Camptothecin delivery systems: enhanced efficacy and tumor accumulation of camptothecin following its conjugation to polyethylene glycol via a glycine linker.

PURPOSE: This study was designed to assess the circulatory retention, antitumor activity and tissue biodistribution of polyethylene glycol (PEG)-conjugated camptothecin-20-O-glycinate, PEG-beta-camptothecin (PEG-beta-CPT). PEG-beta-CPT is a novel water-soluble transport form (macromolecular prodrug) of the naturally derived antitumor drug, 20-(S)-camptothecin (CPT). METHODS: Circulatory retention studies were performed in nontumor-bearing mice injected intravenously (i.v.) with 875 mg/kg of PEG-beta-CPT. Antitumor activity was evaluated both intraperitoneally (i.p.) and i.v. in nude mouse xenograft models. Biodistribution studies were performed in nude mice bearing colorectal carcinoma xenografts with tritium-labelled PEG-beta-CPT and CPT injected i.v. RESULTS: PEG-beta-CPT had a blood t1/2alpha of approximately 6 min and a t1/2beta of 10.2 h. Significant antitumor activity was seen in all treated xenograft models. Biodistribution studies demonstrated that PEG-beta-CPT in saline provided more available labelled CPT in the circulation than unconjugated CPT dissolved in intralipid. In addition, it appeared that more labelled CPT accumulated in solid tumors when delivered in the PEG-beta-CPT form, with greater preference for tumor tissue than normal tissue. CONCLUSION: This soluble transport form of CPT and its underlying technology may have clinical application especially for the treatment of solid tumors.

Camptothecin delivery systems: the antitumor activity of a camptothecin-20-0-polyethylene glycol ester transport form.

BACKGROUND: This study was designed to assess the efficacy of polyethylene glycol(PEG) conjugated camptothecin, PEG-alpha-camptothecin, a novel water soluble transport form (prodrug) of the naturally derived antitumor drug, 20-(S)-camptothecin. MATERIAL AND METHODS: Circulatory retention studies were performed in non-tumor bearing mice injected intravenously with 300 mg/kg of PEG-alpha-camptothecin. Therapeutic efficacy was evaluated in both a murine P388/0 leukemia and a colorectal HT-29 carcinoma xenograft model. RESULTS: PEG-alpha-camptothecin had a blood t1/2 alpha of less than 5 minutes and a t1/2 beta of 3.5 hours. Five intraperitoneal injections of 3.2 mg/kg/day 20-(S)-camptothecin equivalents of PEG-alpha-camptothecin in our leukemia model resulted in significant survival over untreated controls (P < 0.001), with a mean time to death of treated versus control (T/C ratio) of 2.94 and a cure rate of 80% (n = 20). The colorectal carcinoma xenograft model demonstrated that 2-3 mg/kg/day 20-(S)-camptothecin equivalents of PEG-alpha-camptothecin given 5 days a week for 5 weeks could reduce an initial tumor burden of 300 mm3 by more than 90% without any signs of overt toxicity. CONCLUSION: This water soluble transport form of 20-(S)-camptothecin and its underlying technology may have clinical application.

Effect of polyethylene glycol conjugated bovine hemoglobin in both top-load and exchange transfusion rat models.

The purpose of this study was to determine the effect of the hemoglobin based oxygen carrier, polyethylene glycol conjugated bovine hemoglobin (PEG-Hb) on the physiology of the rat. This study was divided into the following 3 parts: pharmacokinetics, cardiovascular, and histopathology. Pharmacokinetic studies evaluated the PEG-Hb circulatory life and the resultant effect on urine composition. Telemetric intravascular blood pressure probes monitored the heart rate and mean arterial pressure. Renal arterial blood flow was determined by intraoperative perivascular ultrasound. Tissue histology was evaluated for both time and model dependent responses. The mean circulatory half-life of PEG-Hb was 17.7+/-0.3 h. Proteinuria and hemoglobinuria were greatly reduced with PEG conjugation. PEG-Hb treated rats produced 8.5 times and 49 times less proteinuria and hemoglobinuria, respectively, than unmodified bovine Hb treated animals. The mean arterial pressure (MAP) in PEG-Hb treated rats was insignificantly different from sham controls undergoing a 30% exchange transfusion while dextran caused an initial reduction and bovine Hb produced a prolonged elevation in the MAP. In these same anesthetized rats, PEG-Hb slightly decreased the heart rate while dextran caused an increase and bovine Hb had no effect. In addition, PEG-Hb was able to maintain the renal arterial blood flow while both Ringer's lactate and bovine Hb caused a reduction in the blood flow. Finally, PEG-Hb treated rats showed a dose and time dependent formation of vacuoles within the renal proximal convoluted tubules and splenic macrophages in both top-load and exchange transfusion models, but no other morphological changes. In conclusion, PEG-Hb had a relatively long vascular persistence that did not cause any significant alterations in the urinalysis, cardiovascular function, or tissue histopathology in the rat.

The impact of polyethylene glycol conjugation on bovine hemoglobin's circulatory half-life and renal effects in a rabbit top-loaded transfusion model.

This study compares the effects of polyethylene glycol (PEG) modified bovine hemoglobin on vascular half-life and renal function in rabbits to those of unmodified bovine hemoglobin. Renal function was assessed by the measurement of the glomerular filtration rate, urinalysis, blood chemistries, hemoglobin (Hb) excretion rates, and tissue histology. The influence of infusion rates on hemoglobin excretion rates and organ morphology was also examined. The mean half-life of unmodified bovine hemoglobin was 3.0 +/- 0.1 (mean +/- SEM) h, which was extended 14-fold to 43.2 +/- 1.7 h following PEG conjugation. The glomerular filtration rate, urinalysis, and blood chemistries were not greatly affected by either the unmodified bovine hemoglobin or the PEG modified bovine hemoglobin. However, unmodified bovine hemoglobin did demonstrate significant hemoglobinuria (Hb excretion levels in excess of 1.0% of the infused dose [p < 0.05]) at all infusion rates given while PEG modified bovine hemoglobin did not. In addition, histological examination by light microscopy indicated that the most severe morphological changes occurred in animals that received unmodified bovine hemoglobin. This data suggests that PEG modification of bovine hemoglobin significantly reduced some of the adverse effects of bovine hemoglobin on renal physiology and morphology.

Drug delivery systems: water soluble taxol 2'-poly(ethylene glycol) ester prodrugs-design and in vivo effectiveness.

Water soluble 2'-taxol poly(ethylene glycol) (PEG) esters have been synthesized and shown to function in vitro as prodrugs. However, in vivo experiments clearly establish that in order for these prodrugs to behave in a predictable fashion, the molecular weight of PEG must be of such magnitude so as to maintain a t1/2(circulation) > t1/2(hydrolysis). When PEG derivatives of molecular weight approximately 40 kDa were employed with paclitaxel, ca. 4% by weight of paclitaxel was carried by the water soluble prodrug form, and equivalent in vivo toxicity and increased life expectancy in the P388-treated mouse was observed. An effective method for prescreening prodrugs was found to be the acute murine lethality, which reflects the equivalency of the solubilized transport form and the native drug.

Phase I trial of ImmTher, a new liposome-incorporated lipophilic disaccharide tripeptide.

A phase I clinical trial was conducted to evaluate the toxicology and biological activity of a new liposome-incorporated lipophilic disaccharide tripeptide, ImmTher. Twelve patients with advanced nonhematological malignant disease received 13 courses of therapy at dose levels of 200-1,200 micrograms/m2. A course of therapy consisted of once-weekly administration of the drug for 2-12 weeks. The major clinical toxicities observed were chills and hypotension. No renal, hepatic, cardiac, or hematological toxicity was observed. A small decrease in pulmonary diffusion capacity was observed. Biological activity was demonstrated by changes in plasma cytokine levels, changes in in vitro monocyte cytotoxicity, and by a decrease in tumor size. Improvement was observed in three of three patients with metastatic disease to the liver. Response in these three patients correlated with an increase in their tumor necrosis factor and neopterin levels compared with nonresponders. These preliminary indications of biological and clinical activity of a liposome-incorporated lipophilic disaccharide tripeptide in patients with advanced metastatic hepatic disease suggest a potential new therapeutic approach to this common problem.

Immunologic and toxicologic study of disaccharide tripeptide glycerol dipalmitoyl: a new lipophilic immunomodulator.

A new lipophilic immunomodulator, disaccharide tripeptide glycerol dipalmitoyl (DTP-GDP), has been synthesized and evaluated for its immunologic activity and toxicology. DTP-GDP alone or in liposomes is more effective as an adjuvant and in activating macrophages compared with muramyldipeptide (MDP). Preclinical studies demonstrate no evidence of toxicity, including vasculitis. DTP-GDP in liposomes has shown antitumor activity in phase I clinical trials.

Poly-N-acetyllactosamine structures on murine cell surface T200 glycoprotein participate in natural killer cell binding to YAC-1 targets.

Cell-surface murine T200 glycoprotein has been implicated in the binding of NK cells to certain susceptible tumor targets. The existence of poly-N-acetyllactosamine structures on T200 glycoprotein and the ability of lactosamine-type oligosaccharides to inhibit NK cell-mediated cytotoxicity suggest that these structures may also be important in NK-target binding. To further identify and characterize these structures, relevant saccharides and reconstituted membrane liposomes containing fractionated effector cell membrane proteins were tested for their ability to block conjugate formation. Under base line conditions, the majority of plastic-non-adherent, Percoll-fractionated, NK-enriched splenocytes that formed conjugates with NK-susceptible YAC-1 targets functioned as lytic effectors in a single-cell cytotoxicity assay. These effectors were blocked in their ability to bind to YAC-1 targets by the addition of N-acetyllactosamine [Gal(beta 1,4)-GlcNAc] and chitobiose [GlcNAc(beta 1,4)GlcNAc], but not by saccharides lacking lactosamine-type linkages. Liposomes prepared from octyl-beta-D-glucopyranoside-extracted YAC-1 and NK-enriched effector cell membranes interfered with conjugate formation, whereas liposomes prepared from NK-insensitive P815 cells were inconsequential. Surface radiolabeled effector cell membrane proteins were fractionated by tomato lectin-Sepharose 4B (poly-N-acetyllactosamine-specific) column chromatography. Tomato lectin-bound material was enriched in a glycoprotein identical with T200, which, when incorporated into liposomes, was a potent inhibitor of effector-target binding. This inhibitory capacity was abrogated by treatment of liposomes with Ly-5 mAb (T200 mAb) or the lactosamine-specific enzyme endo-beta-galactosidase. When T200 was purified by mAb affinity chromatography and incorporated into liposomes, it was a potent inhibitor of conjugate formation, an effect that was blocked by pretreatment of T200-containing liposomes with Ly-5 mAb or endo-beta-galactosidase. These data provide additional evidence that T200 can mediate binding of NK cells to YAC-1 targets, and that poly-N-acetyllactosamine-type structures on NK cell surface T200 glycoprotein are important in the binding process.

Surface Ly-5 glycoprotein in murine natural killer (NK) cell development, target binding and cytotoxicity.

The role of surface Ly-5 glycoprotein expression in the binding and lysis of susceptible tumor targets by natural killer cells was studied using NK cell-enriched splenocytes from 6-8 week old C57BL/6 mice which were reacted with anti-Ly-5 serum in the presence or absence of a source of complement. A conjugate assay was used to demonstrate that abrogation of tumor cell lysis by anti-Ly-5 serum involved the inhibition of NK cell binding to susceptible YAC-1 targets. Additionally, reconstituted membrane vesicles from NK cell-enriched splenocyte populations blocked binding of effector cells to YAC-1 lymphoma targets, a phenomenon which was abrogated by pretreatment of vesicles with anti-Ly-5 serum. Indirect immunofluorescent labeling and cell sorting were used in the physical separation of Ly-5+ and Ly-5- cells to examine the effect of interferon and interleukin preparations on Ly-5 expression and Nk activity. Three hour treatment of sorted Ly-5- cells with murine alpha + beta interferon resulted in conversion of 22% of the cells to an Ly-5+ phenotype, as well as a significant increase in the percent specific lysis of NK-susceptible YAC-1 targets when compared to freshly sorted Ly-5- cells (29.5 +/- 1.9 vs 2.6 +/- 4.0; p less than .001). In vitro proliferation of sorted Ly-5- cells was induced by three week culture in an interferon- and interleukin-containing supernatant from ConA stimulated BALB/c splenocytes (CM), followed by repeat analysis of Ly-5 expression and cytotoxic activity. Cell sorter purified Ly-5- cells cultured in CM acquired substantial surface Ly-5 with concomitant high levels of cytotoxic activity that remained partially susceptible to inhibition by anti-Ly-5 serum. The data presented suggest that surface Ly-5 glycoprotein expression is important for binding of freshly isolated NK cells to YAC-1 targets. In addition, Ly-5- precursors of NK cells are present in murine splenic tissues and can be induced by CM to become highly active effector cells with increased surface Ly-5 expression. The persistent susceptibility of a subset of these cells to inhibition of cytotoxic activity by anti-Ly-5 serum provides additional evidence of an important role for the Ly-5 glycoprotein in the natural killer cell cytolytic mechanism against certain targets.

Temporal Appearance of Chlorophyll-Protein Complexes and the N,N -Dicyclohexylcarbodiimide-Binding Coupling Factor(0)?Subunit III in Forming Thylakoid Membranes of Euglena gracilis.

Dark-grown Euglena gracilis strain Z were exposed to white light which induces chloroplast development including a massive formation of thylakoid membranes. Thylakoid membranes were isolated from greening cells at various times from 12 to 72 h following light-exposure. The temporal appearance in the membranes of the three main chlorophyll-protein complexes (CP1, CPa, LHCP) and the N,N(1)?dicyclohexylcarbodiimide (DCCD)-binding CF(0)-IH subunit of the coupling-factor ATPase was assessed. When the cells were greened on a medium containing the readily-metabolized carbon source, ethanol, LHCP was detected as early as 12 h, and both CP1 and CPa were detected in low quantity up to 36 h and then increased. CP1, CPa and LHCP were detected as green complexes on polyacrylamide gels earalier during greening when cells were greened on resting medium compared to cells greened in the presence of ethanol. Photosystem I activity was detected at 12 h, photosystem II activity at 18 to 24 h, and water-splitting and whole electron transport chain activities after 24 h. Correlations are made between the temporal appearances of these activities and of the chlorophyll-protein complexes. The DCCD- binding CF(0)-IH was detected in the membrane at 12 h and increased in amount thereafter.

Rotational diffusion of calcium-dependent adenosine-5'-triphosphatase in sarcoplasmic reticulum: a detailed study.

The Ca2+-Mg2+ adenosine-5'-triphosphatase (ATPase) in sarcoplasmic reticulum has been covalently labeled with the phosphorescent triplet probe erythrosinyl 5-isothiocyanate. The rotational diffusion of the protein in the membrane at 25 degrees C was examined by measuring the time dependence of the phosphorescence emission anisotropy. Detailed analysis of both the total emission S(t) = Iv(t) + 2IH(t) and anisotropy R(t) = [Iv(t) - IH(t)]/[Iv(t) + 2IH(t)] curves shows the presence of multiple components. The latter is incompatible with a simple model of protein movement. The experimental data are consistent with a model in which the sum of four exponential components defines the phosphorescence decay. The anisotropy decay corresponds to a model in which the phosphor itself or a small phosphor-bearing segment reorients on a sub-microsecond time scale about an axis attached to a larger segment, which in turn reorients on a time scale of a few microseconds about an axis fixed in the frame of the ATPase. A fraction of the protein molecules rotate on a time scale of 100-200 microseconds about the normal to the bilayer, while the rest are rotationally stationary, at least on a sub-millisecond time scale.

Triazine herbicide resistance in the photosynthetic bacterium Rhodopseudomonas sphaeroides.

The photoaffinity herbicide azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-s-triazine) selectively labels the L subunit of the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides. Herbicide-resistant mutants retain the L subunit and have altered binding properties for methylthio- and chloro-substituted triazines as well as altered equilibrium constants for electron transfer between primary and secondary electron acceptors. We suggest that a subtle alteration in the L subunit is responsible for herbicide resistance and that the L subunit is the functional analog of the 32-kDa Q(B) protein of chloroplast membranes.

Polypeptide composition of thylakoid membranes: two-dimensional gel analysis during development of Euglena chloroplasts.

Two-dimensional gel electrophoresis is used to analyse the polypeptide composition of thylakoid membranes during chloroplast development in Euglena gracilis. The number of polypeptide spots resolved on the two-dimensional gels is 3-4 times the number of bands previously resolved on one-dimensional gels. Groups of multiple polypeptides with the same Mr but with differing isoelectric points are detected throughout the period of chloroplast development. The pattern of polypeptide synthesis and insertion into the forming thylakoid membranes of dark-grown Euglena exposed to light conforms with previous biochemical measurements on the assembly of the electron transport chain located in these membranes.

Gel Electrophoresis of Chloroplast Polypeptides: Comparison of One-Dimensional and Two-Dimensional Gel Analyses of Chloroplast Polypeptides From Euglena gracilis.

Euglena chloroplast polypeptides are resolved by an adaptation of the two-dimensional gel electrophoretic technique of O'Farrell (1975 J Biol Chem 250: 4007-4021). The present results are compared with those obtained by our earlier two-dimensional gel analyses as well as those obtained by one-dimensional gel analyses. Up to 75 micrograms of Euglena chloroplast polypeptides are resolved on one-dimensional sodium dodecylsulfate linear gradient 7.5 to 15% polyacrylamide gels into 43 stained polypeptide bands compared to only 33 bands resolved on a similar gel containing only 10% polyacrylamide. In contrast, two-dimensional gel electrophoresis (isoelectric focusing for the first dimension, sodium dodecylsulfate gel electrophoresis for the second dimension) further improves the resolution of the chloroplast polypeptides and especially so when a linear gradient gel is used for the second dimension. Delipidation of Euglena chloroplasts with acetone-ether and subsequent solubilization of polypeptides with Triton X-100 followed by sonication are all necessary for successful resolution of chloroplast polypeptides on two-dimensional gels. Up to 300 micrograms of chloroplast polypeptides can be clearly resolved into 56 to 59 stainable spots by the present two-dimensional gel technique when a linear gradient gel is used for the second dimension. Thus, about 30% of the polypeptide bands on a one-dimensional gel are separated into multiple polypeptides on a two-dimensional gel. The use of two-dimensional gels to separate labeled polypeptides with subsequent detection of labeled spots by autoradiography or fluorography again improves the resolution of the chloroplast polypeptides. For example, when (35)S-labeled chloroplast polypeptides are separated by the present two-dimensional gel technique with a linear gradient polyacrylamide gel in the second dimension, autoradiography or fluorography detects over 80 individual polypeptide spots. This is about twice the number resolved by our previous analyses which used a 10% polyacrylamide gel in the second dimension. Polypeptides detected range in molecular weight from about 8.5 to about 145 kilodaltons with apparent isoelectric points from pH 4.5 to 8.0. Fluorography provides rapid detection of labeled polypeptides and is 10 times more sensitive than autoradiography.

Self-maintenance capacity of CFU-S.

The numbers of CFU-S which developed in spleen colonies were measured 11 days after injection of irradiated mice with marrow from normal mice or mice which had been treated in one of a variety of ways. The broad spread of CFU-S numbers, seen by other authors, in colonies derived from normal marrow was confirmed. However, the range and distribution of CFU-S per colony was generally different in colonies derived from the marrow of mice which were recovering or had recovered from some form of depopulation. From the data obtained, the mean CFU-S/colony, M1, and the probability of self-renewal, p, of the CFU-S were calculated. These values are used to calculate the number of cell cycles undergone during development of the colony and, by making certain assumptions, the cell cycle time of the CFU-S. The plot of p against log M for the various samples measured should be linear if all CFU-S proliferate at the same rate in a growing colony. It is not linear, however, so that CFU-S obtained under different experimental conditions do not all undergo the same number of cycles. In general, treatments given to the mice result in a lowering of the capacity for self-renewal of their CFU-S and also to a shortening of their cell cycle time. Some of the possible implications of these findings are discussed.

Translation of satellite tobacco necrosis virus ribonucleic acid by an in vitro system from wheat germ.

The RNA of satellite tobacco necrosis virus (STNV) is an effective messenger RNA when translated in an in vitro system from wheat germ. This RNA codes for only STNV coat protein, as indicated (1) by coincidence of the tryptic fingerprints of the translation product and of STNV coat protein, (2) by equivalent size of the translation product and STNV coat protein, and (3) by isolation of an initial peptide of the in vitro product containing the amino acid sequence of the N terminus of STNV coat protein. STNV RNA does not contain a 5'-terminal m7G(5')ppp(5')Np---group and translation of STNV RNA by the wheat germ system does not involve prior formation of 5'-terminal m7G(5')ppp(5') nP---groups on STNV RNA. STNV RNA and 125I-labeled STNV RNA form a specific initiation complex when incubated with initiator tRNA, GTP, initiation factors, and wheat germ ribosomes. Treatment of this specific initiation complex with ribonuclease A allows isolation of an 125I-labeled oligonucleotide protected from ribonuclease A by the initiation complex. This specific oligonucleotide contains approximately 38 nucleotides, including nucleotide sequences that coincide with the codons of the N-terminal amino acids of STNV coat proteins.