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Summary From previous studies, it is generally accepted that fibrin potentiates the tissue plasminogen activator (t-PA) mediated activation of plasminogen (PLgN) by substantially lowering the K m of the reaction. However, in the majority of these studies, kinetic constants for PLgN activation by t-PA were derived from experiments where dissimilar PLgN concentration ranges were employed in enzyme assays with and without fibrin. From this study, and employing a similar PLgN substrate range (0.07–1.0μM) in reactions with and without fibrin I (des-AA-), we show that by Michaelis-Menten kinetics, there is little alteration in the K m of the above reaction with fibrin addition, while k cat increases up to 10-fold. In the absence of fibrin I, recombinant two chain t-PA (rTCt-PA) yielded the following kinetic parameters: k cat 0.065s −1 and 0.30s −1 , for Glu- and Lys-PLgN respectively, K m 0.45 μM for both. In the presence of fibrin I between 0.05 and 0.3μM, and with rTCt-PA as the enzyme, the activation of Glu- and Lys-PLgN obeyed Michaelis-Menten kinetics over the substrate range 0.07 to 0.7 μM. For PLgN activation at infinite fibrin concentration, values for k cat were estimated as 0.60s −1 and 1.25s −1 , values for K m as 0.31 and 0.16 μM, and values for K F (the dissociation constant for the fibrin-rTCt-PA complex) as 0.075 and 0.070 μM, for Glu- and Lys-PLgN respectively. To determine whether the apparent Michaelis-Menten parameters for t-PA activation of PLgN are dependent on the employed substrate concentration range, the kinetics of this reaction were reassessed over a broad PLgN range (0.002-30μM). For rTCt-PA activation of Lys-PLgN, reaction kinetics were found to be non-standard, and consistent with cooperativity. The reaction yielded two sets of parameters by Michaelis-Menten kinetics; k cat values were 0.26 and 0.89s −1 , and K m values were 0.30 and 2.7 μM. Taken as a whole, data presented here suggest that in the majority of previous studies, the kinetic constants estimated for reactions in the absence and presence of fibrin might be inappropriate for comparison, owing to the possibility of t-PA exhibiting non-standard Michaelis-Menten kinetics.

A 7kDa protein component, isolated from the venom of the Australian brown snake, Pseudonaja textilis textilis, was found to be an inhibitor of the serine protease plasmin. Its mode of action, inhibitory efficiency and specificity were determined, and compared with those displayed by aprotinin (a Kunitz inhibitor). Using aprotinin as a model small protein protease inhibitor, and with plasmin as the enzyme, the observed slow-onset inhibition was consistent with the two-stage reversible mechanism, E+IEIEI′. Formation of the initial complex (EI) was fast, but binding was relatively loose with an initial K=3.78 nM, while transition from EI to EI′, was slow, and binding was tight, with a final K′=53.2 pM. With snake inhibitor, and in contrast to the above standard mechanism, the observed inhibition was consistent with a competitive, single-stage reversible mechanism, prior to cleavage of the inhibitor to an inactive product. Plasmin and trypsin bound the snake inhibitor via this mechanism, with K values of 0.15 μM and 0.30 μM, respectively. Snake protein concentrations up to 1.0 μM failed to inhibit a number of serine proteases, including recombinant two-chain tissue plasminogen activator, high molecular weight urokinase (55 kDa), α thrombin, elastase and α chymotrypsin. Results demonstrate that the small protein protease inhibitor from the Australian brown snake does not act via the standard slow, tight-binding mechanism common to other small protein serine protease inhibitors.

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.

Preparation of mitomycin C-loaded human serum albumin (HSA) microspheres using a new technique that avoids the use of heat denaturation, which is known chemically to degrade incorporated drug, is described. This method is based on cross-linking of protein by glutaraldehyde (2.2%) during emulsification (W/O) at room temperature. The resultant particles have a mean (s.d.) diameter of 16.9 (0.34) microns (50% weight average), contain mean (s.d.) 1.15 (0.05%) mitomycin C (MMC) (w/w, n = 17) and maintain sustained release of drug over 20 h. High performance liquid chromatography (HPLC) with diode array detection was used to study the chemical integrity of the drug. Two classes of decomposition products were evaluated: chemical degradation products and drug/nucleophile covalent adducts. The HPLC separation was validated by a number of standards of proposed degradation products. To examine incorporated drug, a complete microsphere system was solubilized with 0.4% trypsin for 24 h, while to examine released drug, microspheres were immobilized on a flow-through glass wool column and fractions were collected. No evidence of significant chemical degradation or covalent coupling to protein was detected in microsphere digests. Two candidate decomposition products, representing approximately 10% of drug released from microspheres (assuming similar molar extinction coefficients to MMC), were identified in column fractions. One of these products appeared to be a covalent adduct, the other possibly an isomeric form of intact MMC. Thus, MMC is predominantly incorporated into and released (90%) chemically intact from HSA microspheres prepared by the technique described.

A high-performance liquid chromatographic method is described for the determination of mitomycin C (MMC) and its metabolites 2,7-diaminomitosene (2,7-DM), 1,2-cis-1-hydroxy-2,7-diaminomitosene (cis-hydro) and 1,2-trans-1-hydroxy-2,7-diaminomitosene (trans-hydro) in tumour tissue. N-la-Methylmitomycin C (porfiromycin, PM) was used as an internal standard. Two factors were critical in resolving the metabolites: pH and buffer ionic strength, where the retention times of the four components were affected in the order 2,7-DM >> cis-hydro >> trans-hydro >> MMC. The optimal isocratic conditions (flow-rate 1 ml/min) were 18 mM sodium phosphate pH 5.8-methanol (74:26) and a column temperature of 40 degrees C on a Spherisorb ODS-2 column (25 cm x 4.6 mm I.D.). Liquid-liquid extraction [twice with chloroform-propan-2-ol-ethyl acetate (2:2:1)] is described for tumour tissue. Recoveries varied depending on the component: MMC, 71.9 +/- 12.4%; PM, 85.5 +/- 27%; 2,7-DM, 51.7 +/- 5.4%; cis-hydro, 52.0 +/- 16.8%; trans-hydro, 62 +/- 8%. When applied to the analysis of a rat mammary carcinoma treated intra-tumourally with 450 micrograms of MMC five drug-related "metabolite" peaks were detected. Three of these co-chromatographed with standards of 2,7-DM, cis- and trans-hydro, and had identical absorption maxima to their respective standards, with the possible exception of trans-hydro.

We have reported previously that enzymes present in the Sp 107 rat mammary carcinoma catalyse doxorubicin quinone reduction (QR) to 7-deoxyaglycone metabolites in vivo [Willmott and Cummings, Biochem Pharmacol 36: 521-526, 1987]. In order to provide insights into the role of QR in the antitumour mechanism of action of doxorubicin, we have attempted in this work to identify the enzyme(s) responsible. NAD(P)H: (quinone acceptor) oxidoreductase (DT-diaphorase) was the major quinone reductase in the tumour accounting for approximately 70% of all the activity measured in microsomes and cytosols (microsomal activity, 28.4 +/- 4.6 nmol/min/mg; cytosolic activity, 94.3 +/- 11.9 nmol/min/mg). Its presence was confirmed by western blot analysis. Low levels of NADH cytochrome b5 reductase (15.6 +/- 6.3 nmol/min/mg) and NADPH cytochrome P450 reductase (14.5 +/- 4.0 nmol/min/mg) were detectable in microsomes. The presence of the latter was confirmed by western blot analysis. Pretreatment of tumours with doxorubicin (48 hr) at a therapeutic dose decreased the level of activity of all the reductases studied by at least 2-fold (P0.01, Student's t-test). Doxorubicin was shown not to be a substrate for purified rat Walker 256 tumour DT-diaphorase with either NADH or NADPH as co-factor and utilizing up to 20,000 units of enzyme/incubation but was confirmed to be a substrate for purified rat liver cytochrome P450 reductase. 7-Deoxyaglycone metabolite formation by purified cytochrome P450 reductase had an absolute requirement for NADPH as co-factor, was inhibited by molecular oxygen and dicoumarol (IC50 approx. 50 microM), and modulated by specific reductase antiserum. Reductive deglycoslation of doxorubicin to 7-deoxyaglycones was localized to the microsomal fraction of the Sp 107 tumour, with negligible activity being found in cytosols (NADH, NADPH and hypoxanthine as co-factors) and mitochondria (NADH and NADPH). The tumour microsomal enzyme had an absolute co-factor requirement for NADPH, was inhibited by oxygen and dicoumarol, and modulated by cytochrome P450 reductase antiserum. These data indicate strongly that NADPH cytochrome P450 reductase is the principal enzyme responsible for catalysing doxorubicin QR in the Sp 107 tumour.

(1991). Abnormal Vasculature of Solid Tumours: Significance for Microsphere-based Targeting Strategies. International Journal of Radiation Biology: Vol. 60, No. 1-2, pp. 195-199.

Regionally-administered, drug-loaded microspheres have a potential role in the treatment of renal tumours. Vasoactive agents, for example, angiotensin II, may allow selective delivery of microspheres to tumour. The present study defines the regional advantage that may be obtained from angiotensin II by quantifying tumour and normal kidney blood flow using radiolabelled microsphere renal perfusion studies and per-operative laser-doppler flow measurements. Angiotensin II increased microsphere distribution to tumour, relative to normal kidney, by a factor of four. This enhancement was associated with an absolute increase in tumour blood flow. Images Figure 1

The poor results of systemic chemotherapy for colorectal liver metastases have focussed attention on the use of regional chemotherapy. Embolizing, biodegradable particles can be coadministered with anticancer drugs to slow temporarily the arterial blood flow and increase drug uptake within the tumour-bearing liver. Alternatively, such microspheres can be loaded with cytotoxic agents and act as slow-release mechanisms following embolization. In both instances, size and rate of degradation are important. The aim of this study was to evaluate the biological degradation of albumin microspheres in the liver of patients with colorectal liver metastases. Seven patients with advanced liver metastases had a 200-250 mg bolus dose of customized albumin microspheres (diameter range: 20-40 microns), labelled throughout with covalently bound 131I, injected into the hepatic artery. The abdomen was imaged immediately before injection to give an estimation of background count rate, and daily after injection for five days. Activity-time curves were constructed for the liver region. The median biological half-time of the particles within the whole liver was 2.4 days (range: 1.5-11.7 days), but was longer in tumours than in normal liver in some patients. The rate of microsphere degradation within tumours may be an important factor in the efficacy of microsphere-based regional therapy, and can be studied accurately by the technique we have employed.

This revised Clinical Guideline in Paediatric Dentistry replaces the previously published sixth guideline (Fayle SA. Int J Paediatr Dent 1999; 9: 311-314). The process of guideline production began in 1994, resulting in first publication in 1997. Each guideline has been circulated widely for consultation to all UK consultants in paediatric dentistry, council members of the British Society of Paediatric Dentistry (BSPD), and to people of related specialities recognized to have expertise in the subject. The final version of this guideline is produced from a combination of this input and thorough review of the published literature. The intention is to encourage improvement in clinical practice and to stimulate research and clinical audit in areas where scientific evidence is inadequate. Evidence underlying recommendations is scored according to the SIGN classification and guidelines should be read in this context. Further details regarding the process of paediatric dentistry guideline production in the UK is described in the Int J Paediatr Dent 1997; 7: 267-268.

There is an unmet need for innovative approaches to the treatment of solid lung tumours (both primary and metastatic). Strategies are reviewed that have attempted to address this problem by optimising the localisation and activity of active agents in soluble, liposomal and microspherical form in the lung. Attention is drawn to the biological constraints that must be faced before rational strategies can be devised. Microspherical systems suitable for embolisation (diameter greater than 10 μm) offer most efficient localisation and retention in the lung vasculature: subsequent events relevant to relocation of active agents incorporated in microspheres from vasculature to tumour tissue are also considered.

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.

The relationship between hepatic arterial albumin microsphere distribution and hepatic arterial blood flow and the effects of regional angiotensin II were studied in a rat liver metastases model. Hooded-Lister rats were inoculated subcapsularly with 2 x 10(6) HSN sarcoma cells. At 20 days, hepatic arterial blood flow was measured using the reference microsphere technique. Animals then randomly received 50 microliters hepatic arterial saline or albumin microspheres (40 microns, 20 mg ml-1). Hepatic arterial blood flow measurements were then repeated at 5 min. After 5 min, animals were killed and tissues were weighed and counted in a gamma well counter. There were no significant differences between the hepatic blood flow measurements recorded before and after the control hepatic arterial saline infusion. However, regional albumin microspheres produced a significant reduction in tumour and normal liver blood flow and an 80% reduction in mean T/N blood flow ratio. Regional albumin microspheres were delivered to tumour in greater proportions (mean T/N ratio 3.89, SE 0.49) than would be expected from baseline hepatic arterial blood flow (mean T/N ratio 1.28, SE 0.22. P = 0.006). There was no correlation between T/N for baseline blood flow and albumin microsphere distribution.

Images Figure 1 Figure 2

Cytotoxic microspheres have been developed for intra-arterial use in patients with liver metastases. Following injection, the distribution of microspheres reflects the pattern of hepatic arterial blood-flow. Vasoactive agents, such as angiotensin II, by producing vasoconstriction in normal liver, might divert arterial blood toward tumour and thereby enhance the delivery of drug-loaded particles. Using a double isotope technique, the distribution of radiolabelled microspheres to tumour and normal liver tissue was measured before and after angiotensin II infusion in nine patients with multiple liver metastases. The median increase in tumour: normal ratio following angiotensin II infusion was by a factor of 2.8 (range 0.8-11.7, P less than 0.05). This novel approach to regional chemotherapy, using a combination of angiotensin II infusion and cytotoxic microspheres, increases the exposure of tumour to cytotoxic agents and may, therefore, enhance tumour response rates.

5-Fluorouracil (5-FU) was extracted from plasma and converted to its di-N-ditrifluoromethylbenzyl (DTFMBz) derivative by treatment with DTFMBzBr. Under negative ion chemical ionization (NICI) conditions the derivative yielded a mass spectrum in which most of the ion current was carried by the M(-)-DTFMBz ion (m/z 355). Selected ion monitoring enabled detection of this derivative in amounts less than 1 pg, and the practical limit of detection for 5-FU extracted from plasma was ca 400 pg ml-1. 5-FU was quantified by adding a fixed amount of [15N2]5-FU to samples of plasma before extraction, and comparing the ratio of the ions of m/z 355 and 357 against a calibration curve constructed over the concentration range under investigation. The method was used to measure the variation in the concentration of 5-FU with time during continuous infusion of the drug via three, different protocols in patients with hepatic tumour.

In this study, we describe the activity of CT1746, an orally-active synthetic MMP inhibitor that has a greater specificity for gelatinase A, gelatinase B and stromelysin than for interstitial collagenase and matrilysin, in a nude mouse model that better mimics the clinical development of human colon cancer. The model is constructed by surgical orthotopic implantation (SOI) of histologically-intact tissue of the metastatic human colon tumor cell line Co-3. Animals were gavaged with CT1746 twice a day at 100 mg/kg for 5 days after the SOI of Co-3 for 43 days. In this model CT1746 significantly prolonged the median survival time of the tumor-bearing animals from 51 to 78 days. Significant efficacy of CT1746 was observed on primary tumor growth (32% reduction in mean tumor area at day 36), total spread and metastasis (6/20 treated animals had no detectable spread and metastasis at autopsy compared to 100% incidence of secondaries in control groups). Efficacy of CT1746 could also be seen on reducing tumor spread and metastasis to individual organ sites such as the abdominal wall, cecum and lymph nodes compared to vehicle and untreated controls. We conclude that chronic administration of a peptidomimetic MMP inhibitor via the oral route is feasible and results in inhibition of solid tumor growth, spread and metastasis with increase in survival in this model of human cancer, thus converting aggressive cancer to a more controlled indolent disease.

A strategy is outlined for the removal of reactive dye colour from textile wastewaters using bacteria, capable of dye degradation, attached to environmentally-acceptable cellulose copolymeric supports. A mixed population of bacteria have been isolated from dyehouse effluent and tested on ten different reactive azo dyes. All of the tests resulted in the removal of the original colour and the production of unidentified, colourless or differently coloured, species within the time-scale provided. Immobilisation of these bacterial colonies onto characterised cellulosic supports, as a route to enhance their dye degrading activity, is described.

Summary Regional chemotherapy is an attractive but underevaluated method of treating locally advanced breast cancer. We have combined two novel methods of targeting by delivering a single pulse of adriamycin-loaded albumin microspheres down a radiologically placed internal mammary artery catheter. A complete response was observed and prolonged local control achieved.

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.

There is increasing interest in the use of microspheres, loaded with chemotherapeutic agents, for regional therapy to hepatic metastases. It is necessary to deliver these particles predominately to tumour rather than to normal liver. This study investigates factors influencing the distribution of regionally injected microspheres. Discreet tumour was induced in rats by subcapsular hepatic inoculations of HSN cells. At 20 days, 12.5 microns, 25 microns or 40 microns diameter, radiolabelled albumin microspheres were administered, in various concentrations, via the gastroduodenal artery. Tumour to normal liver microsphere distribution ratios were determined and median values ranged from 0.1 (0.2 mg ml-1 12.5 microns microspheres) to 1.8 (20 mg ml 40 microns microspheres). Concentrated suspensions (20 mg ml-1) of large microspheres (40 microns) produced the most favourable tumour to normal liver distribution ratios. These results not only have implications for the therapeutic administration of microspheres but also for their use in blood-flow studies.

Doxorubicin is shown to be present in albumin microspheres (10-40 microns) in two forms: the native drug and a fraction of drug covalently coupled to the protein matrix probably via glutaraldehyde. Upon trypsin digestion the fraction covalently coupled is released and can be resolved from native doxorubicin by high performance liquid chromatography and quantitated either by using 14C-labelled doxorubicin or by measuring the absorption of the doxorubicin chromophore at 480 nm. Albumin microspheres contained 6.9 micrograms/mg protein covalently bound drug versus 11.1 micrograms/mg native drug when 1% glutaraldehyde was used in microsphere preparation. The covalently bound fraction increased significantly with 2% glutaraldehyde. Albumin/polyaspartic acid microspheres lacked a covalently bound fraction when prepared under the same conditions as pure albumin microspheres (35 micrograms/mg native drug, 1% glutaraldehyde) but transferrin microspheres contained similar amounts of bound and native albumin. In vivo, albumin microspheres altered the disposition of doxorubicin in a rat mammary carcinoma (Sp107) compared to albumin/polyaspartic acid microspheres by reducing the rate of parent drug elimination from the tumour and by reducing its biotransformation to 7-deoxyaglycone metabolites. These data indicate that covalent coupling is a key component in the way doxorubicin is handled in tumours after administration of protein microspheres.

Biodegradable albumin microspheres have been prepared incorporating either 131 I or 111 In 113m In . 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 131 I 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.

The pharmacokinetics of 5-fluorouracil (5FU) following its administration via the hepatic artery in conjunction with biodegradable albumin microspheres and angiotensin II have been studied. Peripheral venous concentrations of 5FU are lower and plasma clearance values higher following intrahepatic arterial administration compared with a similar dose administered by intravenous infusion over both 2 h and 24 h. For the 2 h drug infusions, plasma 5FU concentrations following co-treatment with angiotensin II and microspheres via the hepatic artery were intermediate between those of arterial and venous infusions of 5FU alone. There was a trend towards the peak plasma drug concentrations and the area under the plasma concentration-time curve (AUC) being significantly lower following co-treatment with angiotensin II and microspheres compared with intra-arterial and intravenous infusions of 5FU over 24 h. Co-administration of 5FU, angiotensin II and microspheres via the hepatic artery may reduce drug exposure in the systemic compartment and therefore may increase the therapeutic ratio of 5FU administration via the hepatic artery.

By measuring peripheral drug levels in plasma, the effect of combining albumin microspheres with angiotensin II on systemic exposure to 5FU when administered by bolus injection into the hepatic artery in patients with advanced colorectal liver metastases has been assessed. The results suggest that despite hepatic arterial administration of 5FU, there was no reduction in systemic exposure when compared with that associated with intravenous injection of the same dose. Neither albumin microspheres nor angiotensin II appeared to improve the regional advantage. There have been a number of reports relating the plasma levels of cytotoxic agents with pharmacodynamic parameters. We have shown significant direct correlations between 5FU clearance and 1 week post treatment platelet and white cell counts, and an inverse relationship between the area under the 5FU plasma concentration-time curve (AUC) and 1 week post treatment platelet count and white cell count respectively.

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.

We synthesized doxorubicin (Adriamycin, Adria Laboratories, Columbus, OH)-loaded human albumin microspheres (containing approximately 1% doxorubicin w/w) between 15 and 20 micron in diameter. Intrarenal arterial administration of 99mTC-labeled microspheres demonstrated a high renal entrapment ratio (97% of recovered radioactivity). The pharmacokinetics and metabolism of doxorubicin are different when it is administered in microspherical form. Peak plasma levels are lower (16 ng/ml versus 135 ng/ml) compared with treatment by a doxorubicin solution. Histologic studies showed that the microspheres were trapped within capillaries and small arterioles in the renal vascular arcade. It is apparent that chemoembolization with doxorubicin-loaded microspheres significantly reduces systemic exposure to the antineoplastic agent, and maintains intrarenal drug levels.

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.

We describe the application of a high-speed spectrophotometric detector and high-performance liquid chromatography to the determination of anthracycline purity in extracted patient specimens and to the identification of chemical reduction products. Blood contained pure anthracyclines whilst in urine, tissue and tumour there was evidence of co-eluting endogenous peaks and complexation. Aerobic reduction yielded two main products: a C13 alcohol and a fully reduced, non-fluorescent, yellow hydroquinone, Anaerobic reduction in the presence of DNA yielded a 7-deoxyaglycone metabolite end product instead of the fully reduced hydroquinone. Eight other separate chromatographic species were identified, all of which showed unique absorbance characteristics, having a visible λmax at 530 nm and being coloured purple/blue.

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.

Adriamycin-loaded bovine albumin microspheres have been prepared by a technique that allows preparation and administration to animals on the same day. Criteria adopted for injection were that microspheres should be stable and of a size such as to become trapped in capillary beds. These conditions were fulfilled by preparing microspheres using glutaraldehyde concentrations greater than 0.5 per cent and the appropriate combination of stirring speed and continuous phase viscosity. After systemic administration rats were sacrificed at intervals and major visceral organs examined for entrapped microspheres and serum for released drug. Microspheres sieved out in the first capillary bed encountered, the lung, then following biodegradation they disappeared at a rate dependent on the amount of cross-linking agent used in their preparation. In contrast to bolus injection, serum drug levels after microsphere administration indicated an initial rapid release followed by a more protracted phase lasting at least 24 h. This latter observation is consistent with drug release during biodegradation of carrier.

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.

4'-Deoxydoxorubicin (4'-DOX) is an analogue of the anticancer drug Adriamycin (ADR) believed to lack its cardiotoxicity. Bioreduction to a semi-quinone free radical has been implicated in the etiology of ADR induced cardiotoxicity. We have studied (in a rat model) acute cardiotoxicity (after 16 mg/kg i.v. of both drugs), antitumour activity (after 5 mg/kg i.v.) and the relationship to disposition and metabolism in the target tissues (after 5 mg/kg i.v.). 7-Deoxyaglycones, which are considered inactive lipophilic metabolites derived from ADR semi-quinone, were utilised as markers of in vivo tissue free radical generation. Both drugs produced toxicity of equal severity to hearts after 24 hr, associated with high cardiac levels of 7-deoxyaglycones in the case of ADR (AUC0-48 hr, micrograms/g X hr: ADR, 47; ADR 7-deoxyaglycone (ADR-DONE), 24; and adriamycinol 7-deoxyaglycone (AOL-DONE), 35) compared to low cardiac levels of 7-deoxyaglycones but a times five higher peak cardiac concentration of parent drug in the case of 4'-DOX (AUC0-48 hr, micrograms/g X hr: 4'-DOX, 68; 4'-DOX-DONE, 3.8; and 4'-DOL-DONE, 0.8). 4'-DOX displayed superior antitumour activity to ADR against the MC 40A sarcoma growing sub-cutaneously, achieving higher concentrations of parent drug in tumour (AUC0-48 hr, micrograms/g X hr: 4'-DOX, 150; ADR, 60). There was an absence of 7-deoxyaglycones of both drugs in the tumour. These data suggest that drug bioreduction is involved principally only in ADR induced cardiotoxicity and that the level of unchanged parent drug achieved in the tumour is the most important pharmacokinetic determinant of antitumour activity for both ADR and 4'-DOX.

C. parvum (Wellcome CN6134) has been examined for suppression of a range of transplanted rat tumours of spontaneous origin. With five tumours (three mammary carcinomas and two fibrosarcomas) growth of comparatively high cell inocula (with respect to the minimum for growth in control rats) was suppressed by admixture with the vaccine. Equivalent dry weights of Glaxo, Pasteur or Connaught BCGs were relatively ineffective. Intralesional injection of C. parvum into three three established tumours (two mammary carcinomas and one fibrosarcoma) retarded development of only one, the mmamary carcinoma Sp4. With three mammary carcinomas and one fibrosarcoma, active specific immune stimulation with vaccines of viable or irradiated cells admixed with C. parvum was again consistently effective only with carcinoma Sp4, and this tumour was also susceptible to intradermal but not intravenous treatment with C. parvum alone.

In patients receiving locoregional therapy for liver metastases degradable microspheres are being increasingly used in an attempt to improve cytotoxic delivery to the tumour. However, counterproductive arteriovenous shunting through the liver following intrahepatic arterial injection can occur, leading to deposition in the lung. Measurement of the degree of shunting is important in the monitoring of this kind of therapy. In this study we describe the use of technetium-labelled microspheres of serum albumin (99Tcm MSA) to measure baseline shunting during hepatic arterial perfusion scintigraphy in five patients with liver metastases of colorectal origin, and assess the significance of the values obtained for relative lung uptake (RLU) which we define as: RLU = Activity in lung field/Activity in liver + lung fields X 100%. We found that shunting was less than 5% in all cases, zero shunting occurring in 14 of the 16 patients studied. The sources of error in this technique have been assessed, the most important being the presence of free pertechnetate in the injectate. The use of Boots 10 ml vials and glass syringes respectively to dispense and administer this radiopharmaceutical reduces this error to a small predictable level. In our small group of patients, we found no evidence to suggest that use of the vasoactive agent angiotensin II significantly increases baseline shunting in patients with colorectal liver tumour.

When examined by light microscopy, transplanted animal tumors frequently bear little resemblance to the original neoplasm. If such tumors are to be used as models of human cancer they should be characterised as regards extant rather than historical features. Consequently, we have examined, by electron microscopy and immunocytochemistry, five spontaneously arising tumors transplantable in the WAB/Not rat that are currently diagnosed on the basis of historical features only. A typical sarcoma was used for comparison. Of four spontaneously arising tumors previously classified as carcinoma, Sp4 possessed epithelial features on both ultrastructural and immunocytochemical analysis, Sp107 on ultrastructural analysis only and Sp15 and Sp22 by neither technique. Expression of vimentin was most marked with Sp15 and Sp107. The putative sarcoma, Sp24, showed clear evidence of epithelial differentiation but no evidence of vimentin expression. This study (a) records the phenotypic drift of experimental tumors on transplantation (most clearly with Sp107) and the co-expression of cytokeratins and vimentin in putative carcinomas, (b) confirms the inadequacy of routine histology for accurate characterisation of such tumors and (c) details techniques for a more thorough assessment of state of differentiation that should guide the choice of experimental model.

Anti-tumor activity and fate of adriamycin incorporated into biodegradable albumin microspheres was examined in vivo after direct intratumoral injection. Adriamycin in microspherical form displayed superior anti-tumor activity to a comparable dose of drug in solution. This was associated at later time points (40 hr, 50 hr and 72 hr after injection) with higher median parent drug concentrations in tumor tissue (4.1, 3.6, 2.6 micrograms/g respectively for microspheres and 1.6, 1.7 and 1.0 micrograms/g for solution) and the consistent detection of 7-deoxyaglycone metabolites, end products of reduction of adriamycin under anaerobic conditions (1.1, 1.0, 1.0 micrograms/g respectively for microspheres and less than 0.1 micrograms/g at all time points for solution). It is generally considered that the redox properties of anthracyclines are responsible for their toxicity to normal tissues whereas other mechanisms are responsible for antineoplastic activity. In this study we show that inducing metabolism of Adriamycin via reductive pathways is associated with increased anti-tumor effect.

Information regarding in vitro characteristics and in vivo behaviour of microspherical drug delivery systems in animal models has accumulated and many have been developed to the stage of clinical assessment. A vital requirement for clinical trial is a stable product with a long shelf-life and reproducible properties. Thus, we have investigated whether adriamycin-loaded albumin microspheres can be freeze-dried without deterioration of important characteristics. The following properties of this delivery system were compared in the freeze-dried and freshly prepared state: particle size and state of aggregation (by Coulter Counter); drug content (by HPLC) and drug release rate (in a flow-through system). It was observed that freeze-drying and subsequent storage of adriamycin-loaded albumin microspheres resulted in no substantial difference in these properties compared to freshly prepared product. Thus, the freeze-dried product, with its considerable advantages as regards prolonged storage, appears suitable for clinical use.

The responses to alum precipitated bovine serum albumin (alum BSA) and sheep erythrocytes (SRBC) were assessed in Balbc mice bearing transplanted syngeneic myelomas. The growth of all the myelomas tested significantly depressed the response of mice to alum BSA in terms of the antigen-binding capacity of serum, but had no effect on the relative binding affinity of the antibody produced. The influence of early or established tumour on the response to SRBC, in terms of splenic PFCs of IgM, IgG 1 , IgG 2 a and IgG 2 b specificities was variable. Early growth of MOPC 47 A (IgA), MPC 25 (IgG 1 ) and ADJPC 5 (IgG 2 a) myelomas depressed certain IgG responses, especially IgG 1 , while IgM responses were unaffected. In contrast, the early growth of an IgM-secreting myeloma (MOPC 104 EM) enhanced both IgM and IgG responses. The established myelomas had no effect on the IgM responses to SRBC, although MOPC 47 A and ADJPC 5 gave some enhancement of certain IgG responses.

Ten preparations of BCG, six clinical vaccines, and four experimental preparations were compared for suppression of tumor growth by regional application. The preparations differed widely in their proportions of viable bacterial units and in bacterial unit:dry weight ratios. As assessed by their ability to suppress tumor development following direct admixtures with cell inocula of a rat sarcoma, one of the six clinical vaccines (Connaught) was significantly superior to Glaxo on any parameter (dry weight, No. of total units, or No. of viable units), immuno BCG Pasteur F was superior to Glaxo on two parameters (dry weight and No. of viable units), and Pasteur scarification was superior to Glaxo only on a viable unit basis. The Tice and Rijks Institute vaccines were not significantly different from Glaxo on any basis. Experimental vaccines from the Trudeau Mycobacterial Collection, stored as frozen liquid suspensions, showed a less marked variation in physical properties; here too, the Pasteur strain was superior to two other Trudeau preparations examined (Tice and Phipps). Viable organisms were not essential for tumor suppression, gamma-radiation-sterilized vaccine being equally effective. Tests with pulmonary tumor deposits, treated by iv BCG, and tests with pleural deposits, treated by intrapleural BCG, indicated that agents identified as superior in the subcutaneous screening system were also superior in the treatment of thoracic deposits.