Techno-Functional Properties of Crude Extracts from the Green Microalga Tetraselmis suecica.

A mild fractionation process to extract functional biomolecules from green microalgae was implemented. The process includes bead milling, centrifugation, and filtration with several membrane cut-offs. For each fraction, the corresponding composition was measured, and the surface activity and gelation behavior were determined. A maximum protein yield of 12% was obtained in the supernatant after bead milling and between 3.2 and 11.7% after filtration. Compared to whey protein isolate, most of the algae fractions exhibited comparable or enhanced functionality. Surface activity for air-water and oil-water interfaces and gelation activities were notably superior for the retentate fractions compared to the permeates. It is proposed that such functionality in the retentates is due to the presence of hydrophobic compounds and molecular complexes exhibiting a similar behavior as Pickering particles. We demonstrated that excellent functionality can be obtained with crude fractions, requiring minimum processing and, thus, constituting an interesting option for commercial applications.

Energy consumption and water-soluble protein release by cell wall disruption of Nannochloropsis gaditana.

Several cell disruption methods were tested on Nannochloropsis gaditana, to evaluate their efficiency in terms of cell disintegration, energy input and release of soluble proteins. High-pressure homogenization (HPH) and bead milling were the most efficient with >95% cell disintegration, ±50% (w/w) release of total proteins and low energy input (<0.5kWh.kg-1biomass). Enzymatic treatment required low energy input (<0.34kWh.kg-1biomass), but it only released ±35% protein (w/w). Pulsed Electric Field (PEF) was neither energy-efficient (10.44kWh.kg-1biomass) nor successful for protein release (only 10% proteins w/w) and cell disintegration. The release of proteins after applying HPH and bead milling always required less intensive operating conditions for cell disruption. The energy cost per unit of released protein ranged from 0.15-0.25 €.kgProtein-1 in case of HPH, and up to 2-20 €.kgProtein-1 in case of PEF.

Biorefinery of microalgal soluble proteins by sequential processing and membrane filtration.

A mild biorefinery process was investigated on the microalga Nannochloropsis gaditana, to obtain an enriched fraction of water soluble proteins free from chlorophyll. After harvesting, a 100g.L-1 solution of cells was first subjected to cell disruption by either high-pressure homogenization (HPH) or enzymatic treatment (ENZ). HPH resulted in a larger release of proteins (49%) in the aqueous phase compared to the Alcalase incubation (35%). In both cases, an ultrafiltration/diafiltration (UF/DF) was then performed on the supernatant obtained from cell disruption by testing different membrane cut-off (1000kDa, 500kDa and 300kDa). After optimising the process conditions, the combination of ENZ→UF/DF ended in a larger overall yield of water soluble proteins (24.8%) in the permeate compared to the combination of HPH→UF/DF (17.4%). A gel polarization model was implemented to assess the maximum achievable concentration factor during ultrafiltration and the mass transfer coefficient related to the theoretical permeation flux rate.

Cationic polymers for successful flocculation of marine microalgae.

Flocculation of microalgae is a promising technique to reduce the costs and energy required for harvesting microalgae. Harvesting marine microalgae requires suitable flocculants to induce the flocculation under marine conditions. This study demonstrates that cationic polymeric flocculants can be used to harvest marine microalgae. Different organic flocculants were tested to flocculate Phaeodactylum tricornutum and Neochloris oleoabundans grown under marine conditions. Addition of 10 ppm of the commercial available flocculants Zetag 7557 and Synthofloc 5080H to P. tricornutum showed a recovery of, respectively, 98% ± 2.0 and 94% ± 2.9 after flocculation followed by 2h sedimentation. Using the same flocculants and dosage for harvesting N. oleoabundans resulted in a recovery of 52% ± 1.5 and 36% ± 11.3. This study shows that cationic polymeric flocculants are a viable option to pre-concentrate marine cultivated microalgae via flocculation prior to further dewatering.

Physico-chemical and transglucosylation properties of recombinant sucrose phosphorylase from Bifidobacterium adolescentis DSM20083.

Clones of a genomic library of Bifidobacterium adolescentis were grown in minimal medium with sucrose as sole carbon source. An enzymatic fructose dehydrogenase assay was used to identify sucrose-degrading enzymes. Plasmids were isolated from the positive colonies and sequence analysis revealed that two types of insert were present, which only differed with respect to their orientation in the plasmid. An open reading frame of 1,515 nucleotides with high homology for sucrose phosphorylases was detected on these inserts. The gene was designated SucP and encoded a protein of 56,189 Da. SucP was heterologously expressed in Escherichia coli, purified, and characterized. The molecular mass of SucP was 58 kDa, as estimated by SDS-PAGE, while 129 kDa was found with gel permeation, suggesting that the native enzyme was a dimer. The enzyme showed high activity towards sucrose and a lower extent towards alpha-glucose-1-phosphate. The transglucosylation properties were investigated using a broad range of monomeric sugars as acceptor substrate for the recombinant enzyme, while alpha-glucose-1-phosphate served as donor. D- and L-arabinose, D- and L-arabitol, and xylitol showed the highest production of transglucosylation products. The investigated disaccharides and trisaccharides were not suitable as acceptors. The structure of the transglucosylation product obtained with D-arabinose as acceptor was elucidated by NMR. The structure of the synthesized non-reducing dimer was alpha-Glcp(1-->1)beta-Araf.

Cloning and characterization of two alpha-glucosidases from Bifidobacterium adolescentis DSM20083.

Two alpha-glucosidase encoding genes (aglA and aglB) from Bifidobacterium adolescentis DSM 20083 were isolated and characterized. Both alpha-glucosidases belong to family 13 of the glycosyl hydrolases. Recombinant AglA (EC 3.2.1.10) and AglB (EC 3.2.1.20), expressed in Escherichia coli, showed high hydrolytic activity towards isomaltose and pnp-alpha-glucoside. The K(m) for pnp-alpha-glucoside was 1.05 and 0.47 mM and the V(max) was 228 and 113 U mg(-1) for AglA and AglB, respectively. Using pnp-alpha-glucoside as substrate, the pH optimum for AglA was 6.6 and the temperature optimum was 37 degrees C. For AglB, values of pH 6.8 and 47 degrees C were found. AglA also showed high hydrolytic activity towards isomaltotriose and, to a lesser extent, towards trehalose. AglB has a high preference for maltose and less activity towards sucrose; minor activity was observed towards melizitose, low molecular weight dextrin, maltitol, and maltotriose. The recombinant alpha-glucosidases were tested for their transglucosylation activity. AglA was able to synthesize oligosaccharides from trehalose and sucrose. AglB formed oligosaccharides from sucrose, maltose, and melizitose.

Relative abundance and inhibitory distribution of protease inhibitors in potato juice from cv. Elkana.

Protease inhibitors from potato juice of cv. Elkana were purified and quantified. The protease inhibitors represent ca. 50% of the total soluble proteins in potato juice. The protease inhibitors were classified into seven different families: potato inhibitor I (PI-1), potato inhibitor II (PI-2), potato cysteine protease inhibitor (PCPI), potato aspartate protease inhibitor (PAPI), potato Kunitz-type protease inhibitor (PKPI), potato carboxypeptidase inhibitor (PCI), and "other serine protease inhibitors". The most abundant families were the PI-2 and PCPI families, representing 22 and 12% of all proteins in potato juice, respectively. Potato protease inhibitors show a broad spectrum of enzyme inhibition. All the families (except PCI) inhibited trypsin and/or chymotrypsin. PI-2 isoforms exhibit 82 and 50% of the total trypsin and chymotrypsin inhibiting activity, respectively. A strong variation within the latter activities was shown within one family and between protease inhibitor families.

A new family of rhamnogalacturonan lyases contains an enzyme that binds to cellulose.

Pseudomonas cellulosa is an aerobic bacterium that synthesizes an extensive array of modular cellulases and hemicellulases, which have a modular architecture consisting of catalytic domains and distinct non-catalytic carbohydrate-binding modules (CBMs). To investigate whether the main-chain-cleaving pectinases from this bacterium also have a modular structure, a library of P. cellulosa genomic DNA, constructed in lambdaZAPII, was screened for pectinase-encoding sequences. A recombinant phage that attacked arabinan, galactan and rhamnogalacturonan was isolated. The encoded enzyme, designated Rgl11A, had a modular structure comprising an N-terminal domain that exhibited homology to Bacillus and Streptomyces proteins of unknown function, a middle domain that exhibited sequence identity to fibronectin-3 domains, and a C-terminal domain that was homologous to family 2a CBMs. Expression of the three modules of the Pseudomonas protein in Escherichia coli showed that its C-terminal module was a functional cellulose-binding domain, and the N-terminal module consisted of a catalytic domain that hydrolysed rhamnogalacturonan-containing substrates. The activity of Rgl11A against apple- and potato-derived rhamnogalacturonan substrates indicated that the enzyme had a strong preference for rhamnogalacturonans that contained galactose side chains, and which were not esterified. The enzyme had an absolute requirement for calcium, a high optimum pH, and catalysis was associated with an increase in absorbance at 235 nm, indicating that glycosidic bond cleavage was mediated via a beta-elimination mechanism. These data indicate that Rgl11A is a rhamnogalacturonan lyase and, together with the homologous Bacillus and Streptomyces proteins, comprise a new family of polysaccharide lyases. The presence of a family 2a CBM in Rgl11A, and in a P. cellulosa pectate lyase described in the accompanying paper [Brown, Mallen, Charnock, Davies and Black (2001) Biochem. J. 355, 155-165] suggests that the capacity to bind cellulose plays an important role in the activity of main-chain-cleaving Pseudomonas pectinases, in addition to cellulases and hemicellulases.

Purification and characterisation of a beta-galactosidase from Aspergillus aculeatus with activity towards (modified) exopolysaccharides from Lactococcus lactis subsp. cremoris B39 and B891.

Beta-galactosidase from Aspergillus aculeatus was purified from a commercial source for its hydrolytic activity towards (modified) exopolysaccharides (EPSs) produced by Lactococcus lactis subsp. cremoris B39 and B891. The enzyme had a molecular mass of approximately 120 kDa, a pI between 5.3 and 5.7 and was optimally active at pH 5.4 and 55-60 degrees C. Based on the N-terminal amino acid sequence, the enzyme probably belongs to family 35 of the glycosyl hydrolases. The catalytic mechanism was shown to be retaining and transglycosylation products were demonstrated using lactose as a substrate. The beta-galactosidase was also characterised using its activity towards two EPSs having lactosyl side chains attached to different backbone structures. The enzyme degraded O-deacetylated EPS B891 faster than EPS B39. Furthermore, the presence of acetyl groups in EPS B891 slowed down the hydrolysing rate, but the enzyme was still able to release all terminally linked galactose.

Stereochemical course of hydrolysis catalysed by alpha-L-rhamnosyl and alpha-D-galacturonosyl hydrolases from Aspergillus aculeatus.

The stereochemical course of hydrolysis catalysed by four Aspergillus aculeatus enzymes acting on alpha-L-rhamnosyl and alpha-D-galacturonosyl linkages in the hairy regions of pectins has been determined using 1H-NMR. Exogalacturonase acts with inversion of anomeric configuration (e-->a), shown by the initial release of beta-D-GalpA from the non-reducing end of polygalacturonic acid. Similarly, rhamnogalacturonan (RG) hydrolase also acts with inversion of anomeric configuration (e-->a) during hydrolysis of alpha-D-GalpA-(1-->2)-alpha-L-Rhap linkages in RG, initially releasing oligosaccharides with beta-D-GalpA at the reducing end. This result is consistent with the recently solved crystal structure of this enzyme, as well as its classification based on amino acid sequence similarity into glycosyl hydrolase family 28. alpha-L-Rhamnosidase and RG-rhamnohydrolase also act with inversion of configuration (a-->e), initially releasing beta-L-Rhap from p-nitrophenyl alpha-L-rhamnopyranoside and RG oligosaccharides, respectively. Thus, all four enzymes examined are inverting hydrolases which probably catalyse hydrolysis via single displacement mechanisms.

The N-benzyl derivative of the glucosidase inhibitor 1-deoxynojirimycin shows a prolonged half-life and a more complete oral absorption in the rat compared with the N-methyl analog.

The pharmacokinetic behavior of the glucosidase inhibitor and antiviral compound JV-benzyl-1-deoxynojirimycin (BndNM) was studied in rats in vivo and in isolated perfused livers. BndNM is a more lipophilic derivative of the glucosidase inhibitor A'-methyl-1-deoxynojirimycin (MedNM). This was reflected in the n-octanol/Krebs partition coefficients, which were 0.28 and 0.004, respectively. Compared with previously published data on the N-methyl analog, derivatization with the benzyl moiety caused an increase in both elimination half-life (from 32 min for MedNM to 69 min for BndNM) and steady-state distribution volume (164 mL for MedNM and 322 mL for BndNM) in vivo. The fraction of the dose excreted in urine after 2 h was decreased (66% for BndNM compared to 80% for MedNM), the fraction excreted in bile after 2 h was increased, both in vivo (1.5% of BndNM compared to 0.2% for MedNM) and in perfused livers (5% for BndNM compared to 0.5% for MedNM). Clearance decreased from 6.3 (MedNM) to 4.0 mL/min (BndNM). In unanesthetized rats, bioavailability of BndNM was found to be 100% after oral dosing. Fractionation of liver homogenates showed that BndNM was mainly present in the cytosolic fraction, with a slight accumulation in the lysosomal/endosomal fraction. The authors conclude that addition of the lipophilic phenyl group to the deoxynojirimycin backbone leads to more favorable pharmacokinetic properties of the glucosidase inhibitor.

Sparsomycin and its analogues: a new approach for evaluating their potency as inhibitors of peptide bond formation.

The ability of several sparsomycin analogues to inhibit peptide bond formation was studied in vitro. Peptide bonds are formed between puromycin (S) and the acetylPhe-tRNA of acetylPhe-tRNA/70 S ribosome/poly(U) complex (complex C), according to the puromycin reaction: [formula: see text] It was shown that the sparsomycin analogues, like sparsomycin itself, inhibit peptide bond formation in a time-dependent manner; they react with complex C according to the equation [formula: see text] where C*I is a conformationally altered species in which I is bound more tightly than in CI. The determination of the rate constant k(7) for the regeneration of complex C from the C*I complex allows evaluation of these analogues as inhibitors of peptide bond formation. According to their k7 values, these analogues are classified in order of descending potency as follows: n-pentyl-sparsomycin (4) > n-butyl-sparsomycin (3) approximately n-butyl-deshydroxy-sparsomycin (6) > benzyl-sparsomycin (2) > deshydroxy-sparsomycin (5) approximately sparsomycin (1) > n-propyl-desthio-deshydroxy-sparsomycin (7). The analogues with an aromatic or a larger hydrophobic side chain are stronger inhibitors of the puromycin reaction than are those with a smaller side chain or those lacking the bivalent sulfur atoms; replacement of the hydroxymethyl group with a methyl group does not affect the position of the compound in this ranking; compare the positions of compounds 1 and 3 with those of 5 and 6. In the case of compound 7, C*I adsorbed on cellulose nitrate disks was not sufficiently stable to allow examination by the method applied to the other analogues, probably due to a relatively large value of k7. This analogue showed also time-dependent inhibition, but after the isomerization of CI to C*I, the kinetics of inhibition become complex, and C*I interacted further with puromycin, either as C*I or after its dissociation to C*.

Synthesis of alpha-glucosidase I inhibitors showing antiviral (HIV-1) and immunosuppressive activity.

The synthesis of a series of analogues of the monosaccharide alpha-glucosidase I inhibitor N-decyl-1-deoxynojirimycin (1) is described. With the incorporation of a single oxygen atom particularly at position seven in the N-decyl side chain, i.e. to give N-7-oxadecyl-dNM (4), the therapeutic ratio (alpha-glucosidase I inhibitory activity over toxicity in HepG2 cells) increases considerably. N-7-Oxadecyl-dNM inhibits purified porcine liver alpha-glucosidase I with an IC50 value of 0.28 microM. The position of the oxygen atom in the N-decyl side chain is of importance since N-3-oxadecyl-dNM is less active and, moreover, is toxic to HepG2 cells at 3 mM. Subsequently, the synthesis of a disaccharide inhibitor of alpha-glucosidase I is described. The aminodisaccharide ManNH2 alpha 1,2Glc (12) inhibits alpha-glucosidase I with an IC50 value of 15.7 microM. Two closely related monosaccharide derivatives of 12 did not inhibit the enzyme at low microM concentrations (no inhibition at 5 microM), showing the additional effect of binding of the aglycon fragment of the molecule to the active site of alpha-glucosidase I. Next, the N-alkyl-dNM derivatives were analysed for antiviral and immunomodulatory activity in-vitro. It is found that the most potent alpha-glucosidase I inhibitor from this study, N-7-oxadecyl-dNM (4) inhibits HIV-1 induced syncytia formation and lymphocyte proliferation in-vitro. Finally, compound 4 was also investigated in-vivo. N-7-Oxadecyl-dNM (4) reduced adjuvant-induced arthritis in rats making this compound a potential candidate for treating autoimmune diseases like rheumatoid arthritis.

Glucosidase trimming inhibitors preferentially perturb T cell activation induced by CD2 mAb.

Glycosidase trimming inhibitors may be used to study contribution of N-linked glycan moieties in T cell function. We have studied the effects of castanospermine (Cas), swainsonine (Swain), 1-deoxynojirimycin (dNM), and 1-deoxymannojirimycin (dMM) on T cell activation and differentiation. Our analysis included a new dNM derivative, N-pentyl-1-deoxynojirimycin (pentyldNM). Previous reports showed inhibitory action of trimming inhibitors, such as Swain and Cas, on pokeweed mitogen-driven immunoglobulin (Ig) production. We extend these findings for pentyldNM and observed that glucosidase inhibitors, Cas and pentyldNM were effective in inhibiting CD2 and CD3 monoclonal antibody (mAb) driven Ig production. The pattern of inhibition by mannosidase and glucosidase inhibitors correlated with inhibitory action on T cell activation: only glucosidase trimming inhibitors (Cas and pentyldNM with comparable potency) perturbed mAb-induced T cell activation, in particular if induced by CD2 mAb. Expression of the early activation marker CD69 was not decreased in the presence of these inhibitors, while addition of exogenous recombinant interleukin-2 partially overcame inhibitory effects during proliferation. These findings suggest that glucosidase, but not mannosidase, trimming inhibitors interfere with a late phase of T cell activation. In addition, the enhanced sensitivity of CD2 mAb-induced proliferation for glucosidase trimming inhibitors suggests dependence on N-linked glycans during CD2-mediated adhesion and triggering functions.

Correlation of the in vitro cytotoxicity of ethyldeshydroxysparsomycin and cisplatin with the in vivo antitumour activity in murine L1210 leukaemia and two resistant L1210 subclones.

The cultured murine leukaemia L1210 cell populations used in the present study were derived from L1210 cells that had been grown in vivo. Subclones resistant to sparsomycin (L1210/Sm) or cisplatin (L1210/CDDP) were also developed in vivo. The doubling times of the cultured cell populations were identical. Fractions surviving after drug treatment in vitro were determined by colony formation in soft agar. The results, based on the differential sensitivity of the cell populations to ethyldeshydroxysparsomycin (EdSm) and CDDP, indicated that after a short exposure, cultured L1210/CDDP cells were cross-resistant to EdSm. L1210/Sm cells, however, were not cross-resistant to CDDP. The results obtained in cultured cell populations were confirmed in vivo. CD2f1 mice bearing i.p. implants of 1 x 10(5) tumour cells were given EdSm or CDDP and a combination of the two agents. Drugs were given once daily every 4 days for 3 doses starting at 24 h after tumour implantation. Treatment of mice bearing L1210/wt leukaemia with combined EdSm and CDDP caused strongly synergistic antitumour activity. In animals bearing the two resistant subclones, however, combined drug treatment did not improve the antitumour activity. The corresponding median survival of mice receiving combined drug treatment was 60 days in each group containing 6 mice bearing L1210/wt, with 4-6 cures being noted; 19 days in animals harbouring L1210/Sm, with 2 cures being recorded among 6 mice; and 11 days in mice bearing L1210/CDDP, with no cure being obtained. The results of this study indicate that the synergism resulting from combined treatment with CDDP and EdSm is a function of the cellular properties of the target tumour-cell populations and is independent of host factors.

Potentiation of cisplatin antitumour activity by Ethyldeshydroxy-Sparsomycin in L1210 leukemia.

The combination of Ethyldeshydroxy-Sparsomycin (EdSm) with cisdiamminedichloroplatinum(II) (CDDP) caused significant antitumour activity against murine L1210 leukemia. Although single drug treatment by cisplatin generated some cures, all schedules of combined treatment, using nontoxic doses of EdSm (5mg/kg) and cisplatin (3 mg/kg), resulted in the cure of 4 to 6 mice in each group consisting of 6 mice. No differences in antitumour activity were observed between pretreatment, simultaneous treatment or posttreatment of cisplatin with EdSm. Increasing the number of tumour cells implanted i.p. diminished the antitumour effect of both EdSm as well as CDDP, but not for the drug combination. Changing the route of administration from i.p. to i.v. for one of the drugs out of the combination resulted in loss of antitumour activity.

Biochemical and kinetic characteristics of the interaction of the antitumor antibiotic sparsomycin with prokaryotic and eukaryotic ribosomes.

Using 125I-labeled phenol-alanine sparsomycin, an analogue of sparsomycin having higher biological activity than the unmodified antibiotic, we studied the requirements and the characteristics of its interaction with the ribosome. The drug does not bind to either isolated ribosomal subunits or reconstituted whole ribosomes. For sparsomycin binding to 70S and 80S ribosomes, the occupation of the peptidyltransferase P-site by an N-blocked aminoacyl-tRNA is a definitive requirement. The sparsomycin analogue binds to bacterial and yeast ribosomes with Ka values of around 10(6) M-1 and 0.6 x 10(6) M-1, respectively, but its affinity is probably affected by the character of the peptidyl-tRNA bound to the P-site. Chloramphenicol, lincomycin, and 16-atom ring macrolides compete with sparsomycin for binding to bacterial ribosomes, but streptogramins and 14-atom ring macrolides do not. Considering the reported low affinity of puromycin for bacterial ribosomes, this antibiotic is also a surprisingly good competitor of sparsomycin binding to these particles. In the case of yeast ribosomes, blasticidin is a relatively good competitor of sparsomycin interaction, but anisomycin, trichodermin, and narciclasin are not. As expected, puromycin is a poor competitor of the binding in this case. The results from competition studies carried out with different sparsomycin analogues reveal, in some cases, a discrepancy between the drug ribosomal affinity and its biological effects. This suggests that some intermediate step, perhaps a ribosomal conformational change, is required for the inhibition to take place.

Chemical, biochemical and genetic endeavours characterizing the interaction of sparsomycin with the ribosome.

Sparsomycin interaction with the ribosome and characteristics of the drug binding site in the particle were studied using chemical modification of the drug, affinity labeling methods and isolation of drug resistant mutants. The structure-function relationship studies, performed with a large number of drug derivatives, indicate that the drug interacts with the ribosome by its western and eastern moieties. The uracil ring, in the western end of the drug molecule, probably forms hydrogen bonds with the rRNA, while the apolar CH3-S-CH3 group in the eastern end interacts with a hydrophobic ribosomal domain that affinity labeling results seem to indicate is formed by protein. An increase in lipophilicity in this part of the antibiotic results in a dramatic increase in the inhibitory activity of the drug. The sparsomycin binding site is not accessible in free ribosomes, but the presence of an N-blocked amino acyl-tRNA at the P-site turns the particles capable of reversible interaction with the drug. After failure using Escherichia coli, a sparsomycin-resistant mutant was obtained by direct mutagenesis on Halobacterium halobium, a species with a unique copy of rRNA genes, stressing the role of rRNA on the drug interaction site.

Interaction of the antibiotic sparsomycin with the ribosome.

Phenol-alanine-sparsomycin, a derivative of sparsomycin carrying a p-hydroxy-benzyl function easily labeled by iodination, has been used to study the interaction of this drug with the ribosome. Our study indicated that the binding of the drug to the ribosome is sensitive to trichloracetic acid and is equally affected by disintegration of the particle after RNase and protease treatments. The ribosome is not irreversibly inactivated, and the chemical structure of the drug is not affected by interaction with the particle. These data are not compatible with the proposed covalent association of sparsomycin with the ribosome by G. A. Flynn and R. J. Ash (Biochem. Biophys. Res. Commun. 114:1-7, 1983); therefore, the antibiotic must inhibit protein synthesis through a reversible interaction with the ribosome.

Pharmacokinetics of the antitumor antibiotic n-pentyl-sparsomycin in beagle dogs.

N-pentyl-sparsomycin (PSm) is a lipophilic analogue of sparsomycin (Sm), which is a well known inhibitor of protein synthesis. This compound was selected for preclinical pharmacokinetic studies because of its high in vitro and in vivo antitumor activity. In this study in which the drug was evaluated in beagle dogs under anaesthesia, the drug concentrations in plasma, urine and bile samples were determined using high performance liquid chromatography (HPLC). Plasma protein binding was approximately 54%. The mean t1/2 beta was 0.2 hours (12 minutes) and t1/2 tau was 0.75 +/- 0.1 hours (45 +/- 6 minutes). During continuous infusions up to 5.25 hours, the steady state was reached in 3 out of 6 experiments, suggesting that in some cases the real t1/2 tau was longer than measured. PSm was actively reabsorbed from the renal tubuli. This process was saturable at the higher doses. Tubular reabsorption played only a minor role in pharmacokinetics as most of the drug (67%) was eliminated by the non-renal clearance. The non-renal clearance was saturable at higher doses of PSm and was the reason for non-linearity of pharmacokinetics.