[Prevention of dextran anaphylaxis using hapten dextran].
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Anaphylaxis↗
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Binding constants of dextrans and isomaltose oligosaccharides to dextran-specific myeloma proteins determined by affinity electrophoresis.
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Animals↗
Plasma dextran concentrations in trauma patients administered hypertonic saline-dextran-70.
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Dextrans↗
The generation of non-dextran intravenous iron: is iron dextran obsolete?
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Anemia, Iron-Deficiency↗
Anaphylactoid reactions to Dextran 40 and 70: reports to the United States Food and Drug Administration, 1969 to 2004.
BACKGROUND: Clinical dextrans, such as Dextran 40 and Dextran 70, are associated with anaphylactoid reactions caused by dextran-reactive immunoglobulin G antibodies. When infused immediately before clinical dextrans, dextran 1 significantly reduces the incidence of severe anaphylactoid reactions. The objective of the study was to describe the frequency and characteristics of reports submitted to the United States Food and Drug Administration (FDA) for anaphylaxis or anaphylactoid events after clinical dextran administration. METHODS: We searched the FDA's Adverse Event Reporting System for reports associated with a clinical dextran and describing anaphylaxis/anaphylactoid reactions. Our case definition for a probable anaphylaxis/anaphylactoid event required signs or symptoms from at least two body systems, with at least one sign or symptom being hypotension, vasodilation, or respiratory difficulty, and onset within 60 minutes. Other reports were considered possible cases if the reporter specifically described the reaction as anaphylaxis or an anaphylactoid reaction. Premier RxMarket Advisor provided estimates of total US hospitalizations with clinical dextran or dextran 1 administration from 2000 to 2004, based on discharge billing data from a sample of US hospitals. The IMS National Sales Perspective provided estimates of total doses of dextrans sold in the United States from 1999 to 2004, based on volumes of dextrans sold in a sample of retail and nonretail outlets. RESULTS: The FDA received 366 clinical dextran adverse event reports from 1969 to 2004, of which 90 (24.6%) were anaphylaxis/anaphylactoid events. The ratio of hospitalizations where clinical dextran was administered to hospitalizations where dextran 1 was administered was 28.4:1. The expected ratio would be 1:1 if all clinical dextran patients had received dextran 1 pretreatment. The ratio of clinical dextran doses sold to dextran 1 doses sold in the United States was 38.6:1. CONCLUSIONS: A high proportion of adverse event reports for clinical dextrans described anaphylaxis or anaphylactoid reactions. Hospital discharge and product sales data suggest that dextran 1 has not been used consistently before clinical dextran administration in recent years. To reduce the risk of anaphylactoid reactions, physicians should consider routine administration of dextran 1 before the infusion of a clinical dextran.
Adult↗
Unresponsiveness following immunization with the T-cell-independent antigen dextran B512. Can it be abrogated?
The bacterial carbohydrate dextran B512 is a thymus-independent (TI) antigen and a poor immunogen. Humoral responses consist primarily of IgM and no memory response is observed; rather, secondary responses to native dextran are similar to or suppressed compared with primary responses. However, immune responses to dextran can be enhanced. In this study we have used a protein-dextran conjugate that elicits a thymus-dependent (TD) immune response against dextran. Furthermore, we used the potent adjuvant cholera toxin (CT) for the dextran immunizations. This enables us to re-evaluate the phenomenon of poor secondary response to dextran and whether it can be abrogated. We show that native dextran-primed mice were not able to mount IgG anti-dextran antibody responses after repeated immunizations with the TD, protein-dextran conjugate. This was also apparent in the spleen, where almost no dextran-specific germinal centres were detected. However, the anti-protein antibody response was normal in these mice, demonstrating that it is only the anti-dextran-responding cells that are affected. The effect of CT adjuvant on these events was also evaluated. CT enhanced the humoral IgM anti-dextran responses as well as the splenic responses to dextran. But, the isotype profile was not altered, still no IgG was produced. In contrast, mice primed with the TD conjugate and repeatedly re-immunized with native, TI, dextran generated IgG anti-dextran responses. Our results indicate that it is probable that the lack of proper costimulation in the initiation of the response to dextran causes the suppressed secondary dextran responses. Furthermore, these results suggest that TI and TD forms of dextran activate the same type of B cells, since TI dextran-priming abrogated TD dextran IgG responses. The importance of the priming event for the induction of a classical memory response to carbohydrate antigens and the implications for vaccination strategies, are discussed.
Adjuvants, Immunologic↗
Dextran strongly increases the Michaelis constants of oxidative phosphorylation and of mitochondrial creatine kinase in heart mitochondria.
Macromolecules restore the morphological changes which occur upon isolation of mitochondria in normally used isolation media. It was shown that in the presence of dextrans the permeability of mitochondrial outer membrane for adenine nucleotides decreases which may have considerable implications for the transport of ADP into the mitochondria. In this study the effect of dextran on the apparent Michaelis constants of oxidative phosphorylation and mitochondrial creatine kinase (mi-CK) of rat heart mitochondria was investigated. Mitochondria were isolated either in normally used isolation media or in the additional presence of 15% dextran 20 in order to avoid changes in the oncotic conditions on the mitochondria during preparation and investigation. Except for an increased contamination with extramitochondrial ATPases the basic functional properties of these mitochondria were normal. With oxygraphic measurements it was found that Km(ADP) of oxidative phosphorylation increased from 16 +/- 4 microM ADP (without dextran) to 50 +/- 15 microM (15% dextran 20) and to 122 +/- 62 microM (25% dextran 20) irrespective of the mode of preparation of the mitochondria. Using spectrophotometric measurements the effect of dextran on the Km(ATP) of mi-CK was investigated in three systems (a) as soluble enzyme, (b) bound to mitoplasts, (c) and in intact rat heart mitochondria. The addition of 10% dextran had no effect on kinetic properties of solubilized mi-CK. In intact heart mitochondria, however, the addition of dextran caused an augmentation of Km(ATP) from 332 +/- 91 microM (control) to 525 +/- 150 microM ATP (10% dextran) and 641 +/- 160 microM ATP (30% dextran). In mitoplasts the effect of dextran disappeared (control, 230 +/- 19 microM ATP; 10% dextran, 238 +/- 28 microM ATP) indicating that the outer mitochondrial membrane is a prerequisite for the modulation of the transport of adenine nucleotides into the intermembrane space by macromolecules. To investigate the effects of viscosity of dextran solutions on the diffusion of adenine nucleotides across the outer membrane, dextrans with different molecular size (20, 40 70 and 500 kDa) were used. The viscosity of the 10% solutions drastically increased with the molecular size of the dextrans used, but the effects of different dextran solutions on the kinetic constants were the same. From these results it was concluded that neither the viscosity nor the molar concentration but the content of macromolecules (mass/vol.) correlates with restrictions of diffusion into the intermembrane space of mitochondria with intact outer membranes. Assuming that a dextran concentration of 15% mimicks the intracellular oncotic pressure on mitochondria in vivo, the apparent Km(ATP) of oxidative phosphorylation within the intact cell seems to be about 50 microM ADP which is somewhat higher than the cytoplasmic free ADP concentration as reported for the intact heart.
Adenine Nucleotides↗
Gel permeation chromatography of dextrans in parenteral solutions: calibration procedure development and method validation.
We describe development and validation of a gel permeation chromatographic (GPC) method for dextrans in parenteral solutions. The GPC method was adopted from USP monographs on Dextran 40 and Dextran 70 raw materials. The method was optimized with a mobile phase flow rate of 1 mL/min and column temperature of 40 degrees C, to sharpen dextran and dextrose peaks. An easy-to-use, curve-fitting program capable of non-linear regression was developed in-house, using Microsoft Excel and its Solver add-in to successfully meet the GPC calibration requirements for dextrans and dextrose, i.e., the experimental molecular weights within 100+/-5% of the known molecular weights for dextrans and molecular weight of dextrose within 180+/-2 Da. The GPC method was validated in terms of its stability indicating nature, robustness (column temperature of 40+/-3 degrees C), accuracy (lack of effects of pH and concentration of dextrans or matrix components), and precision (repeatability and intermediate). Molecular weight distribution of dextrans were unchanged when the dextran containing test solutions were subjected to forced degradation using heat, light (daylight and UV light), extreme alkaline conditions or oxidative conditions. The method was capable of detecting changes in molecular weight distribution caused by degradation under extreme acidic conditions and heat, thereby confirming the stability indicating nature of the method. The concentration of Dextran 40 and Dextran 70 (75-125% of the nominal assay concentration), matrix components (108-111% of their nominal concentrations), and solution pH (pH 3-7 for Dextran 40 solutions and pH 4-7 for Dextran 70 solutions) did not affect the measured molecular weight distribution of Dextran 40 or Dextran 70. The method was precise with %R.S.D. of less than 1% for M (W) values of Dextran 40 or Dextran 70.
Chromatography, Gel↗
Streptococcus mutans dextransucrase: mode of interaction with high-molecular-weight dextran and role in cellular aggregation.
The interaction between Streptococcus mutans dextransucrase (EC 2.4.1.5) and high-molecular-weight dextran was studied in both the presence and absence of substrate sucrose. Equivalent weight-percent solutions of primer dextrans that differed 200-fold in molecular weight were found to be equally efficient in priming new dextran synthesis. Sodium borohydride reduction of dextran had no effect on its priming ability. These results suggest that dextran synthesis proceeds by addition of glucosyl residues to nonreducing termini of primer dextrans and that several enzyme molecules simultaneously bind to single high-molecular-weight dextran molecules. Kinetic data suggested that dextransucrase contains only one dextran binding site per enzyme molecule. The nature of the commonly observed highly aggregated state of dextransucrase was also studied. Two types of enzyme aggregates were distinguished: (i) oligomeric enzyme aggregates that formed in the absence of dextran and were dissociated by 1 M KCl; and (ii) dextran-induced enzyme aggregates that were stable to 3 M salt. Oligomeric enzyme aggregates were obtained from supernatants of fructose-grown cultures, whereas dextran-induced enzyme aggregates appeared to be present in glucose-grown cultures. The molecular weight of the smallest species of dextran-free detransucrase observed in solutions of 1 M KCl was estimated to be 40,000 by gel column chromatography. Addition of dextran to primer-dependent dextransucrase resulted in formation of complexes that were stable in CsCl density gradients and exhibited a buoyant density of 1.382 g/cm3 as compared with a buoyant density of 1.302 g/cm3 exhibited by dextransucrase. The enzyme-dextran complexes observed in CsCl density gradients contained about 25% dextran. This corresponded to 150 enzyme molecules (molecular weight, 40,000) per dextran molecule (molecular weight, 2 X 10(6)). The implication of these results to the mechanism of sucrose- and dextran-induced aggregation of S. mutans is discussed.
Binding Sites↗
Transglomerular transport of DEAE dextran in the isolated perfused kidney.
OBJECTIVE: The renal fractional clearance of [3H]DEAE dextran has been widely used to substantiate the charge selective model for renal permselectively, although there has only been one reported study on this type of clearance. This study sets out to examine the fractional clearance and glomerular processing of DEAE dextran. METHODS: Fractional clearance studies were performed using isolated perfused rat kidneys. The glomerular processing of DEAE dextran was assessed by examining the kinetics of DEAE dextran uptake in glomeruli isolated post perfusion. RESULTS: The fractional clearance of DEAE dextran used in the concentration range of 15-150 micrograms/ml in the perfusate of the isolated perfused kidney did not produce the classical in vivo facilitated transport of DEAE dextran as compared to dextran as observed by Bohrer et al. The fractional clearance curve displays retarded clearance of low molecular weight (small radii) DEAE dextran, giving the appearance of a 'flat curve'. Similar results were obtained when an oxygen free radical scavenger cocktail was included in the perfusate. These results may be due to the fact that DEAE dextran binds to the glomeruli (at an order of magnitude greater than dextran sulfate). Perfused kidneys with [3H]DEAE dextran for 1 h followed by a five minute perfusion with unlabelled DEAE dextran revealed no significant change in the glomerular levels of [3H]DEAE dextran (unlike dextran sulfate). Perfusion of rat kidneys with 15 micrograms/ml DEAE dextran produced no changes in the electron microscopical morphology of the glomerulus and no changes in the fractional clearance of dextran. CONCLUSIONS: These results do not support the glomerular charge selectivity model that involves a non-binding electrostatic interaction of the charged dextran with the fixed anion charges of the glomerular capillary wall.
Animals↗
Glutathione improves the function of porcine pulmonary grafts stored for twenty-four hours in low-potassium dextran solution.
BACKGROUND: Flush perfusion with low-potassium dextran is the standard strategy in clinical lung preservation. Despite improved outcome, endothelial cell injury and surfactant dysfunction remain a significant problem after lung transplantation. The radical scavenger glutathione has been shown to be responsible for the efficacy of Celsior solution in lung preservation. We tested the hypothesis that the addition of glutathione to low-potassium dextran might further improve graft function by ameliorating ischemia-reperfusion injury. METHODS: In 12 domestic pigs, lungs were flush preserved with either low-potassium dextran (n = 6) or low-potassium dextran supplemented by 5 mmol glutathione (n = 6). Left single lung transplantation was performed after 24-hour storage in low-potassium dextran at 8 degrees C. After 15 minutes of reperfusion the right main bronchus and pulmonary artery were crossclamped. Hemodynamic and respiratory measures were recorded in 30-minute intervals for a total observation period of 7 hours. Bronchoalveolar lavage fluid was obtained from the native lung and 2 hours after reperfusion from the graft. Bronchoalveolar lavage fluid and surfactant composition, and surfactant function analyses were performed. Neutrophil sequestration was assessed by myeloperoxidase activity assay. Tissue water content was calculated from wet/dry weight ratios at the end of the experiment. RESULTS: In the low-potassium dextran group, 2 animals died during reperfusion. After reperfusion, pulmonary vascular resistance (P = .01) and pulmonary artery pressure remained lower in the glutathione/low-potassium dextran group, which was associated with a higher cardiac output (P = .05) in this group. Also, the oxygenation index at the end of the observation period was higher in the glutathione/low-potassium dextran group compared with the low-potassium dextran group (430 +/- 130 vs 338 +/- 184, respectively; P < .05). The graft water content representing postreperfusion lung edema was not different between the 2 study groups. Alteration of surfactant was less in the glutathione/low-potassium dextran group with a significantly decreased small to large aggregate ratio (P = .03) versus low-potassium dextran group. Myeloperoxidase activity was twice as high in the low-potassium dextran group when compared with the glutathione/low-potassium dextran group (glutathione/low-potassium dextran: 134 +/- 110 mU/g vs low-potassium dextran: 274 +/- 168 mU/g, P = .07). CONCLUSION: The addition of glutathione to low-potassium dextran preservation solution reveals beneficial effects on vascular function and surfactant composition in transplanted lungs. Therefore, glutathione ameliorates ischemia-reperfusion injury in a preclinical model of lung transplantation. Future studies are needed to evaluate this promising modification in clinical lung transplantation.
Animals↗
A comparison of analytic procedures for measurement of fractional dextran clearances.
Fractional dextran clearances have been extensively used to study glomerular size selectivity. We report on an analysis of different laboratory procedures involved in measuring fractional dextran clearances. The deproteinization of plasma samples by 20% trichloroacetic acid (TCA) revealed a protein contamination of 0.2% +/- 0.3%, whereas both 5% TCA and zinc sulfate deproteinization revealed a significantly higher remaining sample protein content (2.5% +/- 0.4% and 3.4% +/- 0.1%, respectively). Only zinc sulfate revealed incomplete deproteinization of urine samples (0.6% +/- 0.2%). Dextran recovery in plasma and urine supernatants was significantly lower after 5% TCA and zinc sulfate deproteinization when compared with 20% TCA deproteinization. Gel permeation chromatography (GPC) and high-performance liquid chromatography (HPLC) showed a variance of calibration smaller than 5% over 1 year. The use of 3 different sets of standard dextrans revealed significant differences in calibration. GPC and HPLC followed by anthrone assay showed a comparable variance in dextran concentration in plasma, from 3 to 6 nm (14% to 25%), whereas the variance in urine was lower for the GPC and anthrone assay, especially from 5.4 to 6 nm (23% to 43% versus 50% to 78%). HPLC and online refractometry showed the lowest variance of dextran concentration in plasma, from 3 to 6 nm (<4%), and in urine, from 3 to 5.2 nm (<7%), whereas it showed a higher variance in urine, from 5.4 to 6 nm, in comparison with GPC and HPLC with the anthrone assay. The GPC and anthrone assay revealed higher fractional dextran clearances in comparison with the HPLC and anthrone assay in healthy subjects (3 to 5.4 nm) as well as in patients with nondiabetic proteinuria (4.2 to 5.8 nm), and lower clearances in patients from 3 to 3.4 nm. The HPLC and anthrone assay revealed higher clearances in comparison with HPLC and online refractometry in healthy subjects (3.6 to 5.4 nm) and in patients (3.6 to 5.2 nm). The GPC and anthrone assay revealed characteristic differences in fractional dextran clearances between healthy subjects and patients. The HPLC and anthrone assay showed no significant differences between both groups, whereas HPLC and online refractometry showed only an increased clearance of dextrans from 4.6 to 5.2 nm in patients. Fractional clearances of dextran 5.6 nm as estimated by all 3 dextran assays were not significantly related to the fractional immunoglobulin G clearance or the immunoglobulin-to-albumin clearance index in our patients. Quantitative and qualitative differences in fractional dextran clearances may be induced by differences in laboratory procedures. We recommend sample preparation by 20% TCA deproteinization, frequent calibration with 1 set of dextran standards with low polydispersity, size-exclusion chromatography by GPC, and dextran detection by anthrone assay for optimal measurement of fractional dextran clearances. Even with such an approach, however, the variability in the measurement remains extremely high in the important range of dextrans greater than 5 nm.
Chromatography, Gel↗
A histochemical study of the distribution of dextran 500 in human corneas during organ culture.
PURPOSE: The aim of this histochemical study was to demonstrate the absorption of dextran 500 and its distribution in the cornea after storage under standard eye bank conditions. Furthermore, an attempt was made to distinguish between the soluble and insoluble parts of dextran 500 absorbed by the cornea, in order to see how much dextran remains in the cornea after transplantation. METHODS: Forty-nine fresh and 65 organ-cultured human corneas were investigated. The corneas were cultured for 28 days in a dextran-free medium, followed by a period of 1-14 days in a medium containing 5% dextran 500. Cryosections were stained by aqueous PAS and a modified alcoholic PAS to determine the amount of dextran. RESULTS: Dextran was not found in the epithelium, stroma or endothelium of fresh human corneas. By contrast, extra- and intracellular positive staining reactions were detected in corneas following storage in a medium containing dextran. Dextran 500 absorption was relatively diffuse in the epithelium after storage in a dextran medium. Initial accumulations were found in the stroma near Bowman's and Descemet's membranes and also in the central part of the cornea, as the period of culture in the medium containing dextran lengthened. We also observed interaction between the stroma and endothelium: decreasing amounts in the endothelium were followed by an increase of same in the stroma. Intracellular deposits of dextran were detected after only one day. A much greater part of the extracellular dextran than previously described was found to be insoluble. CONCLUSIONS: As the amount of dextran in the cornea increases over a longer storage time, we conclude that the period of storage in a medium containing dextran should be limited to four days. The fact that the cornea is saturated with dextran after seven days has been shown in further studies to interfere with mitochondrial function and may also cause severe post-operative swelling of the transplant, hence leading to a longer recovery period for the patient.
Cornea↗
Preparation of benzoyl dextran and its use in aqueous two-phase systems.
The graft modification of dextran with benzoyl groups has been studied. The factors that affect the degree of substitution of benzoyl dextran were investigated. Phase diagrams for aqueous two-phase systems composed of polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran have been determined. Phase separation was also obtained in aqueous solution of two benzoyl dextran polymers with different degrees of substitution. A four-phase system was obtained with a mixture of polyethylene glycol, dextran and two kinds of benzoyl dextrans. The partitioning of methylene blue and a Procion yellow HE-3G dextran derivative were studied in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems and in systems of two benzoyl dextrans differing in degree of substitution. The proteins bovine serum albumin and glucose-6-phosphate dehydrogenase were partitioned in polyethylene glycol/benzoyl dextran aqueous two-phase systems and the effect of the degree of substitution of benzoyl dextran was studied. Chlorella pyrenoidosa, thylakoid membrane vesicles, plasma membrane vesicles and chloroplasts were partitioned in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems, and in a polyethylene glycol/dextran/benzoyl dextran four-phase system.
Cell Membrane↗
Dextran augments delayed-type hypersensitivity by interrupting one limb of the suppressor cascade.
We have studied the immunomodulatory effect of dextran on the development of delayed-type contact hypersensitivity to a hapten in mice. Administration of an optimal dose of dextran 2 hours before applying picryl chloride to abdominal skin caused a twofold rise in the level of hapten-specific DTH. A study of the kinetics of development of DTH under the influence of dextran showed that comparable levels of response could be seen 2 days earlier in treated than in untreated mice, i.e., on the third day in contrast to the fifth day after sensitization. The peak of the responses, while greater in dextran-treated mice than in normal controls, remained the same at 5 days. Adoptive transfer studies revealed that comparable levels of DTH were conferred upon recipient mice by half the number of splenic cells from dextran-treated mice than that required from normal sensitized mice. Because several suppressor mechanisms are known to down-regulate DTH, we have studied dextran's effect on the neutralization of these systems as a possible explanation for its enhancing capabilities. Detailed examination was made of dextran's effect on the two suppressor T cells, Ts1 and Ts3, that act in tandem as well as its effect on the Ts1 and macrophage that work in combination. Both systems depress the efferent limb of DTH. We have found that dextran blocks the Ts1-macrophage pathway that controls DTH. Ts1 was found to arise normally in mice pretreated with dextran. Furthermore, Ts1 from dextran-treated mice produced TsF1 normally. However, we have found that dextran interferes with the production of macrophage suppressor factor (M phi-SF). Interference was partial when dextran was introduced during the interval in which macrophages were being armed with TsF1, and it was complete when dextran was put with pre-armed macrophages before they were triggered with antigen for production of M phi-SF. On the other hand, the Ts1-Ts3 limb of suppression remained unaffected by exposure to the immunomodulator. We found Ts3 arose normally in hapten-sensitized mice that had been pretreated with dextran. In addition, Ts3 became armed with TsF1 in vitro in the presence of dextran since the cells functioned properly to suppress mature DTH effector cells. Finally, TsF3 was able to act in vitro upon DTH effector cells despite the presence of dextran.(ABSTRACT TRUNCATED AT 400 WORDS)
Adjuvants, Immunologic↗
Technetium labeling of dextran incorporating cysteamine as a ligand.
INTRODUCTION: Technetium-99m-labeled dextran is a useful imaging agent for procedures such as angiocardiography and lymphoscintigraphy. To improve the availability of 99mTc-labeled dextran, we designed a cysteamine ligand system for dextran labeling. METHODS: Cysteamine derivatized dextran was synthesized as follows. Dextran was oxidized with sodium periodate, coupled with cysteamine and reduced with sodium borohydride to provide the desired amine ligand. The cysteamine-dextran conjugate was then labeled with reduced 99mTc. Whole-body scintigraphy and biodistribution were examined following injection of the 99mTc-labeled cysteamine-conjugated dextran (99mTc-cysteamine-dextran) in ICR mice. Lymphoscintigraphy was performed after intradermal injection of 99mTc-cysteamine-dextran in SD rats. RESULTS: The cysteamine-derived dextran was easily labeled with reduced 99mTc in greater than 96% yield. 99mTc-cysteamine-dextran has a higher chelation stability against diethylenetriamine pentaacetic acid (DTPA) than the 99mTc-dextran. Axillary lymph nodes were clearly visible after intradermal injection of 99mTc-cysteamine-dextran in rats. CONCLUSION: These results suggest that 99mTc-cysteamine-dextran is available for lymphoscintigraphy. This methodology could expand the usage of 99mTc-labeled dextran, particularly for diagnostic purposes.
Animals↗
Covalent coupling of methotrexate to dextran enhances the penetration of cytotoxicity into a tissue-like matrix.
For antitumor agents introduced directly into the intracranial space, the extent of penetration into tissue, and hence the effectiveness of therapy, depends on the rate of drug elimination from the tissue. To test the hypothesis that slowly eliminated agents would penetrate further through tissues, methotrexate (MTX)-dextran conjugates were produced by covalently linking MTX to dextran through a short-lived ester bond (MTX-ester-dextran; t1/2 approximately 3 days in buffered saline) and a longer-lived amide bond (MTX-amide-dextran; t1/2 > 20 days in buffered saline). The ability of these agents to kill cells and to penetrate through tissue was evaluated using: (a) human brain tumor (H80) cells in a standard format; (b) H80 cells in a novel three-dimensional format that mimics many characteristics of intracranial tumors; and (c) 9L gliosarcoma in the rat brain. Penetration into three-dimensional tissue-like matrices was performed by suspending H80 cells in agarose gels within a hollow fiber that was permeable to MTX but not dextran and injecting MTX or MTX-dextran conjugates into one end of the fiber. The cytotoxicity of MTX-ester-dextran and MTX-amide-dextran against H80 was equivalent to unmodified MTX (50% inhibitory concentration, approximately 0.01 microgram/ml). When released from a biodegradable polyanhydride polymer matrix, MTX and MTX-dextran conjugates retained their ability to inhibit dihydrofolate reductase activity. When MTX or MTX-dextran was diffused into the three-dimensional tumor cell matrix for 10 days, cytotoxic activity penetrated > 2 cm for MTX-amide-dextran and approximately 1 cm for MTX or MTX-ester-dextran; this enhanced penetration correlated with the stability of the MTX-dextran linkage. Intracranial polymeric delivery of MTX or MTX-amide-dextran to rats with intracranial 9L gliosarcoma produced modest but significant increases in survival; conjugation of MTX to dextran appeared to shift the dose-response curve to a lower dosage.
Animals↗
Internalization and excretion of epidermal growth factor-dextran-associated radioactivity in cultured human squamous-carcinoma cells.
Certain tumor cells, such as squamous carcinomas and gliomas, can have an increased number of epidermal-growth-factor (EGF) receptors. The EGF receptors can in these cases be targets for toxic conjugates with specific binding. EGF-based toxic conjugates are potential targeting agents. We have analyzed the internalization and excretion of 125I administered in the form of 125I-EGF-dextran in squamous-carcinoma A431 cells. 125I-EGF without dextran was used for comparison. A431 cells have large numbers of EGF receptors and are capable both of recycling and of degradation of internalized receptor-ligand complexes. The binding of 125I-EGF-dextran and 125I-EGF was receptor-specific, since both ligands competed with non-radioactive EGF for binding. The amount of internalized 125I as a function of time increased continuously within 24 hr following administration of radioactivity as 125I-EGF-dextran. The time pattern was quite different when 125I-EGF without dextran was applied. In the latter case, the amount of internalized radioactivity decreased already after a few hours, probably depending on degradation of EGF. Pre-incubation of the cells with 125I-EGF-dextran or 125I-EGF and analysis of retained and released 125I activity at different times after washing showed that the 125I activity was retained for longer periods of time when EGF-dextran was used instead of EGF. About 30% of the internalized 125I activity was retained after 24 hr when EGF-dextran was used, compared with excretion of nearly all the radioactivity within 5 hr when EGF was used. In some experiments a high concentration of non-radioactive EGF, 5 micrograms/ml, was given to the cells after pre-incubation with 125I-EGF-dextran. This changed the retention and excretion patterns, so that a larger amount of 125I was excreted in the macromolecular fraction and a smaller amount of 125I activity was retained in the cells. Gel chromatography of the 125I activity released into the culture medium showed that the variations in molecular weight were larger after administration of a high concentration of non-radioactive EGF, most likely due to partial degradation of EGF-dextran. The results regarding excretion are in conformity with a model of competition between recycling of EGF-dextran-EGF-receptor complexes and "trapping" of EGF-dextran in the lysosomes followed by slow degradation. For targeting purposes, it is worth noting that the radioactivity administered in the form of 125I-EGF-dextran had a longer retention time than when 125I-EGF without dextran was used, and that the retention and excretion rates could be modified by post-treatment with the receptor ligand itself.
Binding, Competitive↗