albumin (HSA) and mouse serum albumin were entrapped in biodegradable microparticles of polyacrylstarch. When HSA entrapped in microparticles was injected i.v. in Balb/c mice a dose-dependent immune response was elicited. No detectable response was obtained when mouse serum albumin entrapped in microparticles was given to the mice. Neither was any response detected when free HSA or free HSA in combination with empty microparticles were injected, as measured by indirect plaque-forming cells in the spleen and serum antibody titers. The immune response to entrapped HSA had a long duration and was mainly T-cell-dependent as athymic nude mice (Nu/Nu) only generated a weak immune response upon injection of the particle entrapped antigen.

Biodegradable microspheres. I. Duration of action of dextranase entrapped in polyacrylstarch microparticles in vivo.

Dextranase was entrapped in polyacryl starch microspheres of different compositions by emulsion polymerization. After i.v. injection in mice and rats, the particles were removed from the blood circulation by macrophages of the reticuloendothelial system. In these cells, the particles are accumulated in the lysosomes. The degradation of different 14C-labeled microparticles and their entrapped dextranase was followed in an isolated lysosomal fraction in vitro and in liver and spleen after i.v. injection in mice. The duration of entrapped dextranase in vivo was followed directly, i.e., by an enzyme assay, and indirectly by following the decrease of a stored material, [3H]dextran in the liver. The degradation of the entrapped enzyme was dependent on the composition of the particle matrix. More cross-linked spheres could better protect the entrapped enzyme in vitro and in vivo. The half-life of free dextranase in the lysosomal fraction was estimated to be about 4 hr, whereas the duration of entrapped dextranase in the liver was at least 48 hr, as measured with [3H]dextran. Finally, the effect of entrapped and free dextranase on an artificially induced storage disease was studied. The stored [3H]dextran was eliminated completely when dextranase was used in microparticles, whereas free dextranase had no effect in vivo.