Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat.

The mononuclear phagocyte system (MPS) which provides protection against infection is made up of phagocytic cells that engulf and digest bacteria or other foreign substances. Suppression of the MPS may lead to decreased clearance of pathogenic microbes. Drug delivery systems and immunomodulatory therapeutics that target phagocytes have a potential to inhibit MPS function. Available methods to measure inhibition of MPS function use uptake of radioactively-labeled cells or labor-intensive semi-quantitative histologic techniques. The objective of this work was to develop a non-radioactive quantitative method to measure MPS function in vivo by administering heat-killed E. coli conjugated to a pH-sensitive fluorescent dye (Bioparticles(®)). Fluorescence of the Bioparticles(®) is increased at low pH when they are in phagocytic lysosomes. The amount of Bioparticles(®) phagocytosed by MPS organs in rats was determined by measuring fluorescence intensity in livers and spleens ex vivo using an IVIS(®) Spectrum Pre-clinical In Vivo Imaging System. Phagocytosis of the particles by peripheral blood neutrophils was measured by flow cytometry. To assess method sensitivity, compounds likely to suppress the MPS [clodronate-containing liposomes, carboxylate-modified latex particles, maleic vinyl ether (MVE) polymer] were administered to rats prior to injection of the Bioparticles(®). The E. coli particles consistently co-localized with macrophage markers in the liver but not in the spleen. All of the compounds tested decreased phagocytosis in the liver, but had no consistent effects on phagocytic activity in the spleen. In addition, administration of clodronate liposomes and MVE polymer increased the percentage of peripheral blood neutrophils that phagocytosed the Bioparticles(®). In conclusion, an in vivo rat model was developed that measures phagocytosis of E. coli particles in the liver and may be used to assess the impact of test compounds on MPS function. Still, the detection of inhibition of splenic macrophage function will require further assay development.

Synthesis of Cis-fused pyran indolocarbazole derivatives that inhibit FLT3 kinase and the DNA damage kinase, checkpoint kinase 1.

Protein kinases are important enzymes in solid tumour and leukaemia pathologies. Their structures are well understood at the atomic level and their key role in the progression of certain cancers makes them valuable targets for anti-cancer therapy. Through medicinal chemical approaches, we developed an efficient preparative stereospecific synthesis of N12, N13 pyran-bridged indolocarbazoles that opens access to functional diversity within this previously challenging series. We focused upon the indolocarbazole class of chemical inhibitors, which includes S27888, an inhibitor we previously identified. We used biochemical and cell-based assays to identify small molecule inhibitors of Checkpoint kinase 1 (Chk1), a serine/threonine protein kinase that is activated when cancer cells are treated with genotoxic agents. These compounds show a promising inhibitory profile on Chk1. Furthermore, these compounds are active against FLT3, which is a tyrosine kinase that is frequently activated in human leukaemias. These data suggest that this chemical class may provide a source of therapeutic compounds for a broad range of human cancers.

Potential drug delivery system: study of the association of a model nitroimidazole drug with aggregates of amphiphilic polymers on aqueous solution

This study evaluated the association of N-hexyl-2-methyl-4-nitroimidazol, a model drug, to aggregates formed by anionic polyelectrolytes on aqueous solution. The alternating copolymers of maleic anhydride and N-vinyl-2-pyrrolidone were synthesized and then modified by reaction of the anhydride groups with aliphatic amines and alcohols of varying length of the alkyl chain. The partition of the model drug between water and the hydrophobic microdomains provided by the copolymers was studied using the pseudo-phase model to determinate the distribution coefficient K S, and the standard free energy of transfer ∆µ°t. The results indicate that all copolymers assessed are potential pharmaceutical reservoirs of the model drug. Nevertheless, the solubility of N-hexyl-2-methyl-4-nitroimidazol on the polymeric solutions is independent from the length of the alkyl chain of the copolymer.

Realizou-se estudo sobre a associação da N-hexil-2-metil-4-nitroimidazol, fármaco modelo, aos agregados formados por polieletrólitos aniônicos em solução aquosa. Os copolímeros alternados de anidrido maléico e N-vinil-2-pirrolidona foram sintetizados e, em seguida, modificados pela reação dos grupos de anidrido com aminas e álcoois alifáticos de duração variável da cadeia alquílica. A partição do fármaco modelo entre a água e os microdomínios hidrofóbicos fornecido pelos copolímeros foi estudada usando o modelo de pseudo-fase, a fim de determinar a distribuição do coeficiente K S e a energia livre padrão de transferência ∆µ°t. Os resultados indicam que todos os copolímeros avaliados são potenciais reservatórios farmacêuticos do fármaco. No entanto, a solubilidade do N-hexil-2-metil-4-nitroimidazol sobre as soluções poliméricas é independente do comprimento da cadeia alquílica do copolímero.

Nonspecific stimulation of the maternal immune system. I. Effects On teratogen-induced fetal malformations.

BACKGROUND: Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice. METHODS: Teratogenic chemicals (2,3,7, 8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], or valproic acid [VA]) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA). Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guérin (BCG), or by footpad injection with Freund's complete adjuvant (FCA). RESULTS: Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically. In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails). Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe. CONCLUSIONS: For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects. Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation.

Effectiveness of a simply designed tumor vaccine in prevention of malignant melanoma development.

We investigated the efficacy of a simple syngeneic tumor vaccine to induce specific antitumor immunity in female C57Bl/6 mice. Tumor vaccine was prepared by mixing irradiated B-16 melanoma tumor cells with the pleiotropic biological response modifier-maleic anhydride divinyl ether (MVE-2). Experimental animals were pretreated with the vaccine in order to prevent the development of intraperitoneal (i.p.) B-16 melanoma tumors after inoculation of viable tumor cells. More than 40% of prevaccinated animals challenged i.p. with 5 x 10(5) viable tumor cells were completely protected from tumor development and remained tumor-free 100 days after tumor cell inoculation. The percentage of tumor-free animals (survivors) rose to as much as 90% when the application of tumor vaccine was repeated two weeks after the first vaccination (i.e. one week after the inoculation of viable tumor cells). The induced antitumor response depended predominantly upon macrophage function, since vaccinated animals which were depleted of peritoneal macrophages died within the same time range as animals in the control group. Also, tumor-type specificity of the vaccine was confirmed by the fact that the animals vaccinated with B-16 melanoma vaccine were not protected from the development of another type of tumor. In conclusion, comparison of the experimental data with the data from the literature suggests that our simple tumor vaccine may be as effective as genetically engineered tumor vaccines. At the same time, this kind of vaccine is easier to control and thus safer to apply in humans when compared to genetically engineered vaccines.

An effective tumor vaccine against malignant melanoma: irradiated autologous tumor cells admixed with MVE-2.

The aim of this study was to develop as effective as possible autologous tumor vaccine which would be at the same time easy to produce, highly controllable, and without undesired side effects on normal tissue. Therefore, irradiated autologous - syngeneic B-16 tumor cells admixed with a non-specific immunomodulator MVE-2 (a polymer fraction of 1,2-co-polymer of divinyl ether and maleic anhydride) were used for subcutaneous (s.c.). or intraperitoneal (i.p.) prevaccination of experimental mice. Compared to the control mice, a statistically significant delay in tumor development of s.c. tumors was achieved in prevaccinated mice (p<0.05). An even better effect was observed in mice challenged i.p. with viable tumor cells. Using a single prevaccination complete protection was obtained in between 40-85% of the experimental mice. When the survivors from the groups injected once with the tumor vaccine were rechallenged with viable tumor cells (101 day after the first tumor challenge, no additional prevaccination), 15.7% remained free of tumor, while the survivors from the groups injected with the tumor vaccine twice and 101 day later rechallenged with viable tumor cells remained free of tumor in 60% of the cases. Based on these results we can postulate that our vaccine is effective for prevention of tumor development. The achieved protection can be augmented with serial vaccinations and can be maintained for a longer period of time.

Bioconjugation of tumor necrosis factor-alpha with the copolymer of divinyl ether and maleic anhydride increasing its antitumor potency.

To enhance the therapeutic usefulness of antitumor cytokines in vivo, we synthesized bioconjugated tumor necrosis factor-alpha (TNF-alpha) with divinyl ether and maleic anhydride copolymer (DIVEMA), which has intrinsic antitumor activity as a synthetic biological response modifier. The degree of modification could be controlled by the addition of 2,3-dimethylmaleic anhydride (DMMAn), which binds to amino groups of TNF-alpha by changing the pH. In addition, the specific activity of DIVEMA-TNF-alpha was hardly decreased in vitro. DIVEMA-TNF-alpha showed a marked antitumor effect compared to native TNF-alpha without any side effects such as sudden death, body-weight reduction, and decrease in platelet count on mice bearing solid tumors. These results suggest that DIVEMA is a useful polymeric modifier for-bioconjugation of TNF-alpha in order to increase its antitumor activity, and multifunctionally bioconjugated TNF-alpha may be a potentiated antitumor agent for therapeutic use.

Antiviral activity of biological response modifiers in a murine model of AIDS. Requirement for augmentation of natural killer cell activity and synergy with oral AZT.

We employed the Rauscher murine leukemia virus (RMuLV) as a murine retrovirus model of AIDS, to test biological response modifiers (BRM) and antiviral agents for potential therapeutic activity against the human immunodeficiency virus (HIV). We examined the relationship between the augmentation of natural killer (NK) cell activity and antiviral efficacy of a series of BRM, most of which are known inducers of interferon, in this model. Poly [I,C]-LC, MVE-2, and CL 246,738, but not Ampligen, soluble glucan, or 7-thia-8-oxoguanosine, consistently produced antiviral activity. In addition, the combination of suboptimal doses of oral 3'-azido-3'-deoxythymidine (AZT) (in drinking water) and poly [I,C]-LC produced a synergistic antiviral effect. With all the BRM tested, a consistent pattern emerged, namely that antiviral activity always correlated with the augmentation of splenic NK cell activity in infected animals. For instance, poly [I,C]-LC boosted NK activity much more in infected mice treated therapeutically (treatment initiated after infection) than prophylactically (treatment initiated before infection), and it had greater antiviral activity therapeutically than prophylactically. For the BRM tested, antiviral activity did not occur without augmentation of NK activity in infected mice. In contrast, augmentation of NK activity in uninfected mice bore no relationship to antiviral activity. Furthermore, elimination of NK cells by treating mice with anti-asialo GM1 abolished the antiviral activity of poly [I,C]-LC. Although splenic NK activity was ablated by anti-asialo GM1, serum interferon levels were not affected by this treatment. These results point to a causal connection between the augmentation of NK cell activity and the antiviral efficacy of these BRM in this murine AIDS model. NK cells thus appear to play a key role in resistance to this retrovirus, as has been suggested for HIV.

[[Therapeutic perspectives in multiple sclerosis]. / Perspectives thérapeutiques dans la sclérose en plaques.

Numerous clinical trials have been performed during the last 5 years in multiple sclerosis patients. Some of the results have been encouraging. However, clinical benefit remains limited. Corticosteroids are indicated during the course of severe relapses but have not proven any long term benefit. Immunosuppressive agents may be of some help during very active stages of the disease. Results of interferon beta-1b trial in relapsing multiple sclerosis have shown a moderate decrease in the frequency of relapses. The same effect has recently been reported with interferon beta-1a. In addition, an effect on disability progression have been suggested with the latter interferon. In France, interferon beta-1b is now authorized in the relapsing forms of the disease. Initial results with copolymer I also suggest an effect on the frequency of relapses. Despite these major therapeutical efforts, further trials, possibly using new therapeutical approaches, are still needed.

Role of macrophages in the immunotherapy of Lewis lung peritoneal carcinomatosis.

The underlying cellular mechanisms for the antitumor effects of biological response modifiers (BRMs) have not been clearly resolved. We have investigated this issue in the Lewis lung (3LL) peritoneal carcinomatosis model in which treatment with the BRM MVE-2 slows tumor growth and enhances survival. MVE-2 is a potent inducer of cytotoxic macrophages (m phi s); however, in the vivo tumoricidal properties of these m phi s remain to be firmly established. To directly establish that m phi s were at least in part responsible for the in vivo efficacy of MVE-2, a novel method of obtaining highly enriched m phi suspensions was developed which gave high purity, satisfactory yield, and excellent viability without affecting antitumor activity. Using the 3LL peritoneal carcinomatosis model and adoptive transfer techniques, we directly demonstrate that the majority of antitumor activity was associated with the adherent cell fraction enriched for m phi s. Histological observations supported this conclusion, indicating that MVE-2 treatment initially activated cells associated with nonspecific immunity, retarding tumor growth in the ascites long enough for a multifaceted immune response to develop.

Protection against murine cytomegalovirus infection in aged mice and mice with severe combined immunodeficiency disease with the biological response modifiers polyribosinic-polycytidylic acid stabilized with L-lysine and carboxymethylcellulose, maleic anhydride divinyl ether and colony stimulating factor 1.

A variety of biological response modifiers (BRMs) have provided antiviral protection to immunocompetent mice, and this prompted us to determine their efficacy against murine cytomegalovirus (MCMV) infection in immunocompromised mice-including the profoundly immunocompromised SCID mice and C57Bl/6 and B6D2F1 aged mice. SCID mice showed a marked decrease (> 20-fold) in resistance to MCMV, while there was a slight decrease (3-fold) in aged mice. In BRM antiviral protection studies, SCID mice were almost completely protected against MCMV infection by the pleiotropic immunomodulators, MVE-2 and pICLC, but much less by the more selective CSF-1. pICLC-induced IFN and NK cell cytotoxicity were maintained in SCID mice, suggesting that pleiotropic immunomodulatory effects may be required for antiviral protection in such a profoundly immunocompromised model. pICLC also effectively protected aged mice against lethal MCMV infection and effectively induced IFN. These results emphasize the potential for BRM treatment in immunocompromised hosts.

Selective depletion of liver and splenic macrophages using liposomes encapsulating the drug dichloromethylene diphosphonate: effects on antimicrobial resistance.

The current results provide direct evidence for a role of tissue macrophages (M phi) in natural immunity and support the use of immunomodulators to enhance antiviral resistance in immunocompromised individuals. In this study, macrophages (M phi) in the spleen and liver were eliminated by intravenous (i.v.) injection of the drug dichloromethylene diphosphonate (DMDP) encapsulated in liposomes. The effect of this depletion system on peritoneal M phi, peripheral blood leukocytes, splenic natural killer (NK) activity, and natural and immunomodulator-induced host resistance was then assessed. Barrier-maintained CD-1 female mice were inoculated i.v. either with DMDP liposomes, free liposomes (containing no DMDP), or saline on day -2 or on days -3 and -1 before cell population analysis or infection. Single or double treatment with DMDP liposomes had no effect on peritoneal M phi as indicated by no changes in total number, differential counts, or ectoenzyme patterns. Double treatment with DMDP liposomes caused a marked leukocytosis in blood, primarily of lymphocytes and polymorphonuclear leukocytes (PMN), and a transient depression of spontaneous and interferon-inducible splenic NK activity. The effects on host resistance to i.v. infection with Listeria monocytogenes or herpes simplex virus type 2 (HSV-2) indicated that i.v. treatment with DMDP liposomes significantly reduced natural resistance to these microorganisms as evidenced by increased mortality and decreased median survival time. When DMDP liposomes-treated mice were given the immunomodulator maleic anhydride divinyl ether copolymer (MVE-2) intraperitoneally the day before infection with HSV-2, the immunosuppressive effect of DMDP liposomes treatment was significantly reversed.

Reduced bone marrow toxicity of neocarzinostatin by conjugation with divinyl ether-maleic acid copolymer.

Neocarzinostatin (NCS) was conjugated with divinyl ether-maleic acid anhydride copolymer (pyran copolymer), and its therapeutic effect was compared with that of NCS. The conjugated NCS (pyran-NCS) with a molecular weight of about 23,000, exhibited in vitro cytotoxic activity against eight cell lines and bone marrow cells that was similar to the cytotoxic activity of NCS on a molar basis. Furthermore, both drugs had similar effects against a multidrug-resistant Chinese hamster ovary cell line (CHR C5) and its parent cell line (AUXB1) in vitro. However, pharmacological analysis showed that pyran-NCS had reduced accumulation in the spleen, and most important was three times less hematotoxic in vivo compared with NCS. Also, pyran-NCS had a 1.7-fold higher 50% lethal dose (LD50). Antitumor activity of pyran-NCS and NCS was tested against two different forms of Meth A tumor. In a solid tumor model, pyran-NCS and NCS suppressed tumor growth at three-fourths of the LD50 to 12.8 and 19.0% of the control tumor as evaluated on day 28, respectively (P less than 0.025). In an ascitic tumor model, the percentage increase in the median life span caused by pyran-NCS and NCS was more than 400 and 150% on day 60, respectively. Pyran-NCS is more effective than NCS because the reduced acute toxicity permits an increased drug dosage.

[Antitumor activity of polyanion and its application for drug delivery system of antitumor drugs].

Polyanionid copolymer of divinyl ether and maleic anhydride (DIVEMA) with narrow molecular weight distribution was synthesized and tested of its antitumor activity. DIVEMA showed a significant antitumor activity against colon 26 adenocarcinoma and FSaI fibrosarcoma transplanted in syngenic mice. Furthermore, DIVEMA was used as a polymeric drug carrier of antitumor drugs to reduce side effects and enhance the antitumor activity of the drugs. Adriamycin and neocarzinostatin were attached covalently to DIVEMA and the polymeric conjugates showed higher antitumor activity than the corresponding mother drugs against P 388 leukemic mice.

Augmentation of mouse liver-associated natural killer activity by biologic response modifiers occurs largely via rapid recruitment of large granular lymphocytes from the bone marrow.

A variety of biologic response modifiers (BRM) can potently augment NK activity in nonlymphoid organs. By using the liver as a model organ, we have shown that this augmentation of organ-associated NK activity is coincident with a 10- to 15-fold increase in the number of large granular lymphocytes (LGL) which can be isolated. The present study was designed to investigate the mechanism by which BRM induce this increase in liver-associated LGL and the coincident increase in hepatic NK activity. Initial studies confirm that a single dose of the pyran copolymer, maleic anhydride divinyl ether (MVE-2), augmented hepatic NK activity and increased the number of liver-associated LGL from 3 x 10(4)/liver to 5 x 10(5)/liver (a 17-fold increase). Multiple injections of MVE-2 further augmented total liver-associated NK activity and LGL number (to 13 x 10(5)/liver). As expected, both the NK activity and detectable LGL were eliminated by treatment of the mice with antiasialo GM1 (asGM1) serum. Three possible mechanisms for the BRM-induced increase in liver-associated LGL have been investigated, including 1) the rapid proliferation of resident hepatic LGL, 2) the redistribution of mature LGL from peripheral sites such as the spleen, or by 3) a rapid output and subsequent hepatic localization of LGL or their precursors recently derived from the bone marrow (BM). Our results demonstrated that the contribution of in situ proliferation to the BRM-induced increase in liver-LGL was relatively small, since the number of cells expressing NK-associated markers (i.e., asGM1, Thy-1.2, and NK1.1) and in G2/M phase (as assessed by propidium iodide uptake) was only 4 to 8%. Further experiments demonstrated that splenectomy before the administration of MVE-2 did not inhibit the augmentation of liver-associated NK activity. This result argued against a recruitment of mature LGL from the spleen. In contrast, selective depletion of the BM following administration of 89Sr decreased the ability of MVE-2 to augment liver-associated NK activity by greater than 80%. This procedure also significantly decreased the ability of Propionibacterium acnes (85%) and multiple doses of IL-2 (49%) to augment liver-associated NK activity. These results demonstrate that the rapid augmentation of liver-associated NK activity by BRM is largely due to localization and accumulation in the liver of LGL recently derived from the BM.

Maleic vinyl ether anhydride nephropathy: altered glomerular permeability due to an immunomodulating agent.

The polyanionic immunomodulatory polymer, maleic vinyl ether anhydride (MVE-2), enhances antitumor macrophage activity and causes heavy proteinuria. The effects of this compound on renal function and renal morphology were investigated in a rat model. Rats were given daily intravenous infusions of MVE-2, 100 mg/kg, over 2-4 hr on each of three consecutive days. Renal function and morphology in MVE-2-infused rats were examined by standard techniques and compared to control rats given saline. On the average, MVE-2 rats had a significant reduction in inulin clearance to 62% of control values. MVE-2 rats developed heavy proteinuria 1-3 days after the first infusion (mean +/- 1 SEM, 387 +/- 91 mg/24 hr). By light microscopy, the only finding was intratubular protein casts; glomeruli were normal. Immunofluorescence showed no deposition of antibody, complement, or fibrin. Electron microscopy revealed foot process effacement, epithelial cell vacuolization, and subepithelial ring-shaped structures; no immune-complex deposits were present. MVE-2 rats had no increase in the number of glomerular Ia(+) cells. To examine further the mechanism of MVE-2 nephropathy, the ability of MVE-2 to induce proteinuria in animals pretreated with radiation (750 rad), methylprednisolone (MP) or cyclosporine (CyA) was determined. Animals pretreated with radiation or MP had significantly less proteinuria after MVE-2 treatment compared to animals receiving no immunosuppressive therapy, while CyA pretreated rats developed heavy proteinuria. These results are compatible with the hypothesis that MVE-2 induces proteinuria via an effect on steroid- and radiation-sensitive cells, perhaps related to production of circulating factors which alter glomerular permeability.

Liver-associated macrophage precursor cells proliferate under impairment of regular hemopoiesis.

We reported previously that immature macrophage precursor cells can be isolated from spleen and liver of cyclophosphamide or pyran copolymer-pretreated mice. We now extended our investigations to livers of normal, untreated specific pathogen-free mice. Using the response to the macrophage growth factor colony-stimulating factor-1 (CSF-1) and the presence of the mouse macrophage-specific F4/80 antigen as criteria of definition, in the liver of normal mice we could demonstrate macrophage precursor (M phi P) cells by means of proliferation assays and flow cytometric analysis. The amount of M phi P present in the normal liver was significantly increased after administration of pyran copolymer. Also an enhanced proliferative response to CSF-1 as well as augmented natural killer activity and cytostasis of Candida albicans was noted in liver nonparenychymal cells (LNPC) after treatment of bone marrow (BM)-irradiated, splenectomized mice with pyran copolymer. Since the irradiated BM was actually proven to be silent by assessment of BM number and proliferative capacity and by scoring white blood cells, our findings suggest a response of endogenous liver M phi P under the applied conditions. Further evidence for the presence of endogenous liver hemopoietic cells was obtained from transplantation experiments in which LNPC brought about the survival of lethally irradiated mice. The data point towards a significance of the liver in disposing hemopoietic cells to the organism under impairment of regular hemopoiesis.

Enhancement of lung colony formation by admixing irradiated with viable tumor cells: dependence on host status.

The study was designed to determine whether whole-body irradiation or stimulation of the reticuloendothelial system of mice influences the ability of heavily irradiated tumor cells to enhance formation of artificial metastases when given simultaneously with viable tumor cells. Experiments were performed with a nonimmunogenic sarcoma syngeneic to C3Hf/Kam mice. Whole-body irradiation augmented and stimulation of the reticuloendothelial system abolished the metastasis-enhancing effect of tumor cells. Another observation was that heavily irradiated tumor cells can enhance formation of metastases if given i.v. within several hours before or after i.v. injection of tumor cells.

Increased therapeutic efficacy and reduced toxicity of doxorubicin linked to pyran copolymer via the side chain of the drug.

Doxorubicin was covalently linked to divinyl ether-maleic anhydride copolymer (pyran copolymer) in its polycarboxylate form via the methylketone side chain through a nucleophilic substitution reaction of the 14-bromo derivative of the drug. The drug conjugated to the synthetic polyanionic polymer was tested for antitumor activity in a range of experimental murine tumor systems. When administered ip to mice bearing ip implanted tumors (P388 leukemia or macrophage tumor J774), the polymer-linked drug was superior to free doxorubicin and daunorubicin in increasing the life span of treated animals. Treatment with the conjugate also resulted in an improvement in survival time of mice bearing ascitic M50 tumor, although the effects of a single dose of free drug, in the range of maximum tolerated doses, were marginal. When given iv, the conjugate was more effective than free drug against systemic Gross leukemia. The therapeutic advantage of the polymer-linked doxorubicin over free drug was more marked when a multiple treatment schedule was used. Studies in vitro showed that the drug following covalent fixation to the polymer had only marginally decreased cytotoxicity against HeLa and P388 cells when compared with that of free anthracycline. This effect paralleled the lack of reduction in in vivo potency. Moreover, the covalent linkage of the drug to synthetic polymer reduced drug toxicity. This effect was more marked with the ip route of administration than with the iv route.