Placebo Microspheres Research Articles

20-Week intramuscular toxicity study of rotigotine behenate extended-release microspheres for injection via intramuscular injection in cynomolgus monkeys

Continuous dopaminergic stimulation (CDS) has become an important strategy for the development of drugs to treat Parkinson's disease (PD). Rotigotine behenate extended-release microspheres (RBEM) for injection represents a new treatment regime for CDS and is being applied for clinical trial. Our study in cynomolgus monkeys was a 20-week repeat dose toxicity investigation with RBEM at dosages of 90, 180, 360, with a 12-week recovery period. The results observed some irritations in the application site and surrounding tissues in Placebo microspheres and each dose of RBEM, was accompanied with increased white blood count and fibrinogen. RBEM-treated monkeys were additionally noted with a pharmacological action-related decrease in prolactin. These findings showed certain reversibility after the 12-week recovery phase. No clear sex difference was noted in the plasma exposure to rotigotine. The exposure generally increased in a dose-proportional manner. In summary, major toxicological effects are associated with the dopamine agonist-related properties of rotigotine, and the removal of foreign bodies caused by p oly (lactide-co-glycolide) (PLGA)and sodium carboxymethyl cellulose (SCMC), and the no-observed-adverse-effect-level (NOAEL) was 360 mg/kg.

Intracoronary Administration of Microencapsulated HGF in a Reperfused Myocardial Infarction Swine Model.

Therapy microencapsulation allows minimally invasive, safe, and effective administration. Hepatocyte growth factor (HGF) has angiogenic, anti-inflammatory, anti-apoptotic, and anti-fibrotic properties. Our objective was to evaluate the cardiac safety and effectiveness of intracoronary (IC) administration of HGF-loaded extended release microspheres in an acute myocardial infarction (AMI) swine model. An IC infusion of 5 × 106 HGF-loaded microspheres (MS+HGF, n = 7), 5 × 106 placebo microspheres (MS, n = 7), or saline (SAL, n = 7) was performed two days after AMI. TIMI flow and Troponin I (TnI) values were assessed pre- and post-treatment. Cardiac function was evaluated with magnetic resonance imaging (cMR) before injection and at 10 weeks. Plasma cytokines were determined to evaluate the inflammatory profile and hearts were subjected to histopathological evaluation. Post-treatment coronary flow was impaired in five animals (MS+HGF and MS group) without significant increases in TnI. One animal (MS group) died during treatment. There were no significant differences between groups in cMR parameters at any time (p > 0.05). No statistically significant changes were found between groups neither in cytokines nor in histological analyses. The IC administration of 5 × 106 HGF-loaded-microspheres 48 h post-AMI did not improve cardiac function, nor did it decrease inflammation or cardiac fibrosis in this experimental setting.

Evaluating the osteogenic potential of insulin-like growth factor-1 microspheres on osteoblastic activity around dental implants in patients with type 2 diabetes mellitus using bone scintigraphy: A split-mouth randomized controlled trial

Statement of problemDental implants are susceptible to early failure when placed in patients diagnosed with type 2 diabetes mellitus. The osteoinductive potential of insulin-like growth factor-1 (IGF-1) has been widely investigated in animals with type 2 diabetes mellitus, but studies investigating the osteoinductive potential of IGF-1 around dental implants in patients diagnosed with type 2 diabetes mellitus are lacking. PurposeThis randomized controlled trial was conducted to assess the osteogenic efficacy of poly(lactide-co-glycolide)– (PLGA) encapsulated IGF-1 microspheres around dental implants placed in patients diagnosed with type 2 diabetes mellitus. Material and methodsA split-mouth, randomized controlled trial was conducted in 10 participants diagnosed with type 2 diabetes mellitus and with bilaterally missing mandibular posterior teeth. The 20 sites were randomly allotted to receive the PLGA encapsulated IGF-1 or placebo microspheres followed by the placement of Ø3.8×11-mm implants. Osteoblastic activity was quantitatively assessed with bone scintigraphy scanning on the thirtieth, sixtieth, and 90th day after implant placement. The Shapiro-Wilks test was used to analyze the normality of data, followed by the independent t test to compare the experimental and placebo groups. Intragroup comparison was performed by using repeated-measures ANOVA and the post hoc Bonferroni test (α=.05). ResultsStatistical analysis revealed that the mean osteoblastic activity was higher in the experimental group which received the PLGA–encapsulated IGF-1 than in the placebo group at the 30th, 60th, and 90th day after implant placement (P≤.001). ConclusionsThis randomized controlled trial indicated that the PLGA–encapsulated sustained release of IGF-1 microspheres enhanced the process of osseointegration in patients diagnosed with type 2 diabetes mellitus until the 90th day after implant placement.

Enhanced Degradation of Lactide-co-Glycolide Polymer with Basic Nucleophilic Drugs

The purpose of this study was to examine the degradative effect of weakly basic nucleophilic drugs on a lactide-co-glycolide (PLGA) polymer in a microsphere formulation. Biodegradable PLGA microspheres of two second-generation atypical antipsychotics, Risperidone and Olanzapine, were manufactured using a solvent extraction/evaporation technique. The effect of drug content, buffer pH and temperature on polymer molecular weight and degradation, were examined via a series of experiments and compared against a control (Placebo PLGA microspheres). In comparison to Placebo microspheres, significant polymer molecular weight reduction was observed upon encapsulation of varying levels of either Risperidone or Olanzapine. There was excellent correlation between the extent of molecular weight reduction during manufacture and the amount of encapsulated drug in the microspheres. Subsequent studies on polymer degradation showed: the following (a) the Placebo and Olanzapine microspheres followed pseudo first order kinetics, (b) Risperidone microspheres exhibited biphasic degradation profiles, and (c) polymer degradation was dependent on temperature, not pH. The findings of these studies show that encapsulation of weakly basic nucleophile type drugs into PLGA can accelerate the biodegradation of the PLGA and have major implications on the design of polymeric microsphere drug delivery systems.

Casein as a carrier matrix for 5-fluorouracil: drug release from microspheres, drug-protein conjugates and in-vivo degradation of microspheres in rat muscle.

Glutaraldehyde cross-linked casein microspheres were loaded with 5-fluorouracil (5-FU) from concentrated aqueous solutions of the drug after the microspheres were synthesized and cleaned. In-vitro release of the drug was examined in phosphate buffer in the absence and in the presence of protease at 37 degrees C. Drug release data showed that only about 20% of the drug is released in the absence of protease even after 5 days, while digestion of the matrix with protease released the entrapped drug completely in about 24 h. A protein-drug conjugate was synthesized via carbamoyl linkage using 6-(5-FU-1-yl)hexyl isocyanate and the drug release was examined in phosphate buffer at 37 degrees C. Release from the protein-5-FU conjugate was slower compared with the release from microspheres in the presence of protease. Implantation of placebo microspheres of different cross-linking densities in the gluteal muscle of rats showed no adverse tissue reactions over a one-year period. Histopathological examination of the tissues containing injected microspheres suggested that the biological life of casein microspheres in muscle is about 6 months, which is three times that of cross-linked albumin microspheres.

Route of administration affects the ability of naltrexone to reduce amphetamine-potentiated brain stimulation reward in rats

Opioid receptor antagonism has been shown to attenuate behavioral and neurochemical effects of amphetamine in humans and rodents. The effects of acute (oral or subcutaneous) or extended-release naltrexone (XR-NTX) were tested on the reward-enhancing effects of amphetamine using the intracranial self-stimulation (ICSS) paradigm. Acute exposure to drugs of abuse reduces the locus of rise (LOR) in the ICSS procedure, reflecting enhanced brain stimulation reward (BSR). Rats were treated once a day with naltrexone orally (PO; 5.0 mg/kg) or subcutaneously (SC; 0.5 mg/kg) for four consecutive days and tested with D-amphetamine (0.5 mg/kg, intraperitoneal) in the ICSS paradigm 30 minutes later on days 1 and 4. Separate groups of rats received XR-NTX (50 mg/kg, SC) or placebo microspheres (similar mass to XR-NTX, SC) on day 0 and tested with D-amphetamine in the ICSS paradigm on days 4, 14, 21, 28 and 41 after administration. Naltrexone plasma concentrations were determined for each amphetamine testing session using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). In rats pretreated with naltrexone acutely, amphetamine-potentiated BSR did not differ from vehicle-pretreated rats on either day 1 or day 4 (25-30% decrease in LOR). In XR-NTX-pretreated rats, amphetamine-potentiated BSR was reduced by 64 and 70% on days 4 and 14, respectively, compared to placebo microsphere-treated controls. This effect dissipated by day 21. Naltrexone plasma concentrations were comparable across all treatment groups (14-30 ng/ml) on days 1, 4 and 14. In summary, an extended-release formulation of naltrexone results in significant attenuation of psychostimulant-enhanced BSR that is not observed with acute naltrexone.

Design of Methylprednisolone Biodegradable Microspheres Intended for Intra-articular Administration

This study aimed to design methyprednisolone (MP)-loaded poly(D,L lactide-co-glycolide) (PLGA) microspheres (MS) intended for intra-articular administration. MP was encapsulated in four different types of PLGA by using an S/O/W technique. The effects of beta-irradiation at the dose of 25 kGy were evaluated on the chemical and physicochemical properties of MS and the drug release profiles. The S/O/W technique with hydroxypropylmethylcellulose (HPMC) as surfactant allowed obtaining MS in the tolerability size (7-50 microm) for intra-articular administration. The MP encapsulation efficiency ranged 56-60%. HPMC traces were evidenced in the loaded and placebo MS by attenuated total reflectance Fourier transform infrared spectroscopy. MS made of the capped PLGA DL5050 2M (MS 2M) and uncapped PLGA DL5050 3A (MS 3A) prolonged the release of MP over a 2- to 3-month period with a triphasic (burst release-dormant period-second release pulse) and biphasic release pattern, respectively. The beta-irradiation did not significantly alter the morphology, chemical, and physicochemical properties of MS. The only variation was evidenced in the drug release for MS 2M in term of shorting of the dormant period. The minimal variations in the properties of irradiated PLGA MS, which are in disagreement with literature data, may be attributed to a radioprotecting effect exerted by HPMC.

Microencapsulation of doxycycline into poly(lactide‐co‐glycolide) by spray drying technique: Effect of polymer molecular weight on process parameters

AbstractPoly(lactide‐co‐glycolide) (PLGA) polymers with three different molecular weights were prepared, and microparticles were produced by spray drying and water‐in‐oil‐water (w/o/w) double emulsion techniques to encapsulate 86% of doxycycline (DXY), an antibiotic drug, for the use of periodontitis. Placebo and drug‐loaded microspheres and pristine DXY were analyzed by Fourier transform infrared spectroscopy, which indicated no chemical interactions between DXY and PLGA. X‐ray diffraction of drug‐loaded microspheres confirmed the molecular level dispersion of DXY in PLGA. Scanning electron microscopy confirmed spherical nature and smooth surfaces of the microspheres. Mean particle size as measured by laser light scattering technique ranged between 10 and 25 μm. In vitro release of DXY performed in 7.4 pH media continued up to 72 h and depended on molecular weight of PLGA and extent of DXY loading. Antimicrobial studies performed on one formulation and placebo microspheres suggested that drug concentrations during in vitro release are above the minimum inhibitory concentration (MIC) for Staphylococcus aureus growth. Overall, the release studies depended on the molecular weight of PLGA, extent of drug loading, and the method used to prepare microspheres. Statistical analyses of release data performed using the analysis of variance (ANOVA) method agreed well with experimental observations. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Development and evaluation of novel biodegradable microspheres based on poly( d, l-lactide- co-glycolide) and poly(ε-caprolactone) for controlled delivery of doxycycline in the treatment of human periodontal pocket: In vitro and in vivo studies

This study reports on the development of novel biodegradable microspheres prepared by water-in-oil–water (W/O/W) double emulsion technique using the blends of poly( d, l-lactide- co-glycolide) (PLGA) and poly(ε-caprolactone) (PCL) in different ratios for the controlled delivery of doxycycline (DXY). Doxycycline encapsulation of up to 24% was achieved within the polymeric microspheres. Blend placebo microspheres, drug-loaded microspheres and pristine DXY were analyzed by Fourier transform infrared spectroscopy (FT-IR), which indicated no interaction between drug and polymers. Differential scanning calorimetry (DSC) on drug-loaded microspheres confirmed the polymorphism of DXY and indicated a molecular level dispersion of DXY in the microspheres. Scanning electron microscopy (SEM) confirmed the spherical nature and smooth surfaces of the microspheres produced. Mean particle size of the microspheres as measured by dynamic laser light scattering method ranged between 90 and 200 μm. In vitro release studies performed in 7.4 pH media indicated the release of DXY from 7 to 11 days, depending upon the blend ratio of the matrix. Up to 11 days, DXY concentrations in the gingival crevicular fluid were higher than the minimum inhibitory concentration of DXY against most of the periodontal pathogens. One of the developed formulations was subjected to in vivo efficacy studies in thirty sites of human periodontal pockets. Significant results were obtained with respect to both microbiological and clinical parameters up to 3 months even as compared to commercial DXY gel. Statistical analyses of the release data and in vivo results were performed using the analysis of variance (ANOVA) method.

Progesterone bearing mucoadhesive carriers for nasal delivery

The cross-linked microspheres of chitosan have been investigated for the nasal delivery of progesterone. An emulsification method involving the dispersion of aqueous solution of chitosan in paraffin oil was used to prepare spherical microspheres. The effect of stirring time, stirring rate, and citric acid concentration on the various physicochemical attributes such as shape, surface morphology, particle size, size distribution, percent drug payload, swelling ability and the mucoadhesion properties of microspheres were studied. Shape and surface morphology were examined using scanning electron microscopy. Placebo microspheres exhibited the smooth surface while the incorporation of drug imparted a slight roughness to the surface. Optimized process and formulation parameters resulted in spherical shape and rigid surface, homogenous population of microspheres in the size range of 15.34 ± 2.34 μm. The percent drug payload in the optimized formulation DC*S*T*E was observed to be 80.41 ± 1.26%. The drug release from the microspheres follows non-Fickian diffusion pattern. Mucoadhesive chitosan microsphere-bearing progesterone was evaluated for in vitro skin permeation using she goat intestinal mucosa. It is also observed that the optimized formulation DC*S*T* (without permeation enhancer) exhibited 53.29 ± 5.23% drug permeation in 24 h, whereas the similar formulation with permeation enhancer i.e. DC*S*T*E exhibited 75.96 ± 2.45% permeation. The in vivo performance of progesterone loaded mucoadhesive microspheres performed on albino rabbits exhibited better bioavailability (AUC 0 -24 ≈ 1.386 mg h/mL) than oral progesterone that could be due to protection of drug from hepatic first pass. From these results it can be concluded that bioavailability of the progesterone may be improved by delivering progesterone through nasal route using mucoadhesive microspheres.

Potential of Guar Gum Microspheres for Target Specific Drug Release to Colon

Various approaches for colon targeted drug delivery have been studied over the last decade including, pro-drugs, timed-released systems, coating of pH-dependant polymer and the use of polysaccharides. In the present work, a novel formulation consisting of cross-linked microspheres of guar gum has been investigated for colon-targeted delivery of metronidazole. An emulsification method involving the dispersion of aqueous solution of guar gum in castor oil was used to prepare spherical microspheres. Process parameters were analyzed in order to optimize the formulation. Shape and surface morphology of the microspheres were examined using scanning electron microscopy. Placebo microspheres exhibited a smooth surface while the incorporation of drug imparted a slight roughness to the surface texture. Particle size of the microspheres was determined using laser diffraction particle size analyzer. The in vitro drug release studies were performed in simulated gastric fluid for 2 h and intestinal fluid for 3 h, which revealed that the drug was retained comfortably inside the microspheres and that only 15.27±0.56% of the drug was released in 5 h. In vitro release rate studies were also carried out in simulated colonic fluid (SCF) in the presence of rat cecal contents, which showed improved drug release. Moreover, to induce the enzymes that specifically act on guar gum, the rats were treated with 1 ml of 1% w/v dispersion of guar gum for 2, 4 and 6 days and release rate studies were repeated in SCF in the presence of 2 and 4% w/v of cecal matter. A marked improvement in the drug release was observed in presence of cecal matter obtained after induction when compared to those without induction. In vitro release studies exhibited 31.23±1.49% drug release in 24 h in dissolution medium without rat cecal matter. However, the incorporation of 4% w/v cecal matter obtained after 6 days of enzymes induction increased the drug release to 96.24±4.77%.

Anti-tumor effect of combination therapy with intratumoral controlled-release paclitaxel (PACLIMER microspheres) and radiation.

Paclitaxel is one of the few chemotherapeutics effective in patients with advanced protstate cancer. Paclitaxel has also been reported to have radiosensitizing effects in prostate cancer. Local delivery of a controlled-release paclitaxel product may allow for increase local concentrations of paclitaxel at the tumor site and, in conjunction with radiation, may enhance cell kill by its radiosensitization mechanism. Orthotopic LNCaP tumors were injected with 40% PACLIMER Microspheres (40% loading; w:w) when tumors were 100-200 mm(3). Twenty-eight days post cell injection, mice were sacrificed, tumors weighed, and serum measured for PSA. TSU-xenografts were injected with PACLIMER Microspheres (10% and 40% loaded; w:w) or placebo microspheres when the tumors were approximately 100 mm(3). Half of xenograft tumors were irradiated with a single dose (10 Gy) of radiation. Tumor volume was followed over time. Forty percent PACLIMER Microspheres significantly reduced tumor growth in the LNCaP orthotopic model. PSA was a good indicator of response. Forty percent PACLIMER Microspheres had a significant effect on slowing TSU growth compared to placebo microspheres. Addition of a single acute dose of radiation significantly enhanced the effect of 10% PACLIMER Microspheres (P < 0.05), had minimal effect on 40% PACLIMER Microspheres, and no enhancing effect on tumors treated with placebo microspheres. A controlled-release formulation of paclitaxel can be very effective in the treatment of prostate cancer. Additionally, PACLIMER Microspheres may be effectively used as a radiosensitizer in genitourinary cancers.

IL-2 loaded dextran microspheres with attractive histocompatibility properties for local IL-2 cancer therapy.

Biodegradable dextran microspheres (MS) were developed as a slow-release system for interleukin-2 (IL-2) to apply them for local IL-2 therapy of cancer. We describe the tissue reactions induced by these MS without or with IL-2 in rats. Dextran MS stain bright red-purple with the periodic acid Schiff (PAS), visualising the exact spot of IL-2 release and its relation to the histological reaction pattern. Subcutaneously injected MS always form a well-circumscribed deposit. In the first 2 days there is a PMN inflammation within the MS-deposit, but the surroundings show only a scanty inflammatory reaction. The PMN reaction is replaced by an abundant macrophage reaction in particular in the MS-deposit. At day 21 a fibrous capsule of about 50 mum surrounds the deposit. The effect of IL-2 administered in its free form is mainly vascular, with vascular dilatation, vascular leakage and oedema. It is remarkable that lymphocytes are present in the injection area already at day 2. When IL-2 releasing MS were used, the various reactions induced by IL-2 and MS were amplified leading to local necrosis. We conclude that neither placebo MS nor IL-2 leads to necrosis after subcutaneous injection in rats. In contrast, when IL-2 was released from MS, then massive necrosis was induced. This might be due to increased phagocytosis or changes in the micro-niche due to the release of humoral factors by the infiltrating cells. This is probably fortuitous for local IL-2 therapy of cancer, as massive necrosis of tumour cells can be expected to lead to an increased antitumour reaction.

Vivitrex, an injectable, extended-release formulation of naltrexone, provides pharmacokinetic and pharmacodynamic evidence of efficacy for 1 month in rats.

While oral naltrexone is effective in treating alcohol and opiate dependencies, poor patient adherence and widely fluctuating plasma levels limit its efficacy. To overcome these problems, an extended-release formulation of naltrexone (Vivitrex) was developed by encapsulating naltrexone into injectable, biodegradable polymer microspheres. Pharmacokinetic studies in rats demonstrated that this formulation produced stable, pharmacologically relevant plasma levels of naltrexone for approximately 1 month following either subcutaneous or intramuscular injections. While rats receiving placebo microspheres demonstrated a pronounced analgesic response to morphine in the hot-plate test, morphine analgesia was completely blocked in rats treated with extended-release naltrexone. This antagonism began on day 1 following administration and lasted for 28 days. Rats reinjected with extended-release naltrexone 34 days after the initial dose and tested for another 35 days showed consistent suppression of morphine analgesia for an additional 28 days. mu-Opioid receptor density, as measured by [(3)H]DAMGO autoradiography, increased up to two-fold following a single injection of extended-release naltrexone. Saturation binding assays using [(3)H]DAMGO showed changes in the midbrain and striatum at 1 week after extended-release naltrexone administration, and after 1 month in the neocortex. These receptor increases persisted for 2-4 weeks after dissipation of the morphine antagonist actions of naltrexone. These data suggest that therapeutically relevant plasma levels of naltrexone can be maintained using monthly injections of an extended-release microsphere formulation, and that changes in mu-opioid receptor density do not impact its efficacy in suppressing morphine-induced analgesia in the rat. Clinical trials of extended release naltrexone for treating alcohol and opiate dependency are currently ongoing.

Ultrasonic atomization for spray drying: a versatile technique for the preparation of protein loaded biodegradable microspheres

Bovine serum albumin (BDA) loaded microspheres with a spherical shape and smooth surface structure were successfully prepared from poly(lactide-coglycolide) using an ultrasonic nozzle installed in a Niro laboratory spray dryer. Process and formulation parameters were investigated with respect to their influence on microsphere characteristics, such as particle size, loading capacity, and release properties. Preparation of microspheres in yields of more than 50% was achieved using an ultrasonic atomizer connected to a stream of carrier air. Microsphere characteristics could be modified by changing several technological parameters. An increased polymer concentration of the feed generated larger particles with a significantly reduced initial release of the protein. Moreover, microspheres with a smooth surface structure were obtained from the organic polymer solution with the highest viscosity. Microparticles with a low BSA loading showed a large central cavity surrounded by a thin polymer layer in scanning electron microspheres. A high protein loading led to an enlargement of the shell layer, or even to dense particles without any cavities. A continuous in vitro release pattern of BSA was obtained from the particles with low protein loading. Glass transition temperatures (T g) of the microspheres before and after lyophilization did not differ from those of the BSA loaded particles prepared by spray drying with a rotary atomizer. Analysis of the polymer by gel permeation chromatography indicated that ultrasonication had no effect on polymer molecular weight. Molecular weight and polydispersity of the pure polymer, placebo microspheres prepared by spray drying, and placebo microspheres prepared using the ultrasonic nozzle were in the same range. In conclusion, ultrasonic atomization represents a versatile and reliable technique for the production of protein loaded biodegradable microspheres without inducing a degradation of the polymer matrix. Particle characteristics can be modified by adjusting formulation parameters and atomization conditions in a simple manner.

Effect of microencapsulated ampicillin on cell-mediated immune responses in mice.

The effects of free ampicillin, microencapsulated ampicillin anhydrate (MEAA) and antibiotic-free microspheres on the cell-mediated immune response in Balb/c mice were measured by lymphoproliferation assay, delayed-type hypersensitivity (DTH) and cytokine production. Injection into mice for seven consecutive days with equivalent subcutaneous doses of ampicillin, MEAA or placebo microspheres did not produce any consistent change in lymphocyte proliferation nor did it affect DTH responses or interleukin-2 production. Although the production of interleukin-4 in mice treated with ampicillin or MEAA increased compared with the control mice, this increase was not statistically significant. These results indicate that ampicillin and MEAA have similar effects on cell-mediated immunity in mice.

Evaluation of hypersensitivity to microencapsulated ampicillin in guinea pigs.

The purpose of this study was to determine if the sustained release of ampicillin from a biodegradable drug-delivery system (microencapsulated ampicillin anhydrate (MEAA)) will increase or decrease the intensity of a hypersensitivity reaction compared with that observed with free drug. Ovalbumin, which is known to elicit a marked hypersensitivity reaction in guinea pigs, and microencapsulated ovalbumin (MOVA) were tested in parallel with ampicillin and MEAA. Guinea pigs were sensitized biweekly by subcutaneous and intramuscular injections of ampicillin, MEAA, ovalbumin, MOVA or placebo microspheres (test articles), each mixed with Freund's adjuvant, and challenged 2 weeks later, intradermally, with the free compounds. In a separate set of experiments, guinea pigs were sensitized by implantation of the same agents in the caudal thigh of anaesthetized animals. Skin allergic reactions were tested at 1 and 3 weeks following local implantation of the test articles. Sera of sensitized guinea pigs were tested for specific IgG antibodies by enzyme-linked immunosorbent assay, and skin samples from the site of the inflammatory reaction were fixed, stained and evaluated histologically. Guinea pigs sensitized systemically with MEAA or MOVA showed smaller, but not statistically different skin allergic response than animals given corresponding free compounds. However, guinea pigs sensitized by local implantation of MEAA showed a significantly lower inflammatory response (P < 0.0001) than those given an equivalent dose of the free drug. Guinea pigs sensitized with placebo microspheres showed a low inflammatory skin reaction which was similar to those sensitized with all doses of MEAA. There was no significant difference in specific IgG antibody response in the sera of guinea pigs sensitized locally with either free or microencapsulated ampicillin or ovalbumin. Histology of skin revealed a milder inflammatory reaction with MEAA or MOVA than with ampicillin or ovalbumin, respectively. We conclude that the encapsulated ampicillin or ovalbumin and subsequent release of each agent will elicit a reduced hypersensitivity reaction in guinea pigs than will the free agent.

Solid tumor chemotherapy using injectable gelatin microspheres containing free methotrexate and conjugated methotrexate

Biodegradable gelatin microspheres containing free methotrexate (MTX) (GMFM) and gelatin microspheres containing MTX covalently conjugated to gelatin using carbodiimide (GMCM-I) and two types of azide coupling-grafting methods (GMCM-II and GMCM-III) have been prepared and evaluated for their efficacy towards the solid tumor fibrosarcoma in Wistar rats. The tumor was induced by inoculation of a 10% tumor cell suspension in the anterior aspect of the hind limb. Seven groups were studied—untreated control, intratumoral (i.t.) injection of free MTX, i.t. injection of placebo microspheres, i.t. injection of GMFM, i.t. injection of GMCM-I, i.t. injection of GMCM-II and i.t. injection of GMCM-III of mean particle size 10–20 μm. The gelatin-microspheres-entrapped MTX showed an improved antitumor activity over free MTX as evidenced by the gross tumor weight assessments and [ 3H]thymidine incorporation studies in vitro. This was attributed to the slow and controlled release of MTX from gelatin microspheres, which was more effective compared with free MTX given over the same period of treatment. The drug release from microspheres is due to gradual degradation of the spheres and concomitant diffusion of drug into the external medium. GMCM showed higher antitumor activity than GMFM. The antitumor activity of the various GMCM preparations was of the order: GMCM-I > GMCM-II > GMCM-III. The injectable MTX-containing gelatin microspheres can thus be a potential alternative to conventional therapy using free MTX in the treatment of solid tumors such as fibrosarcoma.

Cross-linked chitosan microspheres as carriers for prolonged delivery of macromolecular drugs.

Bovine serum albumin (BSA) and diphtheria toxoid (DT) were loaded by passive absorption from aqueous solutions into preformed glutaraldehyde cross-linked chitosan microspheres. In vitro release of BSA under sink conditions at 37 degrees C showed that even though there was a large burst effect, there was a more or less steady increase with time thereafter for several days. Coating the BSA-loaded particles with paraffin oil or with a polymer, such as polylactic acid, modulated drug release. After the initial burst from PLA coated particles, the release rate increased with time for nearly 2 months. Preliminary immunogenicity studies on Wistar rats using DT loaded chitosan spheres showed that the antibody titres were fairly constant over a 5-month period, although very low compared to DT given on alum as control. Histological studies of placebo microspheres intramuscularly injected into rats demonstrated their tissue compatibility. Biodegradation was not complete in 6 months demonstrating the potential of cross-linked chitosan spheres as a long-acting drug delivery vehicle. The study demonstrated the possibility of incorporating biological macromolecules which are very sensitive to organic solvents, pH, temperature, ultrasound, etc. by a passive absorption technique to degradable biopolymer matrices thereby preserving their biological integrity. It is also shown that drugs passively absorbed into such matrices by taking advantage of their swelling behaviour need not necessarily be released completely in the initial 'burst' and a sustained release may be possible for macromolecules thus incorporated.

In vitro and in vivo evaluation of clarithromycin/poly(lactic acid) microspheres for intramuscular drug delivery

Clarithromycin is a macrolide antibiotic recommended for the treatment of upper respiratory tract infections. This report summarizes the results of in vitro and in vivo studies conducted to evaluate the feasibility of developing an intramuscular (i.m.) formulation of this compound by encapsulating in a biodegradable polymer, poly(lactic acid) (PLA). Drug/PLA microspheres were prepared by solvent evaporation and recovered by filtration or spray-drying. Variations in processing parameters, e.g., polymer concentration in the dispersed phase, type of continuous phase and the recovery process, did not appreciably alter microsphere particle shape, size or drug loading. In 0.05 M phosphate buffer (pH 7.4, at 37°C), compared to the dissolution of bulk drug, the microsphere formulations demonstrated good sustained release properties. In a preliminary in vivo study, 10 mg/kg drug was administered i.m. to groups of three dogs as a suspension of bulk drug or via PLA microspheres. Blood samples were collected over time to monitor drug and creatinine phosphokinase ( CPK ) concentrations. Both groups demonstrated drug concentrations ≥ 1 μg/ml for about 4 days after dosing. Also, the serum CPK concentrations in both groups were low, and compared well with those observed following the i.m. administration of placebo microspheres. The animals receiving drug/PLA microspheres demonstrated minor swelling at the site of injection which lasted for 12–24 h; however, the bulk drug treatment group exhibited much greater swelling lasting for 2–3 days. Since these observations do not correlate with serum CPK levels, alternative quantitative models are required to conclusively evaluate the potentials of microsphere formulations for the i.m. delivery of painful/irritating compounds like clarithromycin.