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PubMed2006

Hydrolytic and enzymatic degradation of nanoparticles based on amphiphilic poly(gamma-glutamic acid)-graft-L-phenylalanine copolymers.

Amphiphilic graft copolymers consisting of poly(gamma-glutamic acid) (gamma-PGA) as the hydrophilic backbone and L-phenylalanine ethylester (L-PAE) as the hydrophobic side chain were synthesized by grafting L-PAE to gamma-PGA. The nanoparticles were prepared by a precipitation method, and about 200 nm-sized nanoparticles were obtained due to their amphiphilic properties. The hydrolytic and enzymatic degradation of these gamma-PGA nanoparticles was studied by gel permeation chromatography (GPC), scanning electron microscopy (SEM), dynamic light scattering (DLS) and (1)H NMR measurements. The hydrolysis ratio of gamma-PGA and these hydrophobic derivatives was found to decrease upon increasing the hydrophobicity of the gamma-PGA derivates. The pH had an effect on the hydrolytic degradation of the polymer. The hydrolysis of the polymer could be accelerated by alkaline conditions. The degradation of the gamma-PGA backbone by gamma-glutamyl transpeptidase (gamma-GTP) resulted in a dramatic change in nanoparticle morphology. With increasing time, the gamma-PGA nanoparticles began to decrease in size and finally disappeared completely. Moreover, the gamma-PGA nanoparticles were degraded by four different enzymes (Pronase E, protease, cathepsin B and lipase) with different degradation patterns. The enzymatic degradation of the nanoparticles occurred via the hydrolysis of gamma-PGA as the main chain and L-PAE as the side chain. In the case of the enzymatic degradation of gamma-PGA nanoparticles with Pronase E, the size of the nanoparticles increased during the initial degradation stage and decreased gradually when the degradation time was extended. Nanoparticles composed of biodegradable amphiphilic gamma-PGA with reactive function groups can undergo further modification and are expected to have a variety of potential pharmaceutical and biomedical applications, such as drug and vaccine carriers.

Guanosine Triphosphate↗
PubMed1998

Complete regression of well-established tumors using a novel water-soluble poly(L-glutamic acid)-paclitaxel conjugate.

Despite an intensive search, few water-soluble paclitaxel derivatives have been shown to have a therapeutic index superior to paclitaxel itself. We now report a water-soluble poly(L-glutamic acid)-paclitaxel conjugate (PG-TXL) that produces striking antitumor effects with diminished toxicity. A single i.v. injection of PG-TXL at its maximum tolerated dose (defined as that dose that produces a maximum 12-15% body weight loss within 2 weeks after a single i.v. injection) equivalent to 60 mg of paclitaxel/kg and at even a lower dose equivalent to 40 mg of paclitaxel/kg resulted in the disappearance of an established implanted 13762F mammary adenocarcinoma (mean size, 2000 mm3) in rats. (An equivalent dose of PG-TXL is the amount of conjugate that contains the stated amount of paclitaxel.) Similarly, mice bearing syngeneic OCA-1 ovarian carcinoma (mean size, 500 mm3) were tumor-free within 2 weeks after a single i.v. injection of the conjugate at a dose equivalent to 160 mg of paclitaxel/kg. The conjugate has little if any intrinsic tubulin polymerization activity in vitro and is >20 times less potent in supporting the growth of a paclitaxel-dependent CHO mutant cell line. PG-TXL has a prolonged half-life in plasma and greater uptake in tumor as compared with paclitaxel. Furthermore, only a small amount of total radioactivity from PG-[3H]TXL was recovered as free [3H]paclitaxel in either the plasma or the tumor tissue within 144 h after drug injection. Histological studies of tumor tissues obtained from mice treated with PG-TXL show fewer apoptotic cells but more extensive tumor necrosis as compared with paclitaxel treatment. These data suggest that in addition to its role as a carrier for selective delivery of paclitaxel to the tumor, PG-TXL exerts distinct pharmacological actions of its own that may contribute to its remarkable antitumor efficacy.

Animals↗
PubMed2001

Effectiveness of water soluble poly(L-glutamic acid)-camptothecin conjugate against resistant human lung cancer xenografted in nude mice.

A camptothecin (CPT) formulation that can be easily administered, is less toxic, and has greater antitumor effect is needed. In this study, a water-soluble CPT derivative was obtained by direct coupling of CPT to poly(L-glutamic acid) (PG) through the C20(S)-hydroxyl group. CPT was released from the resulting conjugate, PG-CPT, in phosphate-buffered saline with a zero-order kinetics in the initial 50 days. The release rates were 0.623% per day, 1.081% per day, and 1.396% per day at pH 5.3, 7.4, and 9.0, respectively. In vitro, PG-CPT was less potent in inhibiting cell growth than was free CPT in all human tumor cell lines tested. However, PG-CPT showed better antitumor activity and tolerability than did CPT in vivo. When H322 human lung tumor cells were inoculated subcutaneously in nude mice, PG-CPT delayed the growth of these well-established tumors with an absolute growth delay of 32 days when given as 4 doses with 4-day intervals between injections at an equivalent CPT dose of 40 mg/kg. When H322 cells were inoculated intratracheally in nude mice, 5 doses of intravenous injection of PG-CPT at an equivalent CPT dose of 10 mg/kg on days 4, 8, 12, 16, and 20 after inoculation significantly prolonged the median survival of treated mice, averaging 1.8-fold that of untreated mice (p=0.01). Increasing the dose of PG-CPT to an equivalent CPT dose of 40 mg/kg per injection administered in 4 doses on days 4, 8, 12, and 16 prolonged the median survival of treated mice by 4-fold (p=0.0008). Significantly, mice with intratracheally inoculated H322 tumors were resistant to both CPT and cisplatin treatments. These studies demonstrated that PG may be used as an effective solubilizing carrier for CPT and that PG-CPT may have potential application in the treatment of lung cancer.

Animals↗
PubMed1983

In vitro formation of polyglutamyl derivatives of methotrexate and 7-hydroxymethotrexate in human lymphoblastic leukemia cells.

The intracellular synthesis of polyglutamyl derivatives of both methotrexate (4-amino-N-10-methylpteroylglutamic acid) and 7-hydroxymethotrexate, the primary plasma metabolite of methotrexate in humans, was evaluated in a methotrexate-sensitive, acute lymphoblastic leukemia cell line, MOLT 4. These studies were performed using a highly specific ion-pairing high-pressure liquid chromatography method which permits the simultaneous determination of methotrexate, 7-hydroxymethotrexate, and their corresponding polyglutamyl derivatives. When MOLT 4 cells were exposed to 1 microM methotrexate, the monoglutamate attained a steady state after 30 min, and polyglutamyl derivatives having from one to 4 additional glutamyl residues were observed over 4 hr. Four additional metabolites were also detected upon incubation with 1 microM 7-hydroxymethotrexate. On the basis of the retention times for these compounds relative to methotrexate polyglutamyl standards and since these metabolites reverted to 7-hydroxymethotrexate upon treatment with a preparation of hog kidney conjugase, they were identified as polyglutamyl derivatives of 7-hydroxymethotrexate. The identification of 7-hydroxymethotrexate polyglutamyl derivatives in vitro raises the possibility of an important new dimension in the pharmacological action of methotrexate. We investigated the effect of extracellular 7-hydroxymethotrexate on net methotrexate uptake and metabolism when cells were exposed simultaneously to 1 microM [3H]-methotrexate and unlabeled 7-hydroxymethotrexate. A decrease in the levels of both intracellular methotrexate and the corresponding polyglutamyl derivatives was noted for cells treated with 1 or 10 microM 7-hydroxymethotrexate. However, no appreciable effect of 7-hydroxymethotrexate on the amount of polyglutamyl derivatives formed relative to the total intracellular antifolate was noted. These studies show that in MOLT 4 cells (a) both methotrexate and 7-hydroxymethotrexate are rapidly converted to polyglutamyl derivatives, and (b) 7-hydroxymethotrexate interferes with net methotrexate accumulation and metabolism when present simultaneously in the extracellular medium. These results, moreover, suggest a potential role for 7-hydroxymethotrexate in modulating the biochemical effects of methotrexate in vivo.

Cell Line↗
PubMed2006

Biodegradable cystamine spacer facilitates the clearance of Gd(III) chelates in poly(glutamic acid) Gd-DO3A conjugates for contrast-enhanced MR imaging.

Poly(L-glutamic acid) (PGA)-cystamine-[gadolinium (Gd)-DO3A] was prepared in high yield with a high Gd-DO3A conjugation efficiency. Approximately 55% of the carboxylic groups in PGA were loaded with Gd-DO3A via cystamine as the spacer. Cystamine can be readily cleaved by endogenous thiols to release the Gd(III) chelates from the conjugate facilitating Gd(III) excretion after the magnetic resonance imaging (MRI). The contrast-enhanced MRI with PGA-cystamine-(Gd-DO3A) was investigated in mice bearing MDA-MB-231 breast carcinoma xenografts. PGA-1,6-hexanediamine-(Gd-DO3A), a paramagnetic polymer conjugate of a nondegradable spacer, was used as a control. Both conjugates resulted in similar contrast enhancement in the heart, vasculature, liver and kidneys in the first hour post injection. More substantial signal intensity reduction was observed for PGA-cystamine-(Gd-DO3A) in these organs than PGA-1,6-hexanediamine-(Gd-DO3A) due to release of the Gd chelates from PGA-cystamine-(Gd-DO3A) after the cleavage of the disulfide spacer by the endogenous thiols. Both conjugates resulted in similar tumor enhancement with approximately 70% increased signal intensity in the tumor periphery and 10-40% increased signal intensity in tumor interstitium. No cross-reaction was observed between PGA-cystamine-(Gd-DO3A) and human serum albumin, a plasma protein containing a cysteine residue. PGA-cystamine-(Gd-DO3A) resulted in significantly lower Gd(III) tissue retention than PGA-1,6-hexanediamine-(Gd-DO3A) 10 days after the injection in the mice (P<.05). The conjugation of Gd(III) chelates to biomedical copolymers via the degradable disulfide spacer resulted in significant contrast enhancement in the blood pool and tumor tissue but minimal long-term Gd(III) tissue retention.

Absorbable Implants↗
PubMed2004

Phase II study of CT-2103 in patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal carcinoma.

PURPOSE: To evaluate the safety and efficacy of CT-2103, a novel conjugate of paclitaxel and poly-L-glutamic acid, in heavily pretreated patients with recurrent ovarian, fallopian tube, or peritoneal cancer. PATIENTS AND METHODS: Ninety-nine patients with measurable disease received intravenous CT-2103 at 175 mg/m2 of conjugated paclitaxel over 10 minutes every 3 weeks without routine premedications. Platinum-sensitive (n = 42) and platinum-refractory or platinum-resistant patients (n = 57) were enrolled. Thirty-nine patients (39%) had received one or two prior regimens, and 60 patients (61%) had received between three and 12 regimens. RESULTS: In 99 patients, the median number of cycles was three (range, one to 14 cycles). The response rate (RR) for all patients was 10% (10 of 99 patients), with median time to disease progression (TTP) of 2 months. The RR (partial response) in platinum-sensitive and platinum-resistant patients was 14% (six of 42 patients) and 7% (four of 57 patients), respectively. In patients with only one or two prior regimens, the RR in platinum-sensitive and platinum-resistant patients was 28% (five of 18 patients) and 10% (two of 21 patients), with a median TTP of 4 and 2 months, respectively. Grade 2 (15 patients) or 3 (15 patients) neuropathy was reported in 30 patients (30%). Grade 2 hypersensitivity occurred in eight patients (8%) who were subsequently treated with premedications; one patient had grade 3 hypersensitivity and was removed. Grade 2 alopecia was absent. CONCLUSION: CT-2103 is active in patients with recurrent ovarian cancer. Neurotoxicity in these heavily pretreated patients was more frequent than predicted from phase I trials. Further study to define toxicity and efficacy in patients with less prior therapy is ongoing.

Adult↗
PubMed2005

Phase I study of CT-2103, a polymer-conjugated paclitaxel, and carboplatin in patients with advanced solid tumors.

PURPOSE: The primary objective of this study was to determine the maximum tolerated dose (MTD) of CT-2103 (poly L-glutamic acid-paclitaxel) in combination with carboplatin in patients with histologically proven solid tumors that were either refractory to conventional treatment or for which no conventional therapy was available. PATIENTS AND METHODS: Twenty-two adult patients with advanced solid tumors were treated in this dose escalation study. Patients were treated every 21 days with CT-2103 at 175, 210, 225, or 250 mg/m2 (doses expressed as units of conjugated-paclitaxel) via 10-20 minute intravenous (IV) infusion, followed one hour later with carboplatin administered at AUC 5 or 6 via 30 minute IV infusion. No prophylaxis for hypersensitivity was administered with initial treatment. Doses were administered every 21 days until progressive disease or dose-limiting toxicity (DLT) was observed. Toxicity was evaluated using NCI Common Toxicity Criteria for Adverse Events v2.0 (CTCAE v2.0); response to treatment was evaluated using Response Criteria in Solid Tumors (RECIST). RESULTS: The MTD was determined to be 225 mg/m2. DLTs observed at 250 mg/m2 were neutropenia and thrombocytopenia. No hypersensitivity reactions were observed. Three patients achieved partial responses (PR). Fifteen patients received at least 3 cycles of treatment without observation of progressive disease. Median survival time was 5.9 months. Patients that demonstrated partial responses were all ovarian cancer patients that had previously failed paclitaxel therapy. The only Grade 4, nonhematologic treatment-related toxicity was febrile neutropenia. Grade 4 neutropenia (9 patients) was observed across all dose groups. Twelve patients developed thrombocytopenia (Grade 3/4) while receiving combination therapy. All had resolution of thrombocytopenia with discontinuation of carboplatin, suggesting that carboplatin, and not CT-2103, contributed mainly to platelet toxicity. CONCLUSION: CT-2103 administered at 225 mg/m2 every 21 days in combination with carboplatin administered at AUC 6 has a manageable safety profile in patients with solid tumors; further clinical investigation is recommended, especially in patients with ovarian or non-small cell lung cancer.

Adult↗
PubMed2003

Poly(gamma-D-glutamic acid) protein conjugates induce IgG antibodies in mice to the capsule of Bacillus anthracis: a potential addition to the anthrax vaccine.

Both the protective antigen (PA) and the poly(gamma-d-glutamic acid) capsule (gamma dPGA) are essential for the virulence of Bacillus anthracis. A critical level of vaccine-induced IgG anti-PA confers immunity to anthrax, but there is no information about the protective action of IgG anti-gamma dPGA. Because the number of spores presented by bioterrorists might be greater than encountered in nature, we sought to induce capsular antibodies to expand the immunity conferred by available anthrax vaccines. The nonimmunogenic gamma dPGA or corresponding synthetic peptides were bound to BSA, recombinant B. anthracis PA (rPA), or recombinant Pseudomonas aeruginosa exotoxin A (rEPA). To identify the optimal construct, conjugates of B. anthracis gamma dPGA, Bacillus pumilus gamma dLPGA, and peptides of varying lengths (5-, 10-, or 20-mers), of the d or l configuration with active groups at the N or C termini, were bound at 5-32 mol per protein. The conjugates were characterized by physico-chemical and immunological assays, including GLC-MS and matrix-assisted laser desorption ionization time-of-flight spectrometry, and immunogenicity in 5- to 6-week-old mice. IgG anti-gamma dPGA and antiprotein were measured by ELISA. The highest levels of IgG anti-gamma dPGA were elicited by decamers of gamma dPGA at 10 -20 mol per protein bound to the N- or C-terminal end. High IgG anti-gamma dPGA levels were elicited by two injections of 2.5 microg of gamma dPGA per mouse, whereas three injections were needed to achieve high levels of protein antibodies. rPA was the most effective carrier. Anti-gamma dPGA induced opsonophagocytic killing of B. anthracis tox-, cap+. gamma dPGA conjugates may enhance the protection conferred by PA alone. gamma dPGA-rPA conjugates induced both anti-PA and anti-gamma dPGA.

Animals↗
PubMedDate not supplied

A heavy metal biotrap for wastewater remediation using poly-gamma-glutamic acid.

Poly-gamma-glutamic acid (gamma-PGA) obtained from Bacillus licheniformis ATCC 9945 was evaluated as a potential biosorbent material for use in the removal of heavy metals from aqueous solution. Copper (Cu(2+)) was chosen as the model heavy metal used in these studies since it is extensively used by electroplating and other industries, has been the model for many other similar studies, and can be easily assayed through a number of convenient methods. Cu(2+)-gamma-PGA binding parameters under varying conditions of pH, temperature, ionic strength, and in the presence of other heavy metal ions were determined for the purified biopolymer using a specially designed dialysis apparatus. Applying the Langmuir adsorption isotherm model showed that gamma-PGA had a copper capacity approaching 77.9 mg/g and a binding constant of 32 mg/L (0.5 mM) at pH 4.0 and 25 degrees C. Cu(2+)-gamma-PGA adsorption was relatively temperature independent between 7 and 40 degrees C, while an increase in ionic strength led to a decrease in metal ion binding. Cd(2+) and Zn(2+) ions compete with Cu(2+) for binding sites on the gamma-PGA biopolymer. Metal uptake by gamma-PGA was further tested using a tangential flow filtration apparatus in a diafiltration mode in which metal was continually processed through a dilute solution of gamma-PGA without allowing for equilibrium to be established. The circulating polymer solution was able to complex metal as well as successfully prevent passage of unbound copper ions present in solution through the membrane. Using 500 mL of a 0.2% gamma-PGA solution, up to 97% of a 50 mg/L copper sulfate solution processed at a flow rate of 115 mL/min was retained by the polymer. For a 10 mg/L solution of Cu(2+) as copper sulfate, filtrate concentrations of Cu(2+) never rose above 0.6 mg/L while processing 2.5 L of dilute copper sulfate.

Cations↗
PubMed1983

Chemical inhibitors of phagosome-lysosome fusion in cultured macrophages also inhibit saltatory lysosomal movements. A combined microscopic and computer study.

The effects on lysosomal movements produced by the weak base ammonium chloride and by a representative polyanion poly-D-glutamic acid (PGA), previously reported to inhibit phagosome-lysosome (P-L) fusion, have been studied in cultured mouse macrophages using direct visual phase-contrast microscopy, a previously described (1, 3, 7) fluorescence assay of fusion, and computer analysis techniques. Treatment of the macrophages with 5-10 mM NH4Cl for 0.5-2 h or with 100 micrograms PGA/ml for 5 d caused a striking inhibition of saltatory lysosomal movements, as well as the expected inhibition of P-L fusion. Two other anionic fusion inhibitors tested, dextran sulphate and suramin, inhibited movements similarly. Removal of the NH4Cl from the cell medium reversed the lysosomal stasis and restored P-L fusion. Computer analyses of changes in lysosomal positions in treated and untreated macrophages during 2, 10, and 30-s intervals, using data from photomicrographs, computer graphics, and quantitative nearest-neighbour techniques developed for this purpose, supported the qualitative visual observation of the inhibition of lysosomal movements by the fusion inhibitors NH4Cl and PGA. Over the chosen intervals, from 80 to 96% of the lysosomes could be paired within 1 micron of each other in the NH4Cl- and PGA-treated cells in comparison with 50-70% in normal cells. The differences between the drug-treated and normal cells were highly significant. In an analogous system, the lysosomal stasis induced by hypertonic sucrose was examined and it was observed that P-L fusion too was inhibited. Both effects were reversible. We conclude that inhibition of P-L fusion and of lysosomal movement are associated. We suggest a causal relationship between these changes, namely, that the lysosomotropic inhibitors of fusion under study produce their effects largely, though perhaps not exclusively, by reducing saltatory lysosomal motion and consequently periphagosomal assembly, rather than directly and independently on P-L contact or on the fusion process itself. The possibility is raised that microtubules may be involved in the effector mechanism of these modulations.

Acridine Orange↗
PubMed2004

Antitumor activity of hydrophilic Paclitaxel copolymer prodrug using locoregional delivery in human orthotopic non-small cell lung cancer xenograft models.

Paclitaxel (Taxol) has demonstrated clinical activity in non-small-cell lung cancer (NSCLC), but its use has not led to marked improvements in survival. This ineffectiveness can in part be attributed to inadequate delivery of effective drug levels to the lung via systemic administration and to drug resistance mechanisms. Locoregional drug administration and the use of drug copolymers are possible approaches to address these issues. In this study, we evaluated the activity of a poly(L-glutamic acid)-paclitaxel (PGA-TXL) formulation administered by intratracheal injection to mice bearing orthotopic human NSCLC tumors (H460, H358). H460 cells were found to be sensitive to paclitaxel and PGA-TXL in vitro, in a time- and concentration-dependent manner. In preliminary acute toxicity studies, PGA-TXL administered by intratracheal injection was found to be much less toxic than paclitaxel, as anticipated. Mice into which H460 cells had been implanted by intratracheal injection were given single-dose intratracheal treatments with paclitaxel (1.2 or 2.4 mg/kg) or with PGA-TXL (15 mg/kg, paclitaxel equivalents) 1 week later. When the mice were sacrificed at up to 65 days after tumor implantation, they were evaluated grossly for tumor at bronchial, neck, and lung sites. Control mice had tumors in 60% of all three sites, and all of the control mice had tumors in at least one site. The low- and high-dose Taxol groups had fewer incidences at these three sites (27-33%) and 60-80% of these mice had tumors in at least one site. The PGA-TXL mice displayed a low (13%) incidence at these sites, and only 40% had detectable tumors. In a subsequent survival study with the intratracheal H358 model, control mice had a mean life span of 95 days, whereas both the intratracheal Taxol (2.5 mg/kg, every 7th day for three doses) and the intratracheal PGA-TXL (20 mg/kg, paclitaxel equivalents, every 7th day for three doses) groups had improved survival (mean life spans: 133.5 and 136.5 days, respectively). In pilot studies intended to compare the feasibility of the development of paclitaxel aerosols suitable for clinical application, based either on Cremophor solutions or on PGA backbones, only the latter gave acceptable particle size distributions and flow rates. These results encourage the development and application of Cremophor-free copolymer formulations of paclitaxel for locoregional treatment (e.g., as aerosol) of endobronchial malignant diseases.

Animals↗
PubMed1999

Antitumor activity of poly(L-glutamic acid)-paclitaxel on syngeneic and xenografted tumors.

Poly(L-glutamic acid)-paclitaxel (PG-TXL) is a new water-soluble paclitaxel derivative that has shown remarkable antitumor activity against both ovarian and breast tumors. The purpose of this study was to test whether the antitumor efficacy of PG-TXL depends on tumor type, as is the case for paclitaxel, and to test whether paclitaxel-resistant tumors could be responsive to PG-TXL. We evaluated the therapeutic activity of PG-TXL against four syngeneic murine tumors (MCa-4, MCa-35, HCa-1, and FSa-II) inoculated i.m. into C3Hf/Kam mice, a human SKOV3ip1 ovarian tumor injected i.p. into nude mice, and a human MDA-MB-435Lung2 breast tumor grown in the mammary fat pad of nude mice. Two paclitaxel-responsive murine tumors, MCa-4 and MCa-35, showed significant growth delay with PG-TXL given as a single i.v. injection at its maximum tolerated dose of 160 mg of equivalent paclitaxel/kg or even at a lower dose of 120 mg of equivalent paclitaxel/kg. The other two murine tumors, HCa-1 and FSa-II, did not respond particularly well to either of the two agents, although significant growth delay was observed for both tumors with PG-TXL. In mice with SKOV3ip1 tumors, the median survival times for mice treated with PG alone and PG-TXL at doses of 60 or 120 mg of equivalent paclitaxel/kg were 43, 61, and 75 days, respectively; no survival difference was found between paclitaxel-treated and Cremophor vehicle-treated mice. In mice with MDA-MB-435Lung2 tumor, PG-TXL at a dose of 120 mg of equivalent paclitaxel/kg produced regression of the tumor in 50% of the animals, and in the remaining mice, micrometastases in the lung were found only in 25% of the animals. In comparison, treatment with paclitaxel at 60 mg/kg did not result in tumor regression, and the rate of lung metastases was 42%. These results clearly demonstrate that PG-TXL has significant therapeutic activity against breast and ovarian tumors tested in this study. Future studies to elucidate the mechanism of action of PG-TXL and to assess its clinical applications are warranted.

Animals↗
PubMed2000

Biodistribution of paclitaxel and poly(L-glutamic acid)-paclitaxel conjugate in mice with ovarian OCa-1 tumor.

PURPOSE: Poly(L-glutamic acid)-paclitaxel (PG-TXL) is a water-soluble paclitaxel (TXL) conjugate made by conjugating TXL to poly(L-glutamic acid) via ester bonds. In preclinical studies, PG-TXL has shown significant antitumor activity against a variety of solid tumors. To elucidate the relationship between tissue distribution and antitumor efficacy of PG-TXL, we studied and compared the biodistribution of PG-TXL and TXL. METHODS: Female C3Hf/Kam mice bearing syngeneic ovarian OCa-1 tumors were injected with either [3H]TXL or PG-[3H]TXL at an equivalent TXL dose of 20 mg/kg. Mice were killed at various times after drug injection, and samples of blood, spleen, liver, kidney, lung, heart, muscle, brain, fat, and tumor were removed and the radioactivity counted. In addition, concentrations of free [3H]TXL released from PG-[3H]TXL in the spleen, liver, kidney, and tumor were analyzed by using high-performance liquid chromatography (HPLC). Whole-body autoradiographs of mice killed 1 day and 6 days after administration of PG-[3H]TXL were obtained to study the intratumoral distribution of PG-TXL. RESULTS: When [3H]TXL was conjugated to polymer, the biodistribution pattern of PG-[3H]TXL differed from that of [3H]TXL. Based on area under the tissue concentration-time curve (AUC) values, tumor exposure to [3H]TXL was five times greater when administered as PG-TXL than as TXL formulated in Cremophor EL/alcohol vehicle. Furthermore, concentrations of free paclitaxel released from PG-[3H]TXL remained relatively constant in tumor tissue, being 489, 949 and 552 ng/g tumor tissue at 5, 48 and 144 h after dosing, respectively. Autoradiographic images of mice injected with PG-[3H]TXL revealed that radioactivity was primarily located in the periphery of the tumor on day 1 after drug administration and was homogeneously diffused into the center of the tumor by day 6. Over the 144-h study period, [3H]TXL concentrations, predominantly as the inactive conjugate, were higher in tissues with a more abundant reticular endothelial system (i.e. liver, kidney, spleen, lung) than in tissues with less abundant or lacking RE systems (i.e. muscle, fat, brain). Both [3H]TXL and PG-[3H]TXL were excreted primarily through the hepatobiliary route, with a small fraction of each drug (5% and 8.7%, respectively) excreted into the urine within 48 h. CONCLUSIONS: This study indicates that the distribution to tumor tissue was enhanced when [3H]TXL was administered as a macromolecular conjugate, and that free TXL was released and maintained within the tumor for a prolonged period. Thus, the antitumor activity of PG-TXL observed in preclinical studies may be attributed in part to enhanced tumor uptake of PG-TXL.

Animals↗
PubMed1998

Glutamylation of centriole and cytoplasmic tubulin in proliferating non-neuronal cells.

We have examined the distribution of glutamylated tubulin in non-neuronal cell lines. A major part of centriole tubulin is highly modified on both the alpha- and beta-tubulin subunits, whereas a minor part of the cytoplasmic tubulin is slightly modified, on the beta-tubulin only. Furthermore, we observed that tubulin glutamylation varies during the cell cycle: an increase occurs during mitosis on both centriole and spindle microtubules. In the spindle, this increase appears more obvious on the pole-to-pole and kinetochore microtubules than on the astral microtubules. The cellular pattern and the temporal variation of this post-translational modification contrast with other previously described tubulin modifications. The functional significance of this distribution is discussed.

Animals↗
PubMed1999

Polyglutamylation of atlantic cod tubulin: immunochemical localization and possible role in pigment granule transport.

In higher organisms, there is a large variety of tubulin isoforms, due to multiple tubulin genes and extensive post-translational modification. The properties of microtubules may be modulated by their tubulin isoform composition. Polyglutamylation is a post-translational modification that is thought to influence binding of both structural microtubule associated proteins (MAPs) and mechano-chemical motors to tubulin. The present study investigates the role of tubulin polyglutamylation in a vesicle transporting system, cod (Gadus morhua) melanophores. We did this by microinjecting an antibody against polyglutamylated tubulin into these cells. To put our results into perspective, and to be able to judge their universal application, we characterized cod tubulin polyglutamylation by Western blotting technique, and compared it to what is known from mammals. We found high levels of polyglutamylation in tissues and cell types whose functions are highly dependent on interactions between microtubules and motor proteins. Microinjection of the anti-polyglutamylation antibody GT335 into cultured melanophores interfered with pigment granule dispersion, while dynein-dependent aggregation was unaffected. Additional experiments showed that GT335-injected cells were able to aggregate pigment even when actin filaments were depolymerized, indicating that the maintained ability of pigment aggregation in these cells was indeed microtubule-based and did not depend upon actin filaments. The results indicate that dynein and the kinesin-like dispersing motor protein in cod melanophores bind to tubulin on slightly different sites, and perhaps depend differentially on polyglutamylation for their interaction with microtubules. The binding site of the dispersing motor may bind directly to the polyglutamate chain, or more closely than dynein.

Animals↗
PubMed1996

Difference in adhesion and proliferation of fibroblast between Langmuir-Blodgett films and cast surfaces of poly (gamma-benzyL L-glutamate)/poly(ethylene oxide) diblock copolymer.

Block copolymers consisting of poly (gamma-benzyl L-glutamate) and poly (ethylene oxide) as the hydrophobic and hydrophilic components, respectively were prepared. Cell attachment onto the surfaces of block copolymers fabricated either as well-defined ordered Langmuir-Blodgett (LB) films or solvent cast microphase-separated structures was studied. On ordered LB surfaces, adherent fibroblasts were larger than on the microphase-separated cast surfaces within 15 min. The difference in cell adhesion between LB films and cast surfaces increased with increasing PEO contents in block copolymer. Adherent cells increased with an increase in surface pressure of LB films in any polymer. Phase-contrast microphotographs of adherent cells showed rapid and extensive morphologic changes associated with the LB surface as compared to cast film surfaces. The number of cells grown on the LB surface is greater than that on the cast film.

3T3 Cells↗
PubMed2000

Distribution of polyglutamylated tubulin in the flagellar apparatus of green flagellates.

Polyglutamylation is a widely distributed posttranslational modification of tubulin that can be demonstrated either by biochemical analysis or by the use of specific antibodies like GT335. Western blotting using GT335 demonstrated that polyglutamylated tubulin is enriched in isolated basal apparatus of Spermatozopsis similis. Single- and double-labeling experiments, using indirect immunofluorescence and immunogold electron microscopy of isolated cytoskeletons of S. similis and Chlamydomonas reinhardtii, revealed that polyglutamylated tubulin was predominately present in the basal bodies and the proximal part of the axonemes. Using immunogold labeling of whole mounts of Spermatozopsis cytoskeletons, we obtained evidence for a predominant occurrence of polyglutamylated tubulin in the B-tubule of the axonemal doublets. Polyglutamylation occurs early during premitotic basal body assembly in S. similis, whereas the probasal bodies of Chlamydomonas, which are present through interphase, showed a reduced staining with GT335 indicating that polyglutamylation is involved in basal body maturation. During flagella regeneration of C. reinhardtii, polyglutamylation preceded detyrosination and became visible shortly after the onset of flagellar regeneration. In C. reinhardtii and S. similis polyglutamylated tubulin was absent or highly reduced in the flagellar transition region, a specialized part of the flagellum linking the basal body to the axoneme. Furthermore, the transition region and the neighboring part of the axoneme showed reduced staining with L3, an antibody directed against detyrosinated tubulin. The results indicate that differences in the modification pattern can occur in a confined area of individual microtubules. The deficiency of polyglutamylated and detyrosinated tubulin in the transition region could have functional implications for flagellar turnover or excision.

Animals↗