Pharmacokinetics of 4'-O-tetrahydropyranyladriamycin given on a weekly schedule in patients with advanced breast cancer.

Improved quality of life has gained importance over shortly lasting remissions in yet incurable metastatic breast cancer. Fractionation of drug administration is one of the possible approaches to reduce the concentration-dependent toxicity of anthracyclines. We evaluated the pharmacokinetics of 4'-O-tetrahydropyranyladriamycin (THP-ADM) under weekly administration in patients with advanced breast cancer (dose escalation, from 20 to 27 mg/m2 THP-ADM). The concentration-time curves of THP-ADM in plasma were best described by an open three-compartment model [half-life of the first disposition phase (t1/2 alpha), 3.15 min; terminal elimination half-life (t1/2 gamma), 13.9 h] with a mean area under the curve (AUC) of 12.2 ng h ml-1mg-1 m-2, resulting in a mean plasma clearance of 86.9 1h-1 m-2. Metabolism included the formation of Adriamycin (ADM), Adriamycinol (ADM-OH), 13-dihydro-4'-O-tetrahydropyranyladriamycin (THP-OH), 7-deoxyadriamycinone (7H-ADn), and 7-deoxy-13-dihydroadriamycinone (7H-ADn-OH), with maximal plasma concentrations ranging from 2.8 to 5.5 ng/ml. The mean total amount of cytotoxic anthracyclines excreted into urine, mainly as the parent drug, was 5% of the delivered dose. ADM and ADM-OH, but not the parent drug, were observed in urine at up to 4 weeks after the last therapeutic cycle. There was a significant correlation between the leukocyte nadir under therapy and the AUC of ADM-OH (r = 0.800, P < 0.05). Since no shift in the plasma kinetics was observed from the first to the sixth cycle, the favorable ratio of the AUCs of THP-ADM and ADM after fractionation of THP-ADM suggests lower toxic side effects attributable to ADM. This hypothesis was confirmed in a clinical study, where no severe cardiotoxicity and only mild alopecia were observed in 19 patients. Thus, pharmacokinetics studies might be helpful in both individualization of therapy with THP-ADM and optimization of the administration schedule.

Effect of free fatty acids and phospholipids on growth of and product formation by recombinant baby hamster kidney (rBHK) and Chinese hamster ovary (rCHO) cells in culture.

Recombinant BHK and CHO cells producing human antithrombin III (rh ATIII) were used to investigate the utilization of phospholipids and free fatty acids from low-serum (0.1% FBS) culture medium. Both cell lines show distinctly different patterns of fatty acid utilization. For rBHK ATIII cells it is shown that under low serum conditions several different combinations of free fatty acids (bound to bovine albumin) elicit an identical growth stimulatory effect although individual consumption and production rates of fatty acids are different. Increased fatty acid concentrations lead to increased uptake rates without any further effect on growth rate being observed. Recombinant antithrombin III formation is found to be a function of combinations and concentrations of fatty acids present in the culture medium.

Repeated batch cultivation of rBHK cells on Cytodex 3 microcarriers: antithrombin III, amino acid, and fatty acid metabolic quotients.

Anchorage-dependent human antithrombin III-producing recombinant baby hamster kidney (rBHK) cells were cultivated on Cytodex 3 microcarriers in repeated batch mode. During a 3-month experiment four different low-serum (0.025% fetal bovine serum) or serum-free medium formulations were evaluated for (a) the initial growth phase of cells and (b) the subsequent production phase, whereby two free fatty acid (FFA) supplements were examined with respect to their growth-promoting and product-formation-enhancing properties. Selected nutrient and (by)product consumption and production rates (including those for antithrombin III, amino acids, and fatty acids) are reported. The calculated metabolic quotients reflect the prevailing slow growth conditions (mu approx. 0.06 day-1) associated with microcarrier cultures. Specific antithrombin III productivities vary significantly as a function of the feed medium supplementation with FFA.

[Isolation of the pregnancy specific beta-glycoprotein (SP1) and antigen-related proteins by means of immunoadsorption]. / Isolierung des schwangerschaftsspezifischen beta1-glykoproteins (SP1) und antigenverwandter Proteine durch Immunadsorption

Purification of human pregnancy-specific beta1-glycoprotein (SP1) and antigenically related proteins of sub-human primates (chimpanzee, rhesus monkey, cynomolgus and baboon) was achieved by means of an immunoadsorbent technique. The immunoglobulins of a rabbit antiserum to human SP1 were isolated on DEAE-cellulose and coupled to CNBr-activated Sepharose. This immunoadsorbent was used to bind human SP1, respectively monkey proteins immunochemically related to SP1 from placental extract fractions. After extensive washing the proteins were eluted by an acidic glycine buffer. Contaminating serum proteins could be removed by chromatography on hydroxyapatite columns. With this method it was possible to obtain SP1 and the antigenically related proteins of monkeys in good yield and in highly purified form. The proteins thus isolated from human and sub-human primate placentae were compared in their physicochemical and immunochemical properties. The amino acid and carbohydrate compositions of human SP1 and rhesus SP1 have been determined. In a biological test certain inhibitory effect of human SP1 on the mixed leukozyte culture (MLC) could be demonstrated.

Molecular aspects of albumin functions: indications for its use in plasma substitution.

The maintenance of the circulating fluid within the vascular system is a major role of plasma albumin. Besides its role as a protein reservoir the third function of albumin is its ability to bind and transport metabolic products, regulatory mediators, nutrients and proteins and to bind and neutralize endogenous or exogenous toxins. Bilirubin binding was chosen as a method to describe functional quality of albumin preparations. Binding at the high affinity site is well preserved in all commercial albumin-containing plasma substitutes investigated with KA values in the range of 3.2 to 4.8 . 10(7) 1/mol. Presence of absence of conventional stabilizers does not influence binding characteristics since heating of products according to various pharmacopoeia levels out any difference. The importance of ligand binding properties of a plasma substitute is stressed either in intoxications, e.g. hyperbilirubinemia of neonates or in surgical situations were multiple drug applications might disturb a balanced binding situation. Though relatively mild in nature and less frequently observed in comparison with artificial plasma substitutes incidence of adverse reactions in connection with albumin was used to search for causes. Biochemical parameters like residual proteolytic activity in general or specified (prekallikrein activator), content of aggregates, anti-complementary activity and influence on granulocyte activity did not give any clear answer in this task, neither did pharmacological testing designed to detect factors leading to histamine and kinin liberation or to detect pyrogens.

Pharmacokinetics of 15-deoxyspergualin studied in renal transplant patients receiving the drug during graft rejection.

The pharmacokinetics of the novel immunosuppressant 15-deoxyspergualin (DSG) were studied in five renal transplant patients who participated in a dose-finding study for the treatment of renal graft rejection. DSG, in a dose of 4 or 6 mg/kg per day, was given in a 3-h i.v. infusion for 5 days, in combination with a 4-day course of i.v. methylprednisolone. Analyses of DSG in plasma and urine were performed by high-performance liquid chromatography (HPLC). Plasma samples were taken up to 12 h following infusion on treatment day 2 and again on day 4 or 5. Urine was collected during the infusion and up to 12 h following the infusion. DSG was rapidly eliminated from the plasma in an apparently biexponential manner. The mean t1/2 alpha was 0.5 h (range 0.1-1.1 h) and the mean t1/2 beta 2.4 h (range 1.0-5.9 h). The mean Cmax was 4117 ng/ml (range 1944-7166 ng/ml) and the mean AUC 12505 ng.ml-1 x h (range 5642-24436 ng.ml-1 x h). Clearance ranged from 375 to 945 ml/min (mean 653 ml/min) and volume of distribution ranged from 0.2 to 1.4 l/kg (mean 0.7 l/kg). A small fraction (mean 1.6%, range 0.1%-2.7%) of the DSG dose given was excreted unmetabolized in the urine. The amount of DSG in the urine correlated strongly to renal function (P = 0.0019). Pharmacokinetics were otherwise not affected by the degree of renal function. There were no significant differences in the pharmacokinetic determinants and no accumulation of the drug on study day 4 or 5, as compared to day 2.(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of IgG with Fc gamma receptors on human neutrophils.

The interaction of IgG with Fc gamma receptors on human PMN was studied by investigating the capacity of IgG and some of its fragments to mediate and inhibit, respectively, binding of immune complexes to these receptors and the subsequent activation of the cells. Fragments included Fab/Fc which is monovalent but contains the complete Fc region, Facb (divalent, C gamma 3 domains removed), Fc, pFc' (a C gamma 3 dimer) and a peptide corresponding to the greater part of one C gamma 2 domain (amino acids 278 through 333) including the carbohydrate side chains. F(ab')2 and Fab served as controls. The results indicate that human IgG interacts with human PMN Fc receptors predominantly through its C gamma 2 domains, while binding of rabbit IgG involves additional sites in C gamma 3. The C gamma 2 binding site on human IgG appears to be located between those for C1 and protein A and to contain lysine residues. For its correct exposure the C gamma 2 domains must be kept in the native conformation, in which both the hinge disulfide bridges and the carbohydrate moieties are involved. A role of the sugars in the actual binding, on the other hand, can be excluded.

On the nature of IgG dimers. I. Dimers in human polyclonal IgG preparations: kinetic studies.

Human polyclonal IgG for therapeutic or prophylactic purposes is usually prepared from pooled plasmas taken from more than 1000 donors. After storage in solution under physiological conditions for a sufficiently long period, a considerable percentage of dimers (approx. 10-30% [w/w] at a protein concentration of approx. 160 mg/ml) represents the main component of aggregates in contrast to the essentially monomeric IgGs of monoclonal or single donor origin. Analysing the kinetics of monomer-dimer equilibration suggests assuming approx. 10(6) different antibody (ab) populations interacting independently and simultaneously with a specific partner of the reaction. Concerning the average apparent (functional) equilibrium constant of association, Kapp., a distinction could be made between two main populations characterized by values in the range if approx. 2.5-3.0 X 10(10) M-1 and 1.0 X 10(12) M-1, respectively. The results were obtained by computer simulation, taking an association rate constant, k+1, of 5 X 10(5) M-1 s-1 as a basis. Since the main part of the individual populations was found to interact Fc-independently via Fab-located binding sites, we suppose that the dimers are for the most part complexes of idiotypic (Ids) and anti-idiotypic abs (anti-Ids). Moreover, dimerization seems to be mainly a bivalent binding reaction, at least at the experimental concentrations. The results are in line with the concept of an idiotypic network regulation in man.

Physiochemical features of monoclonal idiotype-anti-idiotype complexes: importance of inter-chain disulfides for size distribution patterns and molecular geometries.

Cyclic tetramers represent the preferentially formed complexes of a murine monoclonal idiotype-anti-idiotype (Id-anti-Id) system consisting of IgG antibodies or (Fab')2 fragments at micromolar concentrations. The cleavage of inter-chain disulfides of both Id and anti-Id caused the predominant generation of cyclic dimers at the expense of larger aggregates, suggesting with regard to already published data that the hinge located interheavy-chain disulfides are essential for the strain.

Studies of the ligand binding to cholera toxin, I. The lipophilic moiety of sialoglycolipids.

The fixation of cholera toxin by ganglioside GGtet1 is dependent on the nature of the carbohydrate as well as the lipid moiety of the glycolipid. The role of the lipid in binding to the toxin investigated with synthetic ganglioside analogues (gangliosidoides). The interaction between glycolipid and toxin was followed by precipitate formation, by inhibition of toxicity and in polyacrylamide gel electrophoresis. For specific precipitation, an aliphatic hydrocarbon chain at least 14 C-atoms in length is required. Some of the gangliosidoides form high molecular weight complexes with cholera toxin at lower molar ratios of ligand to protein than the natural compound. None of the synthetic gangliosidoides equalled natural ganglioside in its ability to inhibit the effects of the toxin in vivo, but some did show considerable inhibitory activity ih monosialo-gangliotetraose or corresponding sialo-glycolipids prevents the easy degradation of the B-protein of cholera toxin into protein subunits by sodium dodecylsulfate.

Attachment of rhodosaminylanthracyclinone-type anthracyclines to the hinge region of monoclonal antibodies.

We have found that a maleimidobenzoyl spacer attached to OH-4' of the rhodosamine moiety of rhodosaminylanthracyclinone-type anthracyclines is most suitable for the attachment of these drugs to carriers, providing important advantages: The spacer is selectively and most readily introduced into the rhodosamine moiety of the drugs, is stable enough for proper handling of the derivatives, and can easily be attached to thiol groups of carrier systems such as reduced monoclonal antibodies. The anthracyclines can be liberated from the conjugates by mere hydrolysis, requiring neither hydrolytic enzymes nor acidic pH. Liberation of the drugs can, moreover, be affected by the presence of the appropriate substituents Z on the phenylene ring of the spacer, thus allowing slowed or enhanced liberation of the cytostatically active drug. The corresponding p-maleimidobenzoyl derivatives of beta-rhodomycin I, N,N-dimethyldaunorubicin, and rodorubicin have been attached to thiol groups of the hinge region of reduced monoclonal antibody BW 494/32, directed against a pancreatic cancer associated glycoprotein antigen, resulting in MoAb BW 494/32 conjugates, carrying 4.8-6.8 mol of cytotoxic residues/mol of MoAb. Rodorubicin was similarly attached to MoAb BW 575/931/2, directed against a small cell lung cancer associated antigen and to MoAb BW 431/26, recognizing an epitope detectable on carcinoembryonic antigen. The results provide evidence that the newly developed method of coupling of anthracyclines to the hinge region of monoclonal antibodies may be of broader use.