N. Willmott

The Distribution of Doxorubicin in Mice Following Administration in Niosomes

Abstract— Large multilamellar non‐ionic surfactant vesicles (niosomes) with diameters of around 800–900 nm prepared from a C16 triglyceryl ether with and without cholesterol and containing doxorubicin (Adriamycin) were administered to S180 tumour‐bearing NMRI mice by bolus injection. Although in‐vitro drug release from cholesterol‐containing niosomes is delayed, in‐vivo there was little difference between the two preparations when plasma levels were compared. As previously observed, half‐lives of the drug were prolonged compared with free solution profiles. Liver uptake was not significantly affected by niosome encapsulation of doxorubicin. There is minor accumulation of drug in the lung, perhaps because of aggregation of the vesicles and their physical entrapment. Tumour levels of drug were higher following administration of cholesterol‐containing niosomes and this was reflected in the more effective reduction in tumour growth. Metabolism of doxorubicin is altered by niosomal administration, but more studies are required before the significance of the metabolic data can be assessed.

Comparison of albumin and casein microspheres as a carrier for doxorubicin

Doxorubicin (Adriamycin)‐loaded casein and albumin microspheres, with diameters between 14 and 38 μm (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 μg of doxorubicin had a similar inhibitory effect on tumour growth (growth delay = 20ṁ7 days) to 85 μg of drug incorporated into albumin microspheres (growth delay = 18ṁ6 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.

Doxorubicin-loaded casein microspheres : protean nature of drug incorporation

Abstract— 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 μg mg−1) exceeded free drug content (3·2 μg 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 μg g−1 vs 0·7 μg g−1; 24 h, 11·7 μg g−1 vs 1.1 μg g−1;48 h, 11·2 μg g−1 vs 1·2 μg g−1; 72 h, 100 μg g−1 vs 0·8 μg 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.

Biodegradation Rate of Embolized Protein Microspheres in Lung, Liver and Kidney of Rats

Abstract— 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 μm 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.

Haemoglobin, transferrin and albumin/polyaspartic acid microspheres as carriers for the cytotoxic drug adriamycin. I. Ultrastructural appearance and drug content

Abstract Microspheres prepared from transferrin, haemoglobin and polyaspartic acid in admixture with albumin have been evaluated as alternatives to albumin systems as vehicles for the anti-cancer drug adriamycin. Electron microscopy has shown that transferrin and albumin/polyaspartic acid (195 mg/5 mg) microspheres are similar to albumin, possessing neither internal discontinuities nor surface pores, whereas haemoglobin microspheres exhibit both. Assessment of drug content revealed that transferrin (6.9 μ/mg) and haemoglobin microspheres (8.6 μ/mg) contained amounts of adriamycin that were not significantly different to albumin (9.0 μ/mg), whereas incorporation of polyaspartic acid into the albumin system led to an increase of 3–4 fold in native drug content. For albumin/polyaspartic acid microspheres values for drug content were in close agreement when assessed by HPLC and total fluorescence measurements, whereas for microspheres prepared from pure proteins total fluorescence values were 34–100% higher. An adriamycin-derived species was detected in albumin, but not albumin/polyaspartic acid microspheres, that did not co-chromatograph with native drug on thin-layer chromatography. Together these data indicate that a proportion of drug is present in other than native form in microspheres prepared from pure proteins.

Haemoglobin, transferrin and albumin/polyaspartic acid microspheres as carriers for the cytotoxic drug adriamycin. II. in vitro drug release rate

Abstract In vitro release rates of the anti-cancer drug adriamycin have been studied following its incorporation into microspheres prepared from albumin, transferrin, haemoglobin and an albumin/polyaspartic acid mixture. Drug release profiles from albumin, transferrin and haemoglobin microspheres were similar, but significant differences from the albumin/polyaspartic acid (195 mg/5 mg) system were observed. Mathematical modelling, combined with theoretical considerations, led to the conclusion that the release process is best described as triphasic zero order. Using this model it was observed that initial release rates for a particular system were variable ( albuminK 1 = − 1.34 to − 0.44 μg / mg / h ; albumin/polyaspartic acid κ 1 = − 1.52 to − 0.41 μg / mg / h ) whereas the terminal release rates were more reproducible and appeared characteristic of that system ( albumin κ 3 = − 0.10 ± 0.02 μg / mg / h ; albumin / polyaspartic acid κ 3 = − 0.65 ± 0.13 μg / mg / h ). The amounts of adriamycin available for release from this compartment were: albumin 3.5 ± 1.1 μg ; albumin/polyaspartic acid 18.1 ± 3.5 μg . The marked difference in release rates of these closely related systems may prove useful in investigations of the relation between release rate and drug potencye.

Development of a reproduceable in vitro method for assessing the biodegradation of protein microspheres

Abstract Protein microspheres have been used for passive targeting of therapeutic agents by chemoembolisation. The overall efficacy of such systems is influenced by the in vivo degradation of the carrier matrix. In this paper we demonstrate a reproduceable method for assessing the degradation behaviour of protein microspheres using laser light scattering apparatus and a model protease trypsin. Degradation has been quantified in terms of the characteristic time dependent effect of the enzyme on the measured volume concentration of the particles. The latent time (T1) before the particles start to swell and the time taken to reduce them to fifty percent of their original volume (T50) have been measured. Increasing trypsin concentrations up to 0.4% w/v reduce these values for example, the T50 of bovine serum albumin microspheres reduces from 150 min in 0.1% w/v trypsin to 50 min in 0.4% w/v. Degradation rate is also dependent on the protein matrix used for example casein microspheres provide a T50 of 150 min whilst transferrin samples provide values of 400 min. This in vitro method will allow comparison of different matrix proteins and investigation of the effects of changes in the methodology of microsphere preparation.

Development of radiolabelled albumin microspheres: a comparison of gamma-emitting radioisotopes of iodine (131I) and indium (111In/113mIn)

Biodegradable albumin microspheres have been prepared incorporating either 131I or 111In/111In. Using cDTPAA/albumin molar ratios of 1, 3 and 10, approx. 0.7, 2.1 and 7 molecules of DTPA could be coupled to an albumin molecule, labelling efficiency being constant over this range. Because 131I microspheres were more stable in plasma than the system labelled with radionuclides of indium it was selected for clinical evaluation: potential uses of this radiopharmaceutical are illustrated.