Administration Of Microcapsules Research Articles

Targeted Therapy for Glomerulonephritis Using Arterial Delivery of Encapsulated Etanercept.

Complex immunosuppressive therapy is prescribed in medical practice to patients with glomerulonephritis to help them overcome symptoms and prevent chronic renal failure. Such an approach requires long-term systemic administration of strong medications, which causes severe side effects. This work shows the efficiency of polymer capsule accumulation (2.8 ± 0.4 µm) containing labeled etanercept (100 μg per dose) in the kidneys of mice. The comparison of injection into the renal artery and tail vein shows the significant superiority of the intra-arterial administration strategy. The etanercept retention rate of 18% and 8% ID in kidneys was found 1 min and 1 h after injection, respectively. The capsules were predominantly localized in the glomeruli after injection in mice using a model of acute glomerulonephritis. Histological analysis confirmed a significant therapeutic effect only in animals with intra-arterial administration of microcapsules with etanercept. The proposed strategy combines endovascular surgery and the use of polymer microcapsules containing a high molecular weight drug that can be successfully applied to treat a wide range of kidney diseases associated with glomerular pathology.

The effectiveness of marine bacterial microcapsules in controlling vibriosis disease caused by the infection of Vibrio parahaemolyticus in white shrimp Litopenaeus vannamei

Vibriosis is a major bacterial disease in shrimp farming, which is caused by Vibrio spp. It has resulted in substantial shrimp mortality. One strategy to control this disease is to use microcapsules containing marine bacteria. This study was established to evaluate the effectiveness of such microcapsules in controlling vibriosis in white shrimp. The shrimps were fed a diet with microcapsules containing three different bacteria, namely, Bacillus subtilis P2.24, Brevibacterium casei D6.6, and Bacillus altitudinis D6.19, for 30 days. The shrimps were then tested by injecting Vibrio parahaemolyticus (10 6 CFU/mL) on the 31st day. The administration of microcapsules containing the three marine bacteria had a positive effect on white shrimp. The microcapsules with B. subtilis P2.24 showed the best effectiveness as an anti-vibriosis agent, as indicated by the high survival rate after 7 days of challenge test, which was 40% higher than that in the positive control (Duncan's test p < 0.05). Furthermore, the microcapsules containing the three marine bacteria promoted immune responses [i.e., total hemocyte count, percentage of semi-granular/granular cells, phenoloxidase activity, respiratory burst, and immune-related gene expression], microbiota diversity, and total viable bacterial count in shrimp intestinal tract. In addition, the microcapsules reduced the total Vibrio count and total V. parahaemolyticus count in shrimp intestinal tract, as well as the damage to hepatopancreas and muscle. In conclusion, the B. subtilis P2.24 isolate is a potential vibriosis biocontrol agent and an alternative for preventing vibriosis. • The administration of the marine bacterial microcapsule increased the survival rate of white shrimp Litopenaeus vannamei after a challenge test with Vibrio parahaemolyticus , which P2.24 showed the best bacterial strain. • The marine bacterial microcapsule increased the shrimp immunity and the intestinal microbiota diversity. • The marine bacterial microcapsule reduced the V. parahaemolyticus population in intestinal and damage in shrimp hepatopancreas and muscle. • The viability of the marine bacterial microcapsule showed stability after the drying process and storage.

Abstract P121: A Novel Delivery of Alpha-Calcitonin Gene Related Peptide (α-CGRP) Using Alginate Microcapsules Protects Against Pressure-Overload Induced Heart Failure

The cardioprotective role of α-calcitonin gene related peptide (α-CGRP), a 37-amino acid neuropeptide and potent vasodilator, in cardiac diseases has been established by our laboratory and others. Systemic delivery of α-CGRP decreases blood pressure in hypertensive humans, and improves hemodynamic variables in congestive heart failure (CHF) patients. However, the short half-life of the peptide (t 1/2 = ~5.5 min in serum) limits α-CGRP use in any long-term treatment regime. The present study utilized alginate-α-CGRP microcapsules for long-term continuous delivery of α-CGRP in a mouse CHF model. We used an electrospray method to prepare α-CGRP encapsulated alginate microcapsules approximately 200μm in size. The α-CGRP encapsulated microcapsules showed no cytotoxicity when incubated with the cardiac cell lines, HL-1 and H9c2 cells, for 7 days. Subcutaneous administration of microcapsules containing 150, 250, or 500μg α-CGRP lowered the systolic pressure up to 18 h, 3 days, and 7 days, respectively, in wild-type mice (measured by tail-cuff method), indicating that released α-CGRP remains biologically active. The cardioprotective effect of microcapsules released α-CGRP was examined using a transverse aortic constriction (TAC) induced pressure-overload mouse model of CHF. After two days of TAC, 500μl of alginate-α-CGRP microcapsules (containing 150μg α-CGRP) was administered in nine weeks old C57/BL6 mice on alternate days up to 28 days. Echocardiography data showed that microcapsule treatment of α-CGRP significantly preserved left ventricular fraction shortening (FS) and ejection fraction in the TAC mice (FS±SD: sham 47.2±1.5%, sham-alginate-α-CGRP 48.4±1.8%, TAC 29.5±1.4%, and TAC-alginate-α-CGRP 43.2±1.2%). α-CGRP delivery attenuated cardiac hypertrophy in TAC mice when reported as heart wt/tibia length (mg/mm±SD): sham 6.3±0.1, sham-alginate-α-CGRP 6.3±0.2, TAC 8.6±0.3, and TAC-alginate-α-CGRP 6.6±0.2. Alginate-only microcapsules did not affect these cardiac parameters. Our results show that α-CGRP delivery through alginate microcapsules protect hearts from cardiac failure. As alginate is immunologically inactive, the alginate microcapsules offer a new potential delivery paradigm for patients with heart failure.

Physicochemical and preclinical pharmacokinetic and toxicological evaluation of LK-423—a new phthalimido-desmuramyl-dipeptide derivative with immunomodulating activity

Introduction: LK-423 is a new phthalimido-desmuramyl-dipeptide derivative with immunomodulating activity. As optimized delivery to the site of action appears crucial for further preclinical development of LK-423, the aim of this study was to perform a physicochemical and preclinical pharmacokinetic and toxicological evaluation. Methods: The solubility, partition coefficient, permeability, and stability profile were determined. Pharmacokinetics were evaluated in rats following intravenous and oral application of LK-423, and in dogs after intravenous administration and oral administration of microcapsules, designed for colon-specific delivery of LK-423 based on pH-, time-, and enzyme-controlled release mechanisms. Additionally, the acute and subchronic toxicity was examined. Results and discussion: LK-423 is hydrophilic, sparingly to slightly soluble, and poorly permeable. Stability profile in aqueous solution is pH dependent. A pharmacokinetic study following intravenous application to rats and dogs revealed that LK-423 is rapidly eliminated with a short terminal phase half-life, and high plasma clearance, as well as a limited distribution to the peripheral tissue. Oral bioavailability of LK-423 is low, presumably due to low permeability. Debris of insoluble microcapsule coating in feces and obtained plasma concentration profiles confirm that LK-423 microcapsules are a promising approach for local treatment of inflammatory diseases of the large intestine. Acute and a subchronic toxicity results indicate that LK-423 is a safe and nontoxic drug under the applied experimental conditions.

In Vitro Characterization and In Vivo Functionality of Erythropoietin-Secreting Cells Immobilized in Alginate−Poly-l-Lysine−Alginate Microcapsules

The in vitro and in vivo characterization of cell-loaded immobilization devices is an important challenge in cell encapsulation technology for the long-term efficacy of this approach. In the present paper, alginate-poly-l-lysine-alginate (APA) microcapsules containing erythropoietin (Epo)-secreting C2C12 myoblasts have been elaborated, characterized, and tested both in vitro and in vivo. High mechanical and chemical resistance of the elaborated microcapsules was observed. Moreover, the in vitro cultured encapsulated cells released 81.9 +/- 8.2 mIU/mL/24 h (by 100 cell-loaded microcapsules) by day 7, reaching the highest peak at day 21 (161.7 +/- 0.9 mIU/mL/24 h). High and constant hematocrit levels were maintained over 120 days after a single subcutaneous administration of microcapsules and lacking immunosuppressive protocols. No major host reaction was observed. On the basis of the results obtained in our study, cell encapsulation technology might be considered a suitable therapeutic strategy for the long-term delivery of biologically active products, such as Epo.

Immobilization of Mesenchymal Stem Cells and Monocytes in Biocompatible Microcapsules to Cell Therapy

The aim of cell therapy is to replace, repair, or enhance the function of damaged tissues or organs. Several factors complicate the development of cellular therapies. Of primary importance is protection of the implanted cells from the host's immune system. Cells are encapsulated in selectively semipermeable and biocompatible membranes that block entry of immune mediators but allow outward diffusion of active molecules produced by the cells. The immobilization of mesenchymal stem cells and monocytes, in micrometric (30-60 microm) alginate-barium microcapsules based on atomization processes, has been achieved successfully. This size is necessary to the administration of microcapsules via injection (Hamilton syringe with a needle size of 100 microm) and aerosol. Microencapsulated cells survive at least 2 weeks after preparation in vitro.

In-vitro analysis of APA microcapsules for oral delivery of live bacterial cells

Oral administration of microcapsules containing live bacterial cells has potential as an alternative therapy for several diseases. This article evaluates the suitability of the alginate-poly-L-lysine-alginate (APA) microcapsules for oral delivery of live bacterial cells, in-vitro, using a dynamic simulated human gastro-intestinal (GI) model. Results showed that the APA microcapsules were morphologically stable in the simulated stomach conditions, but did not retain their structural integrity after a 3-day exposure in simulated human GI media. The microbial populations of the tested bacterial cells and the activities of the tested enzymes in the simulated human GI suspension were not substantially altered by the presence of the APA microcapsules, suggesting that there were no significant adverse effects of oral administration of the APA microcapsules on the flora of the human gastrointestinal tract. When the APA microcapsules containing Lactobacillus plantarum 80 (LP80) were challenged in the simulated gastric medium (pH = 2.0), 80.0% of the encapsulated cells remained viable after a 5-min incubation; however, the viability decreased considerably (8.3%) after 15 min and dropped to 2.6% after 30 min and lower than 0.2% after 60 min, indicating the limitations of the currently obtainable APA membrane for oral delivery of live bacteria. Further in-vivo studies are required before conclusions can be made concerning the inadequacy of APA microcapsules for oral delivery of live bacterial cells.

Microencapsulated, CYP2B1-transfected cells activating ifosfamide at the site of the tumor: the magic bullets of the 21st century.

Conventional chemotherapy of pancreatic carcinoma is only marginally effective. This is in part due to the severity of side effects following systemic administration of the cytostatic drug. The aim was to create a therapeutic tool allowing the targeting of the conversion site of a cytotoxic prodrug to the site of the tumor. This was realized by transfection of the CYP2B1 gene, the major ifosfamide-converting P450 enzyme, in cells with subsequent microencapsulation and administration of these microcapsules to or into the tumor. The enzyme activity (resorufin assay) remained stable for weeks in vitro and in vivo within the microencapsulated CYP2B1-expressing cells. We demonstrated a significant antitumor effect of the intratumorally injected capsules against xenotransplanted human pancreatic carcinomas in the nude mouse. Angiographic experiments in the pig confirmed the feasibility of an intraarterial placement of the capsules into the pancreas. A clinical protocol was established and approved. L293 cells were transfected with the CYP2B1 gene, microencapsulated (diameter 0.7 mm) under GCP conditions and packed sterile. Patients with confirmed inoperable adenocarcinoma of the pancreas underwent angiography, and capsules were injected into a vessel leading into the tumor. The patients were monitored for 48 h to exclude allergic reactions or pancreatitis. A day later, ifosfamide was administered for three consecutive days to be repeated on days 21-23. The patients were followed up for 5 months. A total of 17 patients were enrolled. The patients tolerated the procedure without any complications. No allergic reactions or pancreatitis were encountered. Chemotherapy was uneventful. All patients had stable disease, and two patients a partial remission. The median survival was 44 weeks which compared favorably with that of a historical control group (22 weeks). The intraarterial administration of microcapsules for targeted chemotherapy was well tolerated. Control of local tumor growth was achieved.

Bioavailability of meclofenamate from experimental sustained-release microcapsules in beagle dogs

Abstract This investigation was carried out to evaluate the absorption characteristics of experimental meclofenamate sustained-release microcapsules, prepared by an emulsion-solvent evaporation method using high molecular weight cellulose propionate polymer and polyethylene glycol (PEG) 2000 in a ratio of 1:2:1 drug:polymer:PEG, using eight male beagle dogs. Meclofenamate was administered intravenously at a dose of 40 mg and orally as a single dose (50 mg) of conventional capsules (Meclomen®), oral solution and microcapsules on four separate occasions. Statistically significant differences were observed between the microcapsules and the other two oral treatments in both the peak plasma concentration ( C max ) and the time of peak concentration ( T max ). No significant difference was found between the three oral treatments in the area under the plasma concentration-time curve (AUC), indicating a comparable extent of absorption. The absorption rate ( C max /AUC) was significantly slower following the administration of microcapsules. Both the mean residence time (MRT) and the mean absorption time (MAT) were dramatically increased following oral administration of the microcapsules compared to the conventional capsules and the oral solution. The mean (in-vivo) dissolution time (MDT) for the prepared microcapsules was 2.14 h and for the conventional capsules 0.3 h, which were found to be consistent with the in-vitro availability of the drug. Duncan's multiple range test indicated no significant difference between the oral solution and the conventional capsules in any of the calculated pharmacokinetic parameters. The absolute bioavailability from the oral solution, the conventional capsules and the experimental microcapsules was 74.6, 67.9 and 72.5%, respectively.

Sustained release of phenytoin following the oral administration of phenytoin sodium/ethylcellulose microcapsules in human subjects and rabbits.

Phenytoin sodium was microencapsulated with ethylcellulose (EC) by a coacervation-phase separation method from ethyl acetate solution to develop a prolonged release dosage form of phenytoin. Release of phenytoin from the microcapsules (phenytoin sodium/EC) was evaluated by the JP dissolution test in JP disintegration media No. 1 and No. 2. The release rates of phenytoin from phenytoin sodium powders were extremely rapid in both media, however, the release rates from the microcapsules were much more retarded. Following the oral administration of microcapsules to rabbits, prolonged plasma concentrations of phenytoin were obtained, while microcapsules orally administered to human subjects showed prolonged urinary excretion of phenytoin metabolites.

Practical Application of Microcapsulation for Toxicity Studies Using Bromodichloromethane as a Model Compound

Gelatin-starch syrup (food grade) microcapsulation was applied for toxicology studies of bromodichloromethane (BDCM). BDCM concentrations were stable for 120 days in the microcapsules and for 9 months when incorporated in the powder diet. BDCM concentration in the blood following the administration of microcapsules in olive oil suspension was retained at higher levels than when BDCM was administered as olive oil solution. Subsequently, the microcapsules were mixed in powder diet and were given at concentrations of microcapsulated BDCM of 0, 0.024, 0.072, and 0.215% to groups of 7 male Wistar rats for 1 month. For comparison, BDCM dissolved in olive oil was administered by gavage to groups of 7 male rats for 1 month at dosage levels adjusted to those in the feeding study (0, 20, 60, and 180 mg/kg body weight). Suppression of body weight gain was seen in the high-dosage groups in both the feeding and the gavage studies and was more severe in the former. Similar histopathologic lesions in the liver were shown in both studies, including vacuolization, swelling, and single necrosis of liver cells. Hepatic cord irregularity and bile duct proliferation were observed in the feeding study but not the gavage study. Serum biochemical changes, such as decreases in glucose, triglyceride, and cholinesterase levels, which reflected the histopathologic findings in the liver, were also observed in both studies. Accordingly, the microcapsulation process was proved to pose no qualitative toxic effects on toxicity of BDCM in short-term toxicity studies. It is concluded that the application of microcapsulation is useful for toxicity tests of volatile chemicals when incorporated into food.

Inhibition of the pituitary-gonadal axis by a single intramuscular administration of D-Trp-6-LH-RH (decapeptyl) in a sustained-release formulation in patients with prostatic carcinoma.

For the past 6 years we used daily injection of luteinizing hormone-releasing hormone (LH-RH) agonists to treat patients with advanced prostate carcinoma. In this study we determined the hormonal response of the pituitary-testicular axis over a 2-month period and evaluated the safety and tolerance of the single intramuscular administration of sustained-release formulations of D-Trp-6-LH-RH microcapsules designed to release 50, 100, or 200 micrograms/day for over 1 month. Serum levels of LH, testosterone, and D-Trp-6-LH-RH were measured by RIA for up to 60 days in 10 patients with advanced prostatic carcinoma who had not received any previous drug therapy. After the administration of the microcapsules there was a biphasic increase in D-Trp-6-LH-RH serum levels. The maximal peak was obtained between 1 and 3 hr, and a second peak occurred between weeks 4 and 6. LH levels increased initially, with a maximal peak at 60 min, and elevated serum LH values persisted for more than 24 hr. LH levels began to fall on the second day, reaching subnormal values after 1 week. Serum testosterone rose during the first week and fell subsequently to less than 100 ng/dl. A rebound in LH and testosterone was seen about the 50th day after the microcapsule administration. Following the first week of therapy, we observed in all patients a significant decrease in bone pain, improvement in urinary flow obstruction, and a reversal of the signs of prostatism. No side effects were observed, and acceptance of the microcapsules was very good. Our results show that a single dose of D-Trp-6-LH-RH microcapsules suppresses of the pituitary-testicular axis for at least 50 days. D-Trp-6-LH-RH microcapsules facilitate the treatment and should lead to an improvement in the therapeutic response.

108 HYPOXANTHINE ACCUMULATION AND DOPAMINE DEPLETION IN LESCH-NYHAN DISEASE: ANIMAL MODELS AND HUMAN STUDIES

In LND, Hx is elevated, especially in brain and CSF. Since Hx accumulation may promote lipid peroxidation and since similar mechanisms may subserve 6-OHDA neurotoxicity, we considered a simple relationship between Hx accumulation and DA depletion in patients with LND. Cannulas packed with solid Hx were implanted in caudate nucleus of rats; turning behavior was stimulated by injection of apomorphine. Treated animals show pronounced time and dose-dependent ipsilateral turning, which peaks 14-18 days after cannula implantation. Turning is blacked or reduced by haloperidol, a general DA antagonists and by SCH 23390, a selective D1 antagonist. Uric acid, purines, nucleosides, catecholamines and metabolites and 5HIAA were monitored by HPLC of brain regions. Depletion of DA was inversely related to purine concentration at peak times of behavioral effect. NE was not reduced in limbic or corticol forebrain. DA receptors were increased 1.3-1.7x in treated caudate. Allopurinol did not block the development of DA depletion. In fact, cannulas packed with allopurinol elicited similar behaviors. In a preliminary clinical trial, we tested the ability of encapsulated and immobilized xanthine oxidase to reduce circulating purines. A single HPRT-null patient (age 18 monghs) stopped allopurinol therapy 3 days before admission to the study. Over a 10 day period of microcapsule administration, his UA/Creat ratio returned to 1.9 and his plasma Hx level fell from 190 uM to 100 uM. Support: Medical Research Council of Canada to RMP & TMSC

Combination of a long-acting delivery system for luteinizing hormone-releasing hormone agonist with Novantrone chemotherapy: increased efficacy in the rat prostate cancer model.

The combination of hormonal treatment based on a long-acting delivery system for the agonist [6-D-tryptophan]luteinizing hormone-releasing hormone ([D-Trp6]-LH-RH) with the chemotherapeutic agent Novantrone (mitoxantrone dihydrochloride) was studied in the Dunning R3327H rat prostate cancer model. Microcapsules of [D-Trp6]-LH-RH formulated from poly(DL-lactide-co-glycolide) and calculated to release a controlled dose of 25 micrograms/day were injected intramuscularly once a month. Novantrone (0.25 mg/kg) was injected intravenously once every 3 weeks. Three separate experiments were carried out. When the therapy was started 45 days after transplantation and continued for 70 days, tumor volume in the presence of the microcapsules (966 +/- 219 mm3) or Novantrone (3606 +/- 785 mm3) given alone was significantly decreased compared to controls (14,476 +/- 3045 mm3). However, the combination of microcapsules and Novantrone caused a greater inhibition of tumor growth (189 +/- 31 mm3) than the single agents. Similar effects were seen when the percent increase in tumor volume was examined. Tumor volume increased 10,527 +/- 1803% for the control group. The inhibition of growth caused by the [D-Trp6]LH-RH microcapsules alone (672 +/- 153% increase in volume) was again greater than that caused by Novantrone alone (2722 +/- 421% increase). The combination of the two agents was again the most effective, resulting in an increase in tumor volume of only 105 +/- 29%. Control tumors weighed 30.0 +/- 6.5 g. Tumor weights were much less in the groups treated with either microcapsules (3.28 +/- 0.69 g) or Novantrone (19.53 +/- 3.3 g) alone. The lowest tumor weights after 70 days of treatment were obtained in the group that received the combination of [D-Trp6]LH-RH microcapsules and Novantrone (1.02 +/- 0.2 g). Testes and ventral prostate weights were significantly diminished by the administration of microcapsules of [D-Trp6]LH-RH alone or in combination with Novantrone. In both of these groups, luteinizing hormone and prolactin levels were reduced and serum testosterone was suppressed to undetectable levels. Similar results were obtained in two other experiments in which the duration of treatment was 60 or 105 days. These results suggest that the overall response could reflect the inhibition of proliferation of hormone-independent cancer cells by Novantrone in addition to the suppressive effect of [D-Trp6]LH-RH on the growth of androgen-dependent tumor cells. The administration of Novantrone in combination with microcapsules of [D-Trp6]LH-RH might produce a better clinical response than LH-RH analog alone in patients with advanced prostate carcinoma.

Effects of a long-acting LHRH agonist preparation on plasma gonadotropin and prolactin levels in castrated male rats and on the release of prolactin from ectopic pituitaries

We have examined the effects of a single subcutaneous injection of an LHRH agonist, d-Trp-6-LHRH, in biodegradable microcapsules of poly( dl-lactide-co-glycolide) on plasma gonadotropin and prolactin (PRL) levels in castrated and in castrated-hypophysectomized-pituitary grafted (CAST-APX-GRAFT) male rats. The results were compared to the effects of daily injections of the same LHRH agonist dissolved in saline. In castrated rats, there were no significant alterations in plasma LH or PRL levels during the 10 days following the injection of LHRH agonist microcapsules, while FSH levels were generally reduced. In castrated males given daily injections of 6 μg of LHRH agonist in saline, plasma LH levels were significantly reduced while plasma PRL levels were not changed. In CAST-APX-GRAFT rats, both d-Trp-6-LHRH microcapsules and daily LHRH agonist injections appeared to increase plasma PRL levels. The pattern of changes in PRL release in both groups was similar, with levels on day 6 being significantly higher than those measured on days 1, 3 and 10 after onset of treatment. As expected, LH and FSH levels in these animals were extremely low. Immunoreactive d-Trp-6-LHRH was consistently detectable in the plasma of CAST-APX-GRAFT animals after microcapsule administration, whereas in animals given daily injections of this agonist in saline, its plasma concentrations were often below the detectability limit of the employed assay. These findings suggest that the LHRH agonist, d-Trp-6-LHRH, is capable of causing a short term stimulation of PRL release from ectopic pituitaries. Elevation of plasma LH levels is apparently not required for this effect.

Investigation of the Combination of the Agonist D-Trp-6-LH-RH and the Antiandrogen Flutamide in the Treatment of Dunning R-3327H Prostate Cancer Model

The therapy for the treatment of prostate cancer and other sex-steroid-dependent tumors based on agonists of LH-RH has been made more practical and efficacious by the development of a long-acting formulation of microcapsules of D-Trp-6-LH-RH for controlled release. Antiandrogens, which neutralize the effect of endogenous androgens, have been used also in the management of prostate cancer in man. The effects of a simultaneous administration of the antiandrogen flutamide and microcapsules of the agonist D-Trp-6-LH-RH were studied in the Dunning R-3327H rat prostate adenocarcinoma model to determine whether the combination of these two drugs might inhibit tumor growth more effectively than single agents. Microcapsules of D-Trp-6-LH-RH, calculated to release a controlled dose of 25 micrograms/day for a period of 30 days were injected intramuscularly once a month. Flutamide was administered SC at a daily dose of 25 mg/kg. The therapy was started 100 days after the tumor transplantation and continued for 60 days. Tumor weights and volumes were significantly reduced in rats treated with microcapsules or flutamide alone, but the former drug inhibited tumor growth more than the latter. The combined treatment of flutamide and microcapsules significantly decreased tumor weight and volume, but did not exert a synergistic effect on tumor growth, the reduction being smaller for the combination than for the microcapsules alone. There was a significant elevation of serum testosterone, LH, and prolactin in rats treated with flutamide. On the other hand, in rats given microcapsules of D-Trp-6-LH-RH, testosterone fell to castration levels within 7 days and remained at nondetectable values, serum LH and prolactin levels being also suppressed in this group. The combined administration of microcapsules and flutamide also significantly decreased serum testosterone to nondetectable levels by day 7 and suppressed serum LH and prolactin. Our findings raise doubts of whether the daily administration of the combination of LH-RH agonist with an antiandrogen offers an advantage over the use of microcapsules of an agonist like D-Trp-6-LH-RH alone in the treatment of prostatic carcinoma.

Radioimmunoassay for 6-D-tryptophan analog of luteinizing hormone-releasing hormone: measurement of serum levels after administration of long-acting microcapsule formulations.

A sensitive and specific radioimmunoassay for [6-D-tryptophan]luteinizing hormone-releasing hormone [( D-Trp6]LH-RH) was developed and used for following the rate of liberation of [D-Trp6]LH-RH from a long-acting delivery system based on a microcapsule formulation. Rabbit antibodies were generated against [D-Trp6]LH-RH conjugated to bovine serum albumin with glutaraldehyde. Crossreactivity with LH-RH was less than 1%; there was no significant crossreactivity with other peptides. The minimal detectable dose of [D-Trp6]LH-RH was 2 pg per tube. Intra- and interassay coefficients of variation were 8% and 10%, respectively. The radioimmunoassay was suitable for direct determination of [D-Trp6]LH-RH in serum, permitting the study of blood levels of the analog after single injections into normal men and after once-a-month administration of microcapsules to rats. In men, 90 min after subcutaneous injection of 250 micrograms of the peptide, serum [D-Trp6]LH-RH rose to 6-12 ng/ml. Luteinizing hormone was increased 90 min and 24 hr after the administration of the analog. Several batches of microcapsules were tested in rats and the rate of release of [D-Trp6]LH-RH was followed. The improved batch of microcapsules of [D-Trp6]LH-RH increased serum concentrations of the analog for 30 days or longer after intramuscular injection. This was accompanied by suppression of testosterone levels for more than 30 days. This radioimmunoassay should be of value for monitoring [D-Trp6]LH-RH during long-term therapy.

Preparation and biopharmaceutical evaluation of microcapsules of ampicillin.

Microencapsulation of ampicillin, an orally administered antibiotic, with different viscosity grades of ethyl cellulose was studied. The preparation of microcapsules was done as follows; the mixture of ethyl cellulose-CH2Cl2-ampicillin was dispersed in purified water containing 0.5% (w/w) sodium lauryl benzenesulfonate, and then CH2Cl2 was dried out by elevating the temperature. The dissolution curves for the release of ampicillin from microcapsules prepared using the four different viscosity grades of ethyl cellulose were quite different. The release of ampicillin increased with decreasing ethyl cellulose viscosity. The evaluation of prepared microcapsules was made using gastric-emptying-controlled rabbits. The plasma concentration of ampicillin obtained by the administration of microcapsules showed a significant sustained-release pattern. The area under the plasma concentration curve (AUC) of ampicillin obtained after a single oral administration of microcapsules prepared using 10 cps ethyl cellulose was 1.8 times greater than that obtained after double oral administration of powder. This fact will be caused by the delaying of gastric-emptying, intestinal-transit and dissolution of ampicillin there. It was confirmed that a large number of microcapsules still remained in the stomach and each microcapsules still contained ampicillin at 24 h after dosing from the experiment using gastric-emptying-controlled rabbits.

Investigation of the combination of the agonist D-Trp-6-LH-RH and the antiandrogen flutamide in the treatment of Dunning R-3327H prostate cancer model.

The therapy for the treatment of prostate cancer and other sex-steroid-dependent tumors based on agonists of LH-RH has been made more practical and efficacious by the development of a long-acting formulation of microcapsules of D-Trp-6-LH-RH for controlled release. Antiandrogens, which neutralize the effect of endogenous androgens, have been used also in the management of prostate cancer in man. The effects of a simultaneous administration of the antiandrogen flutamide and microcapsules of the agonist D-Trp-6-LH-RH were studied in the Dunning R-3327H rat prostate adenocarcinoma model to determine whether the combination of these two drugs might inhibit tumor growth more effectively than single agents. Microcapsules of D-Trp-6-LH-RH, calculated to release a controlled dose of 25 micrograms/day for a period of 30 days were injected intramuscularly once a month. Flutamide was administered SC at a daily dose of 25 mg/kg. The therapy was started 100 days after the tumor transplantation and continued for 60 days. Tumor weights and volumes were significantly reduced in rats treated with microcapsules or flutamide alone, but the former drug inhibited tumor growth more than the latter. The combined treatment of flutamide and microcapsules significantly decreased tumor weight and volume, but did not exert a synergistic effect on tumor growth, the reduction being smaller for the combination than for the microcapsules alone. There was a significant elevation of serum testosterone, LH, and prolactin in rats treated with flutamide. On the other hand, in rats given microcapsules of D-Trp-6-LH-RH, testosterone fell to castration levels within 7 days and remained at nondetectable values, serum LH and prolactin levels being also suppressed in this group. The combined administration of microcapsules and flutamide also significantly decreased serum testosterone to nondetectable levels by day 7 and suppressed serum LH and prolactin. Our findings raise doubts of whether the daily administration of the combination of LH-RH agonist with an antiandrogen offers an advantage over the use of microcapsules of an agonist like D-Trp-6-LH-RH alone in the treatment of prostatic carcinoma.