Comparison of albumin and casein microspheres as a carrier for doxorubicin

Doxorubicin (Adriamycin)-loaded casein and albumin microspheres, with diameters between 14 and 38 micron (50% weight average) were prepared by glutaraldehyde stabilization of the aqueous phase (containing protein and drug) of a water in oil emulsion. Physical properties, drug loading characteristics and release rates from microspheres in-vitro have been compared and correlated with effects on tumour growth when injected intratumourally in rats. Compared with albumin, the surface charge of the casein system was more negative and the microspheres exhibited a slower release of drug in-vitro. Both observations could be explained by the lower drug content of the casein system. There was evidence for the formation of a doxorubicin complex in the microspheres, the significance of which is not yet known. Casein microspheres containing 11 micrograms of doxorubicin had a similar inhibitory effect on tumour growth (growth delay = 20.7 days) to 85 micrograms of drug incorporated into albumin microspheres (growth delay = 18.6 days). The absence of a simple dose-response relationship shows that carrier matrix can influence potency of incorporated drug. The results are consistent with release rate of the drug from microspheres (obversely, rate of drug delivery to the tumour), being a determinant of potency in these systems.     

Biodegradation rate of embolized protein microspheres in lung, liver and kidney of rats

The targeting and sustained release characteristics of cytotoxic drug-loaded protein microspheres may prove useful in the therapeutic chemoembolization of solid tumours. Because biodegradation rate of embolized particles will influence rate of incorporated drug release and duration of exposure, this parameter was studied for microspheres (10-30 microns mean diam.) prepared from the proteins albumin and casein, that we have previously used as carriers for doxorubicin. As a measure of microsphere loss in-vivo the radionuclide 125I was chosen because it can be covalently bound to proteins and also homogeneously distributed throughout the matrix. Radiolabelled microspheres were administered to rats both intravenously (lung as target organ, 1.4-2.2 mg/100 g) and via the hepatic artery (liver as target organ, 0.4-0.8 mg/100 g). In both cases it was observed that the casein system biodegraded more slowly than the albumin in-vivo. Thus, time taken for loss of 50% of embolized microspheres from lung was: albumin 2.0 days; casein 3.5 days and from liver:albumin 3.6 days; casein 6.8 days. Microsphere "debris" did not markedly accumulate in other organs. In-vitro experiments showed that microspheres were stable in serum and that albumin microspheres were not innately more sensitive to enzymic digestion than casein. The results may be useful in estimating duration of exposure of target organs to drug-loaded microsphere systems prepared from these proteins.     

Preparation and in-vitro evaluation of nifedipine loaded albumin microspheres cross-linked by different glutaraldehyde concentrations

Nifedipine-loaded albumin microspheres were prepared by a chemical cross-linking method to develop a sustained release form. The effects of cross-linking agent (glutaraldehyde) on the percentage of drug loading, biodegradability of albumin microspheres and drug release kinetics were investigated in this study. Moreover, the kinetics of nifedipine released from different albumin microspheres were analysed using four different theoretical models, that is, zero order, first order, planar matrix and spherical matrix model. Albumin microspheres prepared with different amounts of glutaraldehyde indicated different release kinetics. Increasing the glutaraldehyde concentration decreased the release rate of nifedipine from albumin microspheres as a result of formation of greater structural strength and more tightly texture. Besides, albumin microspheres gave an adequate fit to either zero order or spherical matrix model, depending on the extent of cross-linking reaction.     

In vitro protein binding behavior of dipyridamole

The in vitro protein binding behavior of dipyridamole in plasma and buffered protein solutions was investigated by equilibrium dialysis. The drug was highly protein bound (approximately 98%) in heparinized human plasma, and the extent of protein binding remained constant for drug concentrations over the range of therapeutic interest of 0.1-10 micrograms/mL. Comparable binding results were obtained with a mixture of 80 mg % of alpha 1-acid glycoprotein and 40 g/L of human serum albumin in pH 7.4 phosphate buffer solution. Pure alpha 1-acid glycoprotein (80-400 mg %) or pure human serum albumin (40 g/L) in phosphate buffer gave significantly (p less than 0.05) lower binding results, indicating that both proteins are responsible for the high binding of dipyridamole in plasma. Addition of alpha 1-acid glycoprotein to heparinized human plasma, to simulate an acute phase increase in the protein, had no effect on the fraction of free drug in plasma. Binding of dipyridamole to heparinized human plasma or human serum albumin in buffer was concentration independent through 40 micrograms/mL. The free fraction of dipyridamole increases with concentrations exceeding 40 micrograms/mL.     

Albumin microspheres as vehicles for the sustained and controlled release of doxorubicin

Biodegradable albumin microspheres have been prepared with the intention of targeting doxorubicin preferentially to tumour tissue. A high-yielding microsphere manufacturing process has been developed that involved the denaturation of an aqueous protein emulsion by chemical and/or thermal crosslinking methods. Microspheres can be closely sized to a diameter of 25.3 +/- 2.6 microns with the aid of micro-sieves. The in-vitro release of doxorubicin from albumin microspheres was measured using a continuous flow system. Doxorubicin release can be sustained for up to 10 days and the rate of release could be controlled by manipulating protein denaturation conditions between the temperatures 110-135 degrees C in the presence of 0-2% glutaraldehyde. Release of doxorubicin was significantly faster in human plasma compared with isotonic saline.     

Relationship between reductive drug metabolism in tumour tissue of anthracyclines in microspherical form and anti-tumour activity

Increased activity against a rat solid tumour of doxorubicin incorporated into protein microspheres and administered intratumourally was associated with both increased duration of exposure of tumour tissue to native drug and anaerobic bioreduction of doxorubicin to 7-deoxyaglycones, indicating formation of reactive drug intermediates within tumour tissue. To investigate which of these aspects of drug disposition determined activity we have compared the in vivo fate (clearance from and metabolism by tumour tissue) of doxorubicin in microspherical form with the analogue 4'-deoxydoxorubicin and related this to the tumour growth delay recorded for these drugs. Within the dose range 42 to 55 micrograms, growth delay (14-18 days) of doxorubicin in microspherical form was markedly superior to drug in solution, whereas growth delay of 4'-deoxydoxorubicin in microspherical form (4.3-7.2 days) was not greater than drug in solution. Metabolism to 7-deoxyaglycones by tumour tissue was not a prominent feature of either drug when administered in solution. However, in microspherical form both drugs were extensively metabolized (peak concentrations: 3.6 micrograms/g doxorubicin 7-deoxyaglycone; 2.5 micrograms/g 4'-deoxydoxorubicin 7-deoxyaglycone). Native drug concentrations in tumour tissue were similar after administration in microspherical form at 48 hr (doxorubicin 3.8 micrograms/g; 4'-deoxydoxorubicin 3.7 micrograms/g) and 72 hr (doxorubicin 2.4 micrograms/g; 4'-deoxydoxorubicin 2.7 micrograms/g). At both time points, following administration in microspherical form, tumour tissue concentrations of doxorubicin were significantly greater than when drug was administered in solution, whereas no significant differences were observed for 4'-deoxydoxorubicin. The results are inconsistent with the process of anaerobic bioreduction of doxorubicin to 7-deoxyaglycones being an important component of its anti-tumour activity in microspherical form and point to the importance of increased duration of exposure to native drug.     

Doxorubicin-loaded casein microspheres: protein nature of drug incorporation.

We have studied incorporation of [14C]doxorubicin within protease-sensitive casein microspheres both by 14C-activity, measuring total drug, and HPLC, measuring free drug only. It was found that total drug content (27.7 micrograms mg-1) exceeded free drug content (3.2 micrograms mg-1) suggesting that the major portion of doxorubicin was incorporated via a covalent linkage to matrix protein. In-vivo drug disposition and activity studies suggested that this fraction of doxorubicin was the major species within tumour tissue (total vs free: 5 min, 14.3 micrograms g-1 vs 0.7 micrograms g-1; 24 h, 11.7 micrograms g-1 vs 1.1 micrograms g-1; 48 h, 11.2 micrograms g-1 vs 1.2 micrograms g-1; 72 h, 10.0 micrograms g-1 vs 0.8 micrograms g-1), did not exhibit a 'burst' effect, was slowly cleared (30% loss over 3 days), and was equiactive (growth delay = 12 days) compared with drug in solution (growth delay = 10 days). This work clearly implicates in-vivo microsphere matrix biodegradation in drug release and subsequent disposition and activity.     

Cytarabine release from comatrices of albumin microspheres in a poly(lactide-co-glycolide) film: in vitro and in vivo studies.

Cytarabine (ara-C) was included in albumin microspheres and these microspheres were immersed in a poly(lactide-co-glycolide) (PLGA) film to constitute a comatrix system to develop a prolonged form of release. Cytarabine-loaded albumin microspheres were synthesized by emulsion, and 25 or 50 mg of drug were included in the disperse phase. Thus, microspheres with 46+/-4 microg drug/mg microspheres and 50+/-5 microg drug/mg microspheres were obtained, which means a percentage of incorporation efficiency of 42+/-4% and 25+/-2%, respectively. These cytarabine-loaded microspheres were used to prepare PLGA-comatrices. Kinetic release studies indicated that total cytarabine release only takes place in the presence of protease, probably due to the fact that glutaraldehyde establishes covalent links with the amine side group of the drug and cross-links it with the protein matrix. A slower kinetic release of the drug was obtained from PLGA-comatrices, although only 80% of the included cytarabine was released on day 7. The comatrices were subcutaneously implanted in the back of rats and in both cases the ara-C administered dose was 36 mg of ara-C per kg of body weight. The drug was detected in plasma 10 days. The mean residence time (MRT) of the drug administered by these comatrices was 87-91 times larger when compared to the value obtained when the drug was administered in solution by intraperitoneal injection. The histological studies show that a degradative process of the comatrices takes place. The comatrices do not damage surrounding tissue; a normal regeneration of the implanted zone was observed.     

The consequences of doxorubicin quinone reduction in vivo in tumour tissue.

A clear role for quinone reduction in the mechanism of action of doxorubicin has still to be established. There are three possible outcomes of this form of doxorubicin metabolism: (1) drug free radical formation, redox cycling and generation of reactive oxygen species (ROS) resulting in lipid peroxidation and DNA damage; (2) covalent binding of reactive drug intermediates to DNA; and (3) formation of an inactive 7-deoxyaglycone metabolite. In this work, the occurrence of each of these pathways has been studied in vivo in a subcutaneously growing rat mammary carcinoma (Sp 107). Doxorubicin was administered by direct intratumoural injection either as the free drug or incorporated in albumin microspheres (10-40 microns diameter). There was no evidence of an increase in lipid peroxidation over background after either treatment at any time point studied. In fact, doxorubicin administration resulted in a statistically significant reduction in lipid peroxidation at the later time points studied compared to control (no drug treatment), e.g. 24 hr: control, 21.7 +/- 2.8 SD nmol malondialdehyde/g tissue; free doxorubicin (70 micrograms drug), 14.5 +/- 4.0 SD nmol/g (P < 0.01 Student's t-test) and doxorubicin microspheres (70 micrograms drug), 17.4 +/- 1.1 nmol/g (P < 0.05). Covalent binding to DNA was measured by a 32P-post-labelling technique. Low levels of four putative drug-DNA adducts were detected; however, there were no qualitative or quantitative differences in profiles between free drug and microspheres. High 7-deoxyaglycone metabolite concentrations comparable to the parent drug itself were detected after administration of microspheres (3.0 micrograms/g +/- 1.7 SD at 24 hr and 3.1 micrograms/g +/- 1.1 SD at 48 hr). In contrast, these metabolites were present at levels close to the limit of detection of our HPLC assay after free drug (0.04 microgram/g +/- 0.03 SD at 24 hr and 0.02 microgram/g +/- 0.03 SD at 48 hr). Thus, 7-deoxyaglycone metabolite formation can occur in tumour tissue (indicating active drug quinone reduction) without concomitant increases in the level of lipid peroxidation or the levels of drug-DNA adducts. In conclusion, the main biological consequence of doxorubicin quinone reduction in vivo in tumour tissue would appear to be drug inactivation to a 7-deoxyaglycone metabolite rather than drug activation to DNA reactive species or ROS.     

Prenatal effect of pyrantel pamoate on several hematological parameter of offsprings in mice

In attempt to develop a drug delivery system using serum albumin microspheres, bovine serum albumin microspheres containing antitumar agent, cytarabine, were prepared. The shape, surface characteristics, size distribution, behavior of in vivo distribution, drug release behavior, and degradation of albumin microspheres in animal liver tissue homogenate and proteolytic enzyme were investigated. The shape of albumin microspheres was spherical and the surface was smooth and compact. The size distribution of the albumin microspheres was effected by dispertion forces during emulsification and albumin concentration. Distribution of albumin microspheres after imtravenous administration in rabbit was achieved immediately.In vitro, albumin microsphere matrix was so hard that it retained most of cytarabine except initial burst during the first 10 minutes, and the level of drug release during the initial burst was affected by heating temperature, drug/albumin concentration ratio and size distribution. After drug release test, the morphology of albumin microspheres was not changed. Albumin microsphere matrix was degraded by the animal liver tissue homogenate and proteolytic enzyme. The degree of degradation was affected by heating temperature.     

Development of specific organ-targeting drug delivery system I: Physico-pharmaceutical characteristics of thermally denatured albumin microspheres containing cytarabine

In attempt to develop a drug delivery system using serum albumin microspheres, bovine serum albumin microspheres containing antitumor agent, cytarabine, were prepared. The shape, surface characteristics, size distribution, behavior ofin vitro distribution, drug release behavior, and degradation of albumin microspheres in animal liver tissue homogenate and proteolytic enzyme were investigated. The shape of albumin microspheres was spherical and the surface was smooth and compact. The size distribution of the albumin microspheres was affected by dispersion forces during emulsification and albumin concentration. Distribution of albumin microspheres after intravenous administration in rabbit was achieved immediately.In vitro, albumin microsphere matrix was so hard that it retained most of cytarabine except initial burst during the first 10 minutes, and the level of drug release during the initial burst was affected by heating temperature, drug/albumin concentration ratio and size distribution. After drug release test, the morphology of albumin microspheres was not changed. Albumin microsphere matrix was degraded by the rabbit liver tissue homogenate and proteolytic enzyme. The degree of degradation was affected by heating temperature.     

The binding of amsacrine to human plasma proteins

Determination of amsacrine plasma protein binding by both equilibrium dialysis and ultracentrifugation gave similar results and indicated that amsacrine is highly bound (approximately 97%) in human plasma. This binding is independent of amsacrine concentration over the range 1-100 mumol litre-1, but is very sensitive to plasma pH and, to a lesser extent, to temperature. Approximately 20% of the drug appeared to be covalently bound to plasma proteins. Amsacrine was bound by all plasma proteins investigated including albumin, alpha 1-acid glycoprotein and various gamma-globulins. The binding to albumin appeared to occur by two processes, a saturable process at a single site with a KD of 13.9 mumol litre-1 and a non-saturable process. Despite differences in individual protein concentrations, no significant difference was observed in the unbound amsacrine fraction in plasma from patients receiving this drug for treatment of acute myelogenous leukaemia and plasma from healthy individuals.     

The protein binding of timegadine determined by equilibrium dialysis.

The protein binding of timegadine to albumin, serum, plasma and plasma enriched with the acute phase reactants alpha 1-acid glycoprotein, alpha 1-anti-trypsin and C-reactive protein was determined by equilibrium dialysis. The effects of other analgesic and anti-inflammatories (indomethacin, ketoprofen, paracetamol and sodium salicylate) and other basic drugs (disopyramide, lignocaine, propranolol and quinidine) on the binding of timegadine were also determined. Timegadine binding was concentration-dependent up to 0.5 micrograms/ml, but independent above this level up to 10.0 micrograms/ml, the mean and standard error being 93.8 +/- 0.5%. Albumin accounted for only 32.4% of timegadine bound to plasma. Plasma enrichment with the acute phase reactants led to significant increases in timegadine binding. Simultaneous dialysis with other drugs caused significant decreases in timegadine binding.     

Targeted delivery of low dose doxorubicin hydrochloride administered via magnetic albumin microspheres in rats

The efficacy of magnetic albumin microspheres in the targeted delivery of an anti-cancer agent, doxorubicin hydrochloride, has been investigated in rats. Using the tail as a target organ, the animals were intra-arterially administered with either 0.12 mg/kg of free drug, or 0.04 mg/kg of microsphere entrapped drug in the presence of a 8000 Gauss magnet applied for 30 min at the target-site. In each group, the animals were sacrificed over a 48 h period and their various tissues analysed for drug concentration using HPLC. It was found that compared to the free drug, a one-third dose of microsphere entrapped drug resulted in almost eight times higher drug exposure (AUC0-infinity) at the target site. In addition, the drug delivery to all the non-target tissues, including liver and heart, was substantially reduced. The study confirms the efficacy of magnetic albumin microspheres in the targeted delivery of chemotherapeutic agents.     

Immobilization of Active Urokinase on Albumin Microspheres: Use of a Chemical Dehydrant and Process Monitoring

A method of immobilizing urokinase on albumin microspheres has been developed. Laser scattering, which was used to follow particle size from the initial emulsification stage to the final aqueous resuspension of the microsphere stage, showed that particle coalescence and crosslinking were critical parameters in manufacturing the microspheres. Chemical dehydration with 2,2-dimethoxypropane was used to convert an albumin emulsion into an albumin suspension and to reduce coalescence. An optimal amount of dehydrant produced 0.3-µm particles which resisted a 50°C temperature challenge. Since oil/glutaraldehyde emulsion resulted in large particles with no urokinase activity, the cross-linking concentration of glutaraldehyde was reduced by solubilizing 25% (w/v) glutaraldehyde in the oil phase with n-propanol. A concentration of 0.015% (v/v) glutaraldehyde effectively immobilized urokinase and stabilized albumin microspheres. Amidolytic activity using the specific chromogenic substrate for urokinase, S-2444, showed that enzyme activity could be retained during this glutaraldehyde cross-linking.     

Serum protein binding of a new oral cephalosporin, CL 284,635, in various species

CL 284,635 is a new third generation oral cephalosporin. Its serum protein binding was investigated in the human, monkey, dog, rat, and rabbit. This study was performed by using an equilibrium dialysis and ultrafiltration method, using radiolabeled and cold CL 284,635. In humans, CL 284,635 was found to have a mean free fraction [fu = concentration of unbound (free) drug divided by total concentration of unbound plus bound to serum proteins] of 31.3 +/- 3.3% with no serum concentration dependency in a range of 0.5 to 26 micrograms/ml. The drug was mainly bound to albumin. In rabbits and monkeys the protein binding profile of CL 284,635 was found to be 36.1 +/- 2.3% and 33.9 +/- 1.5% with no serum concentration dependency. In rats and dogs a non-concentration-dependent fu was observed at serum concentrations ranging from 0.5 to 30 micrograms/ml. A gradual increase in fu values was observed at higher serum concentrations of CL 284,635. Overall, the protein binding profile of CL 284,635 was found to be different in the five investigated species. The protein binding of CL 284,635 in monkeys and rabbits was most similar to that in humans. These species differences in protein binding may have an impact on the disposition of the drug in different species.     

Valproic acid: in vitro plasma protein binding and interaction with phenytoin.

Because valproic acid (VPA) is highly bound to plasma protein, several variables affecting binding will significantly alter the quantity of free drug which is pharmacologically active. Therefore, total VPA plasma concentrations do not reflect the therapeutic strength of the drug in tissue. We have performed equilibrium dialysis and ultrafiltration studies of VPA binding to plasma protein. The converging data in these in vitro studies indicate a clinically significant alteration in the percent of free VPA when total drug concentration exceeds 80 micrograms/ml. Saturation of drug binding sites probably occurs in this range. At 20--60 micrograms/ml VPA there is 5% free drug, with a significant increase to 8% free at 80 micrograms/ml; free drug increases to over 20% at 145 micrograms/ml total VPA. Human plasma, which is low in albumin, has twice the quantity of free VPA as normal plasma (10 versus 5% free). The clinical evidence of interaction between VPA and phenytoin is confirmed in vitro by the increase in the free fraction of both drugs. VPA binding decreases by 3--6%, while phenytoin binding decreases 5--6% as both drugs reach high plasma concentrations. When appropriate, laboratory reports should be available defining concentration of free drug in plasma for optimal interpretation of drug concetrations relative to clinical effects.     

Efficacy and tolerability of an aminopterin-albumin conjugate in tumor-bearing rats

The antifolate aminopterin (AMPT) was developed before methotrexate (MTX), but was not clinically established or generally used due its increased toxicity compared to MTX. Recently, we reported on the increased metabolism of albumin conjugates such as methotrexate-albumin (MTX-SA) in malignant tumors and the feasibility of using albumin as a carrier for drug targeting. Consequently, AMPT was covalently bound to serum albumin (AMPT-SA) at a 1:1 molar ratio. Biodistribution, tolerability and efficacy of this novel conjugate were studied in Walker-256 (W-256) carcinoma-bearing rats. As compared to native albumin, the same biodistribution and plasma clearance were found for AMPT-SA, which achieved 20.1% tumor uptake (estimated uptake per g tumor 6.4%) within 24 h after i.v. administration in rats. In a randomized study, AMPT-SA, repeatedly i.v. injected, was compared with low-molecular-weight AMPT. Depending on the molar concentration, the maximum tolerated dose (MTD) of AMPT covalently bound to SA was twice that of unbound AMPT (three repeated injections of 1.0 mg AMPT-SA/kg body weight versus three repeated injections of 0.5 mg AMPT/kg body weight; p=0.0006). Efficacy was studied at the level of the MTD and MTD/2, and demonstrated that AMPT-SA was significantly more active. At the MTD/2 in W-256 carcinoma-bearing rats, AMPT-SA achieved a 100% volume reduction and an optimal volume reduction during treatment/control (T/C) of 8.3% compared to a 53% volume reduction of AMPT and a T/C of 16.5% (p=0.032). Tumor relapses were reduced and occurred later in the AMPT-SA group (two tumor recurrences for AMPT-SA versus seven for AMPT; p=0.05). In this comparative study, the AMPT-SA conjugate showed high antitumor activity in vivo and a favorable toxicity compared to low-molecular-weight AMPT. These effects are attributed to the albumin carrier which seems to be an effective tool for selective tumor drug targeting.     

In vitro release of adriamycin from drug-loaded albumin and haemoglobin microspheres

Release of adriamycin from albumin and haemoglobin microspheres has been determined in vitro using a flow through system. Release from cross-linked albumin microspheres is controlled by the percentage of glutaraldehyde used but release profiles of spheres of 23, 41 and 60 microns diameter were virtually superimposable. Not all of the adriamycin is released from the microspheres over 20 h; as biodegradation occurs in vivo after 24 h the amount of drug remaining in the system at 20 h is likely to be principally released by degradation of the protein matrix in vivo. Estimates of the retained adriamycin vary from 16-26 per cent for albumin (n = 3) and 30 per cent for haemoglobin (n = 1).     

Studies on the uptake of tomato lectin nanoparticles in everted gut sacs

Tomato lectin (TL) is a bioadhesive glycoprotein that has been shown to bind selectively to the small intestine epithelium. When bound to polystyrene microspheres, intestinal uptake occurs not only through the gut associated lymphoid tissue (GALT) but also through normal enterocytes. In this study, the everted gut sac model was used to compare the rates and quantities of intestinal uptake of tomato lectin and that of TL coupled to microspheres. Using bovine serum albumin (BSA) and BSA coupled to microspheres as comparators. Uptake is time and concentration dependent. Transfer of TL from the lumen to the serosa was 3.9 ng/mg per h whereas that of BSA was 0.5 ng/mg per h. Hence uptake of tomato lectin was 7-fold higher than BSA. The rate of uptake of TL coupled microspheres was 41.5 ng/mg per h, which was 4-fold higher than microspheres coupled to BSA (11.8 ng/mg per h). The uptake of TL conjugated microspheres was shown to be inhibited by N-acetyl-d-glucosamine tetramer [GlcNac]₄.