Effects of transgene expression level per cell in mice livers on induction of transgene-specific immune responses after hydrodynamic gene transfer.

We previously showed that high and sustained transgene expression of antigenic proteins induced transgene-specific immune responses. In the present study, a detailed relationship between the level of transgene expression per cell and immune response after hydrodynamic gene transfer was investigated. Cypridina luciferase (cLuc), a secretory antigenic reporter protein, was selected as a model antigen, and pROSA-cLuc, a plasmid expressing cLuc, was constructed. A fixed dose (30 µg) of pROSA-cLuc was delivered to mice by a single hydrodynamic injection or three injections at 24-h intervals because the number of cells transfected with plasmids is dependent on the number of hydrodynamic injections. Serum cLuc activity, an indicator of the total amount of cLuc transgene expression, was almost equal between these two groups. In contrast, the high-dose single injection induced higher levels of cLuc-specific humoral and cellular immune responses than the three low-dose injections. Moreover, the serum cLuc activity of the high-dose single injection group began to decline ~10 days after injection, whereas the activity remained constant in the three low-dose injection group. These results indicate that it is preferable to reduce the level of transgene expression per cell to avoid induction of the transgene-specific immune response after hydrodynamic gene transfer.

An elevated preoperative serum carbohydrate antigen 19-9 level is a significant predictor for peritoneal dissemination and poor survival in colorectal cancer.

AIM: Many studies support the role of carcinoembryonic antigen (CEA) as a strong indicator of the status of colorectal cancer patients, but evidence for carbohydrate antigen 19-9 (CA19-9) is poor. For this reason the study aimed to evaluate the prognostic value of preoperative serum CA19-9 levels in colorectal cancer patients. METHOD: In all, 1190 colorectal cancer patients were included in this study, of whom 955 underwent a potentially curative resection. These were analysed for recurrence and survival. The 255 patients with Stage IV disease were analysed for metastatic status. RESULTS: Patients with an elevated preoperative CEA with Stage II and III disease showed a significantly poorer survival than those with normal levels. In contrast patients with elevated preoperative CA19-9 levels were associated with a significantly poorer survival irrespective of disease stage. Of the 255 patients with Stage IV disease, 92 (39.1%) had peritoneal dissemination at laparotomy observed more frequently in patients with an elevated CA19-9 (47.9%). Of the 955 patients having a curative resection, 18 (1.9%) developed peritoneal dissemination. In multivariate analysis, an elevated preoperative CA19-9 level was a significant risk factor for postoperative peritoneal recurrence. CONCLUSION: After curative surgery for colorectal cancer the preoperative CA19-9 level is a strong prognostic indicator of higher risk of peritoneal dissemination.

Effects of upregulated indoleamine 2, 3-dioxygenase 1 by interferon γ gene transfer on interferon γ-mediated antitumor activity.

Interferon γ (IFN-γ), an anticancer agent, is a strong inducer of indoleamine 2,3-dioxygenase 1 (IDO1), which is a tryptophan-metabolizing enzyme involved in the induction of tumor immune tolerance. In this study, we investigated the IDO1 expression in organs after IFN-γ gene transfer to mice. IFN-γ gene transfer greatly increased the mRNA expression of IDO1 in many tissues with the highest in the liver. This upregulation was associated with reduced L-tryptophan levels and increased L-kynurenine levels in serum, indicating that IFN-γ gene transfer increased the IDO activity. Then, Lewis lung carcinoma (LLC) tumor-bearing wild-type and IDO1-knockout (IDO1 KO) mice were used to investigate the effects of IDO1 on the antitumor activity of IFN-γ. IFN-γ gene transfer significantly retarded the tumor growth in both strains without any significant difference in tumor size between the two groups. By contrast, the IDO1 activity was increased only in the wild-type mice by IFN-γ gene transfer, suggesting that cells other than LLC cells, such as tumor stromal cells, are the major contributors of IDO1 expression in LLC tumor. Taken together, these results imply that IFN-γ gene transfer mediated IDO1 upregulation in cells other than LLC cells has hardly any effect on the antitumor activity of IFN-γ.

Histological and molecular characterization of the femoral attachment of the human ligamentum capitis femoris.

The ligamentum capitis femoris (LCF) has increased in clinical significance through the development of hip arthroscopy. The histological pathologies and molecular composition of the femoral attachment of the LCF and the degeneration caused by LCF disruption were investigated in the human hip joint. Twenty-four LCFs were retrieved at surgery for femoral neck fracture (age range: 63-87 years). In the "intact" (i.e., intact throughout its length, n = 12) group, the attachment consisted of rich fibrocartilage. Fibrocartilage cells were present in the midsubstance. In contrast, the construction of the attachment in the "disrupted" (i.e., ligament no longer attached to the femoral head, n = 12) group had disappeared. The attachment in the disrupted group was not labeled for type II collagen or aggrecan, while that in the intact group was labeled for types I, II and III collagen, chondroitin 4-sulfate, chondroitin 6-sulfate, aggrecan, and versican. The percentage of single-stranded DNA-positive chondrocytes was significantly higher in the disrupted group than in the intact group. We conclude that the femoral attachment of the LCF has a characteristic fibrocartilaginous structure that is likely to adjust to the mechanical load, and suggest that its degeneration is advanced by disruption and should be regarded as a clinical pathology.

Regulation of immunological balance by sustained interferon-γ gene transfer for acute phase of atopic dermatitis in mice.

Interferon (IFN)-γ, a potent T helper 1 (Th1) cell cytokine, is suggested to suppress Th2 cell responses. Here, we aimed to investigate whether pCpG-Muγ, a plasmid continuously expressing murine IFN-γ, is an effective treatment of atopic dermatitis, a Th2-dominant skin disease. Nishiki-nezumi Cinnamon/Nagoya (NC/Nga) atopic mice with early dermatitis were transfected with pCpG-Muγ by a hydrodynamic tail vein injection at a dose of 0.05 or 0.2 pmol per mouse. The skin lesions improved only in mice receiving the high dose of pCpG-Muγ. IFN-γ gene transfer resulted in a high mRNA expression of IFN-γ and interleukin (IL)-12 and regulatory T cell (Treg) related cytokines, such as IL-10 and transforming growth factor-ß, in the spleen, whereas it reduced the IL-4 mRNA expression, and serum levels of immunoglobulin (Ig) G1 and IgE. In addition, the gene transfer markedly inhibited the epidermal thickening, infiltration of inflammatory cells into the skin, the occurrence of dry skin and pruritus. No exacerbating effect on the Th1-mediated contact dermatitis was observed after IFN-γ gene transfer. Taken together, these results indicate that sustained IFN-γ gene transfer induced polarized Th1 immunity under Th2-dominant conditions in NC/Nga mice, leading to an improvement in the symptoms of acute atopic dermatitis without adverse side effects.

Inhibition of nuclear delivery of plasmid DNA and transcription by interferon γ: hurdles to be overcome for sustained gene therapy.

Sustained expression of murine interferon (IFN)-γ (Muγ) was found to be effective in preventing tumor metastasis and atopic dermatitis in mouse models. However, our preliminary experiments suggested that the time-dependent decrease in the Muγ expression was not compensated for by repeated injections of Muγ-expressing plasmid. To identify the mechanism underlying this observation, a reporter plasmid was hydrodynamically injected into mice and the levels of the plasmid, mRNA and reporter protein were measured in mice receiving a pre- or co-administration of Muγ-expressing plasmid. Co-injection of Muγ-expressing plasmid had no significant effects on transgene expression from the reporter plasmid. In contrast, pre-injection of Muγ-expressing plasmid greatly inhibited the expression of the reporter protein. Moreover, pre-injection of Muγ-expressing plasmid also reduced the amount of the reporter plasmid in the nuclear fraction of mouse liver to < 10%, and that of reporter mRNA to < 1%. The degree of reduction in the expression of reporter protein was comparable with the reduction in mRNA. These results indicate that the difficulty in regaining the expression level of IFN-γ is due to the impaired delivery of plasmid to the nucleus and to the suppression of transcription from the plasmid.

Short disease-free interval is a significant risk factor for intrapulmonary recurrence after resection of pulmonary metastases in colorectal cancer.

OBJECTIVE: Surgical resection has been the first choice of treatment for resectable pulmonary metastases from colorectal cancer. However, early intrapulmonary recurrence is often observed and appropriate assessment of prognostic factors is controversial. The aim of this study was to define the factors affecting survival and intrapulmonary recurrence after surgical treatment of metastatic pulmonary tumours from colorectal cancer. METHOD: A retrospective analysis was performed of 56 consecutive patients who underwent pulmonary resection for colorectal metastases with a focus on prognostic factors and risk factors for intrapulmonary recurrence. RESULTS: The overall 5-year survival rate was 48.2%. In a univariate analysis, a short disease-free interval (DFI), multiple pulmonary metastases and abnormal prethoracotomy carcinoembryonic antigen (CEA) levels were poor prognostic factors. In a multivariate analysis, a short DFI and abnormal prethoracotomy CEA levels were independent prognostic factors. Twenty-two (39.3%) of the 56 patients had recurrences in the remnant lung after resection for pulmonary metastases, and 8 (36.4%) of these 22 patients underwent repeated pulmonary resections. The median DFI between first and second lung metastasis was 13 months. Univariate analyses revealed that multiple and bilateral lung metastases were risk factors for intrapulmonary recurrence. There was also a strong correlation between the DFI for intrapulmonary recurrence after the first pulmonary resection and the DFI for first pulmonary metastases. CONCLUSIONS: A short DFI was a risk factor for early tumour recurrence after resection for pulmonary metastases. The DFI might reflect oncological characteristics such as the tumour doubling time in colorectal cancer.

Spectrocolorimetric evaluation of human articular cartilage.

OBJECTIVE: The aim of this study was to investigate whether human articular cartilage can be quantitatively evaluated using a spectrocolorimeter. MATERIALS AND METHODS: Human articular cartilage specimens were analyzed using a spectrocolorimeter after macroscopic evaluation using the Outerbridge classification. The cartilage characteristics were examined, the L*, a*, b* colorimetric system, the spectral reflectance distribution and the yellow/red spectral reflectance percentage (Y/R SRP). Moreover, the results of the spectrocolorimetric evaluation were compared with the histological score described by Mankin et al. RESULTS: There were significant differences among the macroscopic four grades in the L*, a* and Y/R SRP values. The spectral reflectance distribution of grade 1 cartilage exhibited a gradual increase in the spectral reflectance ratio as the wavelength increased. The spectral reflectance curves of grades 2 to 4 cartilage had dips at a wavelength of around 580 nm. Across all the measured wavelengths, there were lower reflectance ratios with the progression of cartilage degeneration. Moreover, correlations were observed between the spectrocolorimetric values and Mankin score. A strong relationship existed between Mankin score and he Y/R SRP values. CONCLUSIONS: The present study is the first to clearly demonstrate the relationship between spectrocolorimetric evaluation and the degeneration of human articular cartilage. The spectrocolorimeter may be a new quantitative evaluation tool for articular cartilage with clinical potential.

Total ankle arthroplasty incorporating a total talar prosthesis: a comparative study against the standard total ankle arthroplasty.

AIMS: Total ankle arthroplasty (TAA) has become the most reliable surgical solution for patients with end-stage arthritis of the ankle. Aseptic loosening of the talar component is the most common complication. A custom-made artificial talus can be used as the talar component in a combined TAA for patients with poor bone stock of the talus. The purpose of this study was to investigate the functional and clinical outcomes of combined TAA. PATIENTS AND METHODS: Ten patients (two men, eight women; ten ankles) treated using a combined TAA between 2009 and 2013 were matched for age, gender, and length of follow-up with 12 patients (one man, 11 women; 12 ankles) who underwent a standard TAA. All had end-stage arthritis of the ankle. The combined TAA features a tibial component of the TNK ankle (Kyocera, Kyoto, Japan) and an alumina ceramic artificial talus (Kyocera), designed using individualized CT data. The mean age at the time of surgery in the combined TAA and standard TAA groups was 71 years (61 to 82) and 75 years (62 to 82), respectively. The mean follow-up was 58 months (43 to 81) and 64 months (48 to 88), respectively. The outcome was assessed using the Japanese Society for Surgery of the Foot (JSSF) ankle-hindfoot scale, the Ankle Osteoarthritis Scale (AOS), and the Self-Administered Foot Evaluation Questionnaire (SAFE-Q). RESULTS: The mean preoperative JSSF score of the combined TAA and standard TAA groups was 44 (sd 11) and 49 (sd 10), respectively. The mean postoperative JSSF scores were 89 (sd 6.1) and 72 (sd 15), respectively. The mean postoperative JSSF score of the combined TAA group was significantly higher (p = 0.0034). The mean preoperative AOS scores for pain and function in the combined TAA and standard TAA groups were 5.8 (sd 3.3) and 5.5 (sd 3.1), and 8.6 (sd 1.3), and 7.1 (sd 2.9), respectively. The mean postoperative AOS scores of pain and function were 2.5 (sd 2.5) and 2.2 (sd 1.9), and 2.5 (sd 3.3) and 3.4 (sd 2.9), respectively. There were no significant differences between the two groups in terms of postoperative AOS scores. The mean postoperative SAFE-Q scores were: for pain, 76 (sd 23) and 70 (sd 23); for physical function, 66 (sd 25) and 55 (sd 27); for social function, 73 (sd 35) and 62 (sd 34); for shoe-related, 73 (sd 19) and 65 (sd 26); and for general health, 78 (sd 28) and 67 (sd 29), respectively. There were no significant differences between the two groups in terms of postoperative SAFE-Q scores. CONCLUSION: Combined TAA resulted in better clinical results than standard TAA. Cite this article: Bone Joint J 2019;101-B:443-446.

Inhibition of tumor cell growth in the liver by RNA interference-mediated suppression of HIF-1alpha expression in tumor cells and hepatocytes.

Hypoxia-inducible factor-1 (HIF-1) is a ubiquitously expressed oxygen-regulated transcription factor composed of alpha and beta subunits. HIF-1 activates transcription of various genes including those involved in metastatic tumor growth. In the present study, HIF-1alpha expression in tumor-bearing mouse liver was examined after inoculation of tumor cells into portal vein. We found that tumor-bearing liver showed greatly increased HIF-1alpha expression. Plasmid DNA (pDNA) expressing short hairpin RNA targeting HIF-1alpha (pshHIF-1alpha) was effective in suppressing protein expression of HIF-1alpha in vitro. Intravenous injection of pshHIF-1alpha by hydrodynamics-based procedure reduced the HIF-1alpha protein expression in both normal and tumor cells and tumor cell number in the liver. Pre-injection of pshHIF-1alpha to mice, by which pDNA was delivered only to liver cells, not to tumor cells, was also effective in reducing the number of tumor cells inoculated 3 days after pDNA injection. These findings indicate that HIF-1alpha expression is increased in normal liver cells as well as tumor cells, and HIF-1alpha expression plays an important role in tumor progression. Use of the RNA interference (RNAi) of HIF-1 is an effective strategy for inhibiting tumor cell growth, and both tumor and normal cells can be the target for RNAi-based anticancer treatment.Gene Therapy (2008) 15, 572-582; doi:10.1038/sj.gt.3303103; published online 14 February 2008.

Low tibial osteotomy for varus-type osteoarthritis of the ankle.

In this retrospective study we have assessed the results of low tibial valgus osteotomy for varus-type osteoarthritis of the ankle and its indications. We performed an opening wedge osteotomy in 25 women (26 ankles). The mean follow-up was for eight years and three months (2 years 3 months to 17 years 11 months). Of the 26 ankles, 19 showed excellent or good clinical results. Their mean scores for pain, walking, and activities of daily living were significantly improved but there was no change in the range of movement. In the ankles which were classified radiologically as stage 2 according to our own grading system, with narrowing of the medial joint space, and in 11 as stage 3a, with obliteration of the joint space at the medial malleolus only, the joint space recovered. In contrast, such recovery was seen in only two of 12 ankles classified as stage 3b, with obliteration of the joint space advancing to the upper surface of the dome of the talus. Low tibial osteotomy is indicated for varus-type osteoarthritis of stage 2 or stage 3a.

Enhanced lymphatic delivery of mitomycin C conjugated with dextran.

Absorption and lymphatic transfer of a polymeric prodrug of mitomycin C (MMC), mitomycin C-dextran conjugate (MMC-D), following i.m. injection were studied in rats in order to assess the feasibility of a macromolecular prodrug as a lymphotropic delivery system. Three types of MMC-D, conjugates with dextran with molecular weights of 10,000, 70,000 and 500,000, were synthesized, and the disposition of MMC was determined by bioassay. Following i.m. injection of MMC-D, MMC was retained at the injection site for a long period in a conjugated form while MMC administered as a free form disappeared rapidly. The disappearance was markedly influenced by the size of carrier dextran, because the remaining amount of MMC increased with an increase of molecular size. The lymphatic uptake of the drug was evaluated by determining the concentration in the regional lymph nodes and thoracic lymph fluid. In contrast to a slight lymphatic uptake following i.v. and i.m. injection of free MMC, MMC-D exhibited remarkable accumulation in the regional lymph nodes after i.m. injection which persisted up to 48 hr. MMC-D (Mr 10,000) appeared in the thoracic lymph as both the conjugated and the free form. Larger MMC-D gave a persistent supply of free MMC in thoracic lymph, suggesting that it was accumulated in the lymph node and supplying MMC continuously. These MMC-Ds suppressed the lymph node metastases introduced by a s.c. inoculation of L1210 leukemia cells. The usefulness of MMC-D as a lymphotropic delivery system for preventing lymphatic metastasis of cancer was suggested.

Cellular interaction and in vitro antitumor activity of mitomycin C-dextran conjugate.

Cellular interaction and in vitro antitumor activity of a polymeric prodrug of mitomycin C (MMC), mitomycin C-dextran conjugate (MMC-D), were studied in relation to its physicochemical characteristics. MMC-D with cationic and anionic charges were examined. The cationic MMC-D was synthesized using a spacer, epsilon-aminocaproic acid and dextrans with molecular weights of 10,000, 70,000, or 500,000 [MMC(C6)Dcat]. The anionic MMC-D was synthesized using 6-bromohexanoic acid as a spacer and dextran with a molecular weight of 70,000 [MMC(C6)Dan]. Cellular adsorption was determined by measuring the concentration of the drug in the medium after incubation with three tumor cell lines, Ehrlich ascites carcinoma, L1210 leukemia, and AH66 ascites hepatoma cells. MMC(C6)Dcat was adsorbed more readily than MMC or MMC(C6)Dan on the tumor cell surface by an electrostatic force. The percentage of adsorption remained almost constant during the course of incubation and no significant difference was observed between the incubation at 4 degrees C and that at 37 degrees C. A corresponding increase in the amounts of MMC(C6)Dcat adsorbed on with higher molecular weights was noted, which conformed to Langmuir's adsorption isotherm. In vitro antitumor activity was evaluated using L1210 and EAC cell culture systems and human tumor colony forming assay. MMC(C6)Dcat showed growth inhibition essentially equal to that of MMC in continuous drug exposure experiments. In a 1-h drug exposure experiment, MMC(C6)Dcat with a molecular weight of 70,000 or 500,000 was more active than MMC, and a good correlation was observed between the effects of MMC(C6)Dcat and the extent of cellular interaction. These results show that cellular interaction played an important role in the manifestation of the antitumor effect of MMC-D and that these phenomena are governed by the physicochemical properties of macromolecular prodrugs, such as electric charge and molecular weight.

Intratumoral pharmacokinetics and in vivo gene expression of naked plasmid DNA and its cationic liposome complexes after direct gene transfer.

The pharmacokinetic properties and gene expression of naked plasmid DNA and its cationic liposome complexes were studied after direct intratumoral injection. Using a Walker 256 tissue-isolated tumor perfusion system, we quantified the recovery of naked plasmid DNA and cationic liposome complexes in the tumor, leakage from the tumor surface, and the venous outflow after intratumoral injection. Approximately 50% of naked plasmid DNA had been eliminated from the tumor 2 h after injection, whereas more than 90% of plasmid DNA was retained in the tumor when it was complexed with cationic liposomes. However, the distribution of these complexes in the tumor was restricted to the tissue surrounding the injection site. Pharmacokinetic analysis of the venous outflow profiles suggested that the rate-limiting process that determines the retention of plasmid DNA in the tumor is transferred from the injection site in the tumor tissue and that complexation with cationic liposomes may retard this process. Furthermore, we examined the gene expression of chloramphenicol acetyltransferase DNA constructs (naked pCMV-CAT) and the corresponding cationic liposome [3-beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol] complexes. A similar level of gene expression was observed in vivo after direct intratumoral injection of naked DNA and its cationic liposome complexes. In both cases, a great variation was observed between tumors, and localization of gene-transduced cells in the tumor tissue was limited to the area in the vicinity of the injection site. Thus, these pharmacokinetic and gene expression studies have demonstrated that cationic liposomes can enhance the retention of injected DNA in the tumor model, whereas cationic liposome complex does not necessarily improve gene expression because of its poor dissemination in this tumor. The present study also suggested that there is a need to control the behavior of the injected naked plasmid DNA and its cationic liposome complexes to ensure better distribution throughout the tumor.

Disposition of anticancer drugs after bolus arterial administration in a tissue-isolated tumor perfusion system.

Disposition characteristics of various anticancer drugs in a tissue-isolated tumor preparation were studied in Walker 256 carcinosarcoma-bearing rats using an in situ single-pass vascular perfusion technique. Three anticancer drugs, 5-fluorouracil, mitomycin C, and Adriamycin, and two lipophilic prodrugs of mitomycin C were tested in the tumor preparation perfused with Tyrode's solution containing 4.7% bovine serum albumin. After bolus arterial injection of test drugs, their outflow concentration-time curves were analyzed based on statistical moment theory. In each tumor preparation, the injection of drug was paired with that of vascular reference substance, Evans' blue-labeled bovine serum albumin, and disposition parameters of the drug were corrected with those of vascular reference substance. From the mean transit time values of vascular reference substance, the average vascular volume of the tumor preparation was calculated to be 0.063 ml/g, which decreased with tumor growth. All drugs showed significant extraction by the tumor tissue, depending on their physicochemical properties. Distribution volumes of tested drugs were from 1.53 to 3.33 times larger than the vascular volume. Calculated intrinsic clearance values for the protein-unbound fractions increased as the lipophilicity of the drug increased. The potential increase in tumor uptake was observed in lipophilic prodrugs of mitomycin C. The present experimental system is thus suggested to be useful for analyzing drug disposition in tumor tissue.

Disposition characteristics of mitomycin C-dextran conjugate in normal and tumor-bearing muscles of rabbits.

Disposition characteristics of the macromolecular prodrug of mitomycin C (MMC), mitomycin C-dextran conjugate (MMC-D), in normal and tumor (VX2 carcinoma)-bearing rabbit thigh muscles were studied using the in situ vascular perfusion technique. Three types of cationic MMC-D (MMC-Dcat) and two types of anionic MMC-D (MMC-Dan) with different carrier molecular weights were used. After bolus arterial injection in normal muscles, 83-96% of injected MMC-D was recovered in the venous outflow regardless of the carrier size or charge, whereas less than 60% of MMC was recovered in the same system. By applying statistical moment analysis to the outflow pattern of these drugs, pharmacokinetic parameters representing their disposition characteristics were obtained. Smaller intrinsic clearance (CLint) and distribution volume (V) were noted for MMC-D than for MMC, indicating low extravascular diffusion of MMC-D. In the tumor-bearing muscle, blood contamination from other parts of the body increased and a shortage of flow recovery due to the neovascularization of the tumors occurred. The disposition parameters of MMC-Dcat with a molecular weight of 500,000 (T-500) indicated some tissue distribution and sequestration in the tumor preparation. After constant infusion of [14C]MMC-D (T-500) for 4 h, tissue radioactivity concentrations were determined in various tissues. A higher radioactivity was observed in the viable region of the tumor and the lymph node compared with the normal muscle tissue and the necrotic region of the tumors. 131I-Labeled human serum albumin also gave similar results. In conclusion, higher tumor localization of antitumor agents may be made possible by the application of macromolecular prodrugs.

Disposition characteristics of macromolecules in the perfused tissue-isolated tumor preparation.

Disposition characteristics of model macromolecules with different physicochemical characteristics and macromolecular prodrugs of mitomycin C, namely mitomycin C-dextran conjugates, were studied in tissue-isolated tumor preparations of Walker 256 carcinoma with the use of a single-pass vascular perfusion technique. In constant infusion experiments, all radiolabeled macromolecules accumulated in the tumor tissue, but the degree and pattern of distribution greatly varied, depending on their electric charges. Positively charged macromolecules were markedly accumulated compared with those that were neutral or negatively charged. In addition, their concentrations were significantly higher in viable than in necrotic regions, while neutral and negative compounds were distributed in necrotic rather than in viable regions. Pharmacokinetic analysis of tissue concentration-time courses of positively charged diethylaminoethyl and neutral dextrans showed that their movement occurred by convective fluid flow, and that high tissue accumulation of positively charged macromolecules could be explained by strong binding due to electrostatic interaction. For neutral and anionic macromolecules with negligible affinity to the tissue, it was suggested that the final concentration gradient between the viable and necrotic regions was decided by their tissue fluid content. Thus, the present study revealed the basic disposition characteristics of macromolecules in tumor tissue relative to their physicochemical properties.

Pharmacokinetics and disposition characteristics of recombinant decorin after intravenous injection into mice.

The pharmacokinetics and disposition characteristics of recombinant decorin after intravenous administration were investigated in mice. Following bolus injection of 111In-labeled decorin at doses of 0.02 and 0.1 mg/kg, radioactivity rapidly disappeared from the circulation and approximately 70% of the dose accumulated in liver within 10 min. 111In-labeled decorin was preferentially localized in hepatic nonparenchymal cells. At a higher dose of 1 mg/kg, clearance from the circulation and hepatic uptake of [111In]decorin were slower than at lower doses. Both the accumulation in other tissues and urinary excretion of [111In]decorin were 5% or less. Pharmacokinetic analysis demonstrated that hepatic uptake clearance was large and accounted almost completely for total body clearance; in addition the clearance values decreased as the dose increased, suggesting that the hepatic uptake of decorin is mediated by a specific mechanism which becomes saturated at higher doses. In competitive inhibition experiments, hepatic uptake of 111In-labeled decorin was partially inhibited (about 20-30%) by several sulfated glycans such as glycosaminoglycans and dextran sulfate and by mannosylated bovine serum albumin (BSA), mannan and mannose to a lesser extent (about 10%). On the other hand, polyinosinic acid, polycytidylic acid and succinylated BSA were ineffective, suggesting that the scavenger receptor for polyanions in the liver is not involved in the hepatic uptake of decorin. A basic protein, protamine, and a ligand of the apoE receptor, lactoferrin, also had no effect. Taken together, the present results have demonstrated that recombinant decorin is rapidly eliminated from the blood circulation through extensive uptake by the liver, primarily by the nonparenchymal cells, following systemic administration. The sugar structure and mannose residue in decorin have also been suggested to play an important role in the hepatic uptake of decorin. These findings provide useful information for the development of decorin as a therapeutic agent.

Liver uptake and hepato-biliary transfer of galactosylated proteins in rats are determined by the extent of galactosylation.

The effect of molecular mass and surface density of galactose residues on hepatic uptake and subsequent biliary excretion of galactosylated proteins was investigated in rats. Several proteins with different molecular weights (15-70 kDa) and different numbers of galactose units were synthesized and radiolabeled with 111In. Galactosylated proteins were administered i.v. to anaesthetized rats and samples of plasma and bile were collected for 3 h. Liver was harvested at the end of the experiments and the radioactivity of all samples was measured. Galactosylated proteins accumulated primarily in the liver and 2-10% of the administered dose appeared in the bile, mainly in undegraded form. The hepatic uptake clearance (Cl liver) and biliary excretion rate constant (kbile) of galactosylated proteins were calculated. No direct effect of molecular weight was observed, however, on increasing the galactose density, Cl liver increased from about 4 to 400 ml/h whereas kbile gradually decreased from about 0.057 to 0.007 (h-1). In conclusion, both hepatic uptake and biliary excretion of galactosylated proteins were found to be affected by the extent of galactosylation.

Augmented inhibitory effect of superoxide dismutase on superoxide anion release from macrophages by direct cationization.

Superoxide dismutase (SOD) was modified into cationized form (Cat-SOD) in order to enhance its pharmacological efficacy based on an electrostatic interaction. The inhibitory effect of Cat-SOD on superoxide anion release from inflammatory macrophages and its cellular interaction were studied in vitro. Cat-SOD exhibited an excellent inhibitory effect on superoxide anion release from the macrophages, and this effect surpassed those of native SOD and SOD modified with mannose (Man-SOD) which is taken up via mannose receptor-mediated endocytosis by macrophages. In the presence of colchicine, a microtubule-disruptive agent, the inhibitory effect of Cat-SOD was slightly impaired, whereas the effect of Man-SOD completely disappeared. The intracellular localization of fluorescein isothiocyanate-labeled SOD, Cat-SOD and Man-SOD observed by confocal laser microscopy supported the difference in their abilities to eliminate superoxide anions. The different sensitivities of Cat-SOD and Man-SOD to colchicine were also confirmed by the confocal laser microscopic images, suggesting their distinct intracellular trafficking pathways in the macrophages. In conclusion, Cat-SOD is desirable for its pharmacological activity, which is probably the result of its ability to be delivered to the vicinity of NADPH-oxidase which locates in the cell membrane and generates superoxide anions.