Showing papers on "In vivo published in 1995"

A Theoretical Basis for a Biopharmaceutic Drug Classification: The Correlation of in Vitro Drug Product Dissolution and in Vivo Bioavailability

Abstract: A biopharmaceutics drug classification scheme for correlating in vitro drug product dissolution and in vivo bioavailability is proposed based on recognizing that drug dissolution and gastrointestinal permeability are the fundamental parameters controlling rate and extent of drug absorption. This analysis uses a transport model and human permeability results for estimating in vivo drug absorption to illustrate the primary importance of solubility and permeability on drug absorption. The fundamental parameters which define oral drug absorption in humans resulting from this analysis are discussed and used as a basis for this classification scheme. These Biopharmaceutic Drug Classes are defined as: Case 1. High solubility-high permeability drugs, Case 2. Low solubility-high permeability drugs, Case 3. High solubility-low permeability drugs, and Case 4. Low solubility-low permeability drugs. Based on this classification scheme, suggestions are made for setting standards for in vitro drug dissolution testing methodology which will correlate with the in vivo process. This methodology must be based on the physiological and physical chemical properties controlling drug absorption. This analysis points out conditions under which no in vitro-in vivo correlation may be expected e.g. rapidly dissolving low permeability drugs. Furthermore, it is suggested for example that for very rapidly dissolving high solubility drugs, e.g. 85% dissolution in less than 15 minutes, a simple one point dissolution test, is all that may be needed to insure bioavailability. For slowly dissolving drugs a dissolution profile is required with multiple time points in systems which would include low pH, physiological pH, and surfactants and the in vitro conditions should mimic the in vivo processes. This classification scheme provides a basis for establishing in vitro-in vivo correlations and for estimating the absorption of drugs based on the fundamental dissolution and permeability properties of physiologic importance.

Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection

Abstract: Treatment of infected patients with ABT-538, an inhibitor of the protease of human immunodeficiency virus type 1 (HIV-1), causes plasma HIV-1 levels to decrease exponentially (mean half-life, 2.1 +/- 0.4 days) and CD4 lymphocyte counts to rise substantially. Minimum estimates of HIV-1 production and clearance and of CD4 lymphocyte turnover indicate that replication of HIV-1 in vivo is continuous and highly productive, driving the rapid turnover of CD4 lymphocytes.

Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53

Abstract: The gene p53 encodes a transcriptional activator of genes involved in growth arrest, DNA repair and apoptosis. Loss of p53 function contributes to tumour development in vivo. The transcriptional activation function of p53 is inactivated by interaction with the mdm2 gene product. Amplification of mdm2 has been observed in 36% of human sarcomas, indicating that it may represent an alternative mechanism of preventing p53 function in tumour development. To study mdm2 function in vivo, we generated an mdm2 null allele by homologous recombination. Mdm2 null mice are not viable, and further analysis revealed embryonic lethality around implantation. To examine the importance of the interaction of MDM2 with p53 in vivo, we crossed mice heterozygous for mdm2 and p53 and obtained progeny homozygous for both p53 and mdm2 null alleles. Rescue of the mdm2-/- lethality in a p53 null background suggests that a critical in vivo function of MDM2 is the negative regulation of p53 activity.

Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo.

Abstract: In a number of experimental systems in which lymphocyte depletion was induced by apoptosis-inducing manipulations, no apoptotic morphology and ladder-type DNA fragmentation were detected among freshly isolated peripheral lymphocytes ex vivo. Here we report that one alteration that can be detected among splenocytes stimulated with lymphocyte-depleting doses of dexamethasone (DEX) in vivo is a reduced uptake of 3,3'dihexyloxacarbocyanine iodide (DiOC6[3]), a fluorochrome which incorporates into cells dependent upon their mitochondrial transmembrane potential (delta psi m). In contrast, ex vivo isolated splenocytes still lacked established signs of programmed cell death (PCD):DNA degradation into high or low molecular weight fragments, ultrastructural changes of chromatin arrangement and endoplasmatic reticulum, loss in viability, or accumulation of intracellular peroxides. Moreover, no changes in cell membrane potential could be detected. A reduced delta psi m has been observed in response to different agents inducing lymphoid cell depletion in vivo (superantigen and glucocorticoids [GC]), in mature T and B lymphocytes, as well as their precursors. DEX treatment in vivo, followed by cytofluorometric purification of viable delta psi mlow splenic T cells ex vivo, revealed that this fraction of cells is irreversibly committed to undergoing DNA fragmentation. Immediately after purification neither delta psi mlow, nor delta psi mhigh cells, exhibit detectable DNA fragmentation. However, after short-term culture (37 degrees C, 1 h) delta psi mlow cells show endonucleolysis, followed by cytolysis several hours later. Incubation of delta psi mlow cells in the presence of excess amount of the GC receptor antagonist RU38486 (which displaces DEX from the GC receptor), cytokines that inhibit DEX-induced cell death, or cycloheximide fails to prevent cytolysis. The antioxidant, N-acetylcysteine, as well as linomide, an agent that effectively inhibits DEX or superantigen-induced lymphocyte depletion in vivo, also stabilize the DiOC6(3) uptake. In contrast, the endonuclease inhibitor, aurintricarboxylic acid acts at later stages of apoptosis and only retards the transition from the viable delta psi mlow to the nonviable fraction. Altogether, these data suggest a sequence of PCD-associated events in which a reduction in delta psi m constitutes an obligate irreversible step of ongoing lymphocyte death, preceding other alterations of cellular physiology, and thus allowing for the ex vivo assessment of PCD.

Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase.

Abstract: The level of genetic variation of human immunodeficiency virus type 1 (HIV-1), a member of the lentivirus genus of the Retroviridae family, is high relative to that of retroviruses in some other genera. The high error rates of purified HIV-1 reverse transcriptase in cell-free systems suggest an explanation for this high genetic variation. To test whether the in vivo rate of mutation during reverse transcription of HIV-1 is as high as predicted by cell-free studies, and therefore higher than that rates of mutation of retroviruses in other genera, we developed an in vivo assay for detecting forward mutations in HIV-1, using the lacZ alpha peptide gene as a reporter for mutations. This system allows the rates and types of mutations that occur during a single cycle of replication to be studied. We found that the forward mutation rate for HIV-1 was 3.4 x 10(-5) mutations per bp per cycle. Base substitution mutations predominated; G-to-A transition mutations were the most common base substitution. The in vivo mutation rates for HIV-1 are three and seven times higher than those previously reported for two other retroviruses, spleen necrosis virus and bovine leukemia virus, respectively. In contrast, our calculated in vivo mutation rate for HIV-1 is about 20-fold lower than the error rate of purified HIV-1 reverse transcriptase, with the same target sequence. This finding indicates that HIV-1 reverse transcription in vivo is not as error prone as predicted from the fidelity of purified reverse transcriptase in cell-free studies. Our data suggest that the fidelity of purified HIV-1 reverse transcriptase may not accurately reflect the level of genetic variation in a natural infection.

Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model.

Abstract: The epidermal growth factor receptor (EGFR) is a protein tyrosine kinase expressed on many types of tumor cells, including breast, ovarian, bladder, head and neck, and prostatic carcinoma. There seems to be an association between up-regulation of the EGFR and poor clinical prognosis for a number of human cancers. The 225 antibody is a highly specific murine monoclonal antibody that binds specifically to the human EGFR with an affinity equal to its ligand, competes with the ligand for binding, and blocks activation of the receptor tyrosine kinase. In addition, 225 has been shown to inhibit the growth of human tumor xenografts in athymic nude mice. The 225 antibody has recently been chimerized with human IgG1 in its constant region to increase its clinical utility by decreasing the potential for generation of human antimouse antibodies in recipients. This report compares the biological effects of 225 and its chimeric counterpart (designated C225) against established A431 tumor xenografts in nude mice. The results of these experiments indicated that C225 was more effective than 225 in inhibiting tumor growth in this model. In addition, many of the animals treated with C225 were tumor free at the end of each treatment protocol. It was determined that the dissociation constant of C225 was about 5-fold lower than 225. This suggested that the increased capacity of C225 to compete with ligand for binding to the EGFR was responsible for its enhanced in vivo antitumor effect.

Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo

Abstract: The metabolism of glucagon-like peptide-1 (GLP-1) has not been studied in detail, but it is known to be rapidly cleared from the circulation. Measurement by RIA is hampered by the fact that most antisera are side-viewing or C-terminally directed, and recognize both intact GLP-1 and biologically inactive. N-terminally truncated fragments. Using high pressure liquid chromatography in combination with RIAs, methodology allowing specific determination of both intact GLP-1 and its metabolites was developed. Human plasma was shown to degrade GLP-1-(7-36)amide, forming an N-terminally truncated peptide with a t1/2 of 20.4 +/- 1.4 min at 37 C (n = 6). This was unaffected by EDTA or aprotinin. Inhibitors of dipeptidyl peptidase-IV or low temperature (4 C) completely prevented formation of the metabolite, which was confirmed to be GLP-1-(9-36)amide by mass spectrometry and sequence analysis. High pressure liquid chromatography revealed the concentration of GLP-1-(9-36)amide to be 53.5 +/- 13.7% of the concentration of endogenous intact GLP-1 in the fasted state, which increased to 130.8 +/- 10.0% (P < 0.01; n = 6) 1 h postprandially. Metabolism at the C-terminus was not observed. This study suggests that dipeptidyl peptidase-IV is the primary mechanism for GLP-1 degradation in human plasma in vitro and may have a role in inactivating the peptide in vivo.

Human interferon-inducible protein 10 is a potent inhibitor of angiogenesis in vivo.

Abstract: Human interferon-inducible protein 10 (IP-10), a member of the alpha chemokine family, inhibits bone marrow colony formation, has antitumor activity in vivo, is chemoattractant for human monocytes and T cells, and promotes T cell adhesion to endothelial cells. Here we report that IP-10 is a potent inhibitor of angiogenesis in vivo. IP-10 profoundly inhibited basic fibroblast growth factor-induced neovascularization of Matrigel (prepared by H. K. Kleinman) injected subcutaneously into athymic mice. In addition, IP-10, in a dose-dependent fashion, suppressed endothelial cell differentiation into tubular capillary structures in vitro. IP-10 had no effect on endothelial cell growth, attachment, and migration as assayed in vitro. These results document an important biological property of IP-10 and raise the possibility that IP-10 may participate in the regulation of angiogenesis during inflammation and tumorigenesis.

Photonic detection of bacterial pathogens in living hosts

Abstract: The study of pathogenic processes is often limited to ex vivo assays and cell-culture correlates. A greater understanding of infectious diseases would be facilitated by in vivo analyses. Therefore, we have developed a method for detecting bacterial pathogens in a living host and used this method to evaluate disease processes for strains of Salmonella typhimurlum that differ in their virulence for mice. Three strains of Salmonella were marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescent Salmonella allowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive. We observed patterns of bioluminescence that suggested the caecum may play a pivotal role in Salmonella pathogenesis. In vivo efficacy of an antibiotic was monitored using this optical method. This study demonstrates that real time non-invasive analyses of pathogenic events and pharmacological monitoring can be performed in vivo.

Requirement of Mip-1α for an Inflammatory Response to Viral Infection

Abstract: Macrophage inflammatory protein-1 alpha (MIP-1 alpha) is a chemokine that has pro-inflammatory and stem cell inhibitory activities in vitro. Its biologic role in vivo was examined in mice in which the gene encoding MIP-1 alpha had been disrupted. Homozygous MIP-1 alpha mutant (-/-) mice were resistant to Coxsackievirus-induced myocarditis seen in infected wild-type (+/+) mice. Influenza virus-infected -/- mice had reduced pneumonitis and delayed clearance of the virus compared with infected +/+ mice. The -/- mice had no overt hematopoietic abnormalities. These results demonstrate that MIP-1 alpha is an important mediator of virus-induced inflammation in vivo.

Comparison of In Vivo Tissue Temperature Profile and Lesion Geometry for Radiofrequency Ablation With a Saline-Irrigated Electrode Versus Temperature Control in a Canine Thigh Muscle Preparation

Abstract: Background It is thought that only a thin layer of tissue adjacent to the electrode is heated directly by electrical current (resistive heating) during radiofrequency ablation. Most of the thermal ...

Noninvasive MRI thermometry with the proton resonance frequency (PRF) method: in vivo results in human muscle.

Abstract: The noninvasive thermometry method is based on the temperature dependence of the proton resonance frequency (PRF). High-quality temperature images can be obtained from phase information of standard gradient-echo sequences with an accuracy of 0.2 degrees C in phantoms. This work was focused on the in vivo capabilities of this method. An experimental setup was designed that allows a qualitative in vivo verification. The lower-leg muscles of a volunteer were cooled and afterwards reheated with an external water bolus. The temperature of the bolus water varied between 17 degrees C and 37 degrees C. The in vivo temperature images can be used to extract the temperature in muscle tissue. The data in the fat tissue are difficult to interpret because of the predominance of susceptibility effects. The results confirm the method's potential for hyperthermia control.

CD28-B7 blockade after alloantigenic challenge in vivo inhibits Th1 cytokines but spares Th2.

Abstract: Blocking the CD28-B7 T cell costimulatory pathway with the fusion protein CTLA4Ig inhibits alloimmune responses in vitro and in vivo and induces tolerance to cardiac allografts in mice and rats, but the mechanisms mediating the tolerant state in vivo are unknown. Here, we report the effects and potential mechanisms of CTLA4Ig in the rat renal allograft model. LEW rats were nephrectomized and received renal allografts from major histocompatibility complex-incompatible WF rats. While all untreated and control immunoglobulin (Ig)-treated animals acutely rejected their allografts and died, 86% of rats that received a single injection of CTLA4Ig on day 2 after transplantation had prolonged survival (> 60-100 days) with preserved renal function. By contrast, only 29% of animals that received CTLA4Ig on the day of engraftment had prolonged survival. Long-term survivors (> 100 days) exhibited donor-specific tolerance, accepting donor-matched WF but acutely rejecting third-party BN cardiac allografts. Immunohistological analysis of grafts sampled at 1 week after transplantation showed that both control and CTLA4Ig-treated animals had mononuclear cell infiltrates, with a higher percentage of CD4+ cells in the CTLA4Ig-treated group. However, while this was associated with vasculitis and tubulitis in control grafts, there was no evidence of tissue injury in CTLA4Ig-treated animals. The immune response leading to graft rejection in control animals was characterized by expression of the T helper (Th) type 1 cytokines interleukin (IL)-2 and interferon-gamma. In contrast, the persistent CD4+ infiltrate without graft rejection in CTLA4Ig-treated animals was associated with increased staining for the Th2-related cytokines IL-4 and IL-10. Furthermore, grafts from CTLA4Ig-treated animals had marked upregulation of intragraft staining for IgG1, but not IgG2a or IgG2b. Administration of rIL-2 to CTLA4Ig-treated animals restored allograft rejection in 50% of animals tested. These results confirm that blockade of the CD28-B7 pathway after alloantigenic challenge induces donor-specific acceptance of vascularized organ allografts, and indicates in this model that CTLA4Ig inhibits Th1 but spares Th2 cytokines in vivo.

Adhesive sealant composition

Abstract: This invention is related to an adhesive composition which may be used to bond or seal tissue in vivo. The adhesive composition is readily formed from a two component mixture which includes a first part of a protein, preferably a serum albumin protein, in an aqueous buffer having a pH in the range of about 8.0-11.0 and a second part of a water-compatible or water-soluble bifunctional crosslinking agent. When the two parts of the mixture are combined, the mixture is initially a liquid which cures in vivo on the surface of tissue in less than about one minute to give a strong, flexible, pliant substantive composition which bonds to the tissue and is absorbed in about four to sixty days. The adhesive composition may be used either to bond tissue, to seal tissue or to prevent tissue adhesions caused by surgery.

A gene therapy strategy using a transcription factor decoy of the E2F binding site inhibits smooth muscle proliferation in vivo.

Abstract: The application of DNA technology to regulate the transcription of disease-related genes in vivo has important therapeutic potentials. The transcription factor E2F plays a pivotal role in the coordinated transactivation of cell cycle-regulatory genes such as c-myc, cdc2, and the gene encoding proliferating-cell nuclear antigen (PCNA) that are involved in lesion formation after vascular injury. We hypothesized that double-stranded DNA with high affinity for E2F may be introduced in vivo as a decoy to bind E2F and block the activation of genes mediating cell cycle progression and intimal hyperplasia after vascular injury. Gel mobility-shift assays showed complete competition for E2F binding protein by the E2F decoy. Transfection with E2F decoy inhibited expression of c-myc, cdc2, and the PCNA gene as well as vascular smooth muscle cell proliferation both in vitro and in the in vivo model of rat carotid injury. Furthermore, 2 weeks after in vivo transfection, neointimal formation was significantly prevented by the E2F decoy, and this inhibition continued up to 8 weeks after a single transfection in a dose-dependent manner. Transfer of an E2F decoy can therefore modulate gene expression and inhibit smooth muscle proliferation and vascular lesion formation in vivo.

Immunosuppressive Actions of 1,25-Dihydroxyvitamin D3: Preferential Inhibition of Th1 Functions

Abstract: 1,25-Dihydroxyvitamin D3 [1,25-(OH)2-D3] is known to be an immunosuppressive hormone. This review primarily deals with in vitro and in vivo effects of 1,25-(OH)2-D3 and analogue, 1,25-dihydroxy-16ene-vitamin D3 [1,25-(OH)2-16ene-D3], on T helper subsets type 1 (Th1) or type 2 (Th2) that have distinctive functional characteristics in humans. Th1 secrete interferon (IFN-gamma), interleukin (IL-2) and induce B cells to produce immunoglobulin IgG2a while Th2 secrete IL-4, IL-10 and induce the production of IgG1 and IgE by B cells. The sterol inhibits the secretion of IL-12, a cytokine produced by monocytes and B cells, which leads to the activation and differentiation of Th1. In addition, 1,25-(OH)2-D3 directly inhibits IFN-gamma secretion by Th1 clones while it has little effect on IL-4 secretion by Th2 clones. The analogue, 1,25-(OH)2-16ene-D3, is 100-fold more potent than 1,25-(OH)2-D3 in inhibiting IFN-gamma secretion but also has little effect on IL-4 secretion. In mice, when given in vivo, the sterol prevents the induction of spontaneous and induced autoimmune diseases and inhibits Th1 induce IgG2a responses. These actions of the vitamin D3 compounds suggest that it may have potential therapeutic applications in Th1-mediated clinical situations such as autoimmunity and transplantation.

Mice lacking extracellular superoxide dismutase are more sensitive to hyperoxia.

Abstract: Extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) is a secreted Cu- and Zn-containing tetrameric glycoprotein, the bulk of which is bound to heparan sulfate proteoglycans in the interstitium of tissues. To test the function of EC-SOD in vivo, mice carrying a targeted disruption of the EC-SOD gene were generated. The EC-SOD null mutant mice develop normally and remain healthy until at least 14 months of age. No compensatory induction of other SOD isoenzymes or other antioxidant enzymes was observed. When stressed by exposure to > 99% oxygen, the EC-SOD null mutant mice display a considerable reduction in survival time compared to wild-type mice and an earlier onset of severe lung edema. These findings suggest that while under normal physiological conditions other antioxidant systems may substitute for the loss of EC-SOD; when the animal is stressed these systems are unable to provide adequate protection.

The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake.

Abstract: Purpose. We examined the stability and disposition characteristics of a naked plasmid DNA pCAT as a model gene after intravenous injection in mice to construct the strategy of in vivo gene delivery systems. Methods. After the injection of pCAT to the mice, stability, tissue distribution, hepatic cellular localization, and effect of some polyanions on the hepatic uptake were studied. Results. The in vitro study demonstrated that the pCAT was rapidly degraded in mouse whole blood with a half-life of approximately 10 min at a concentration of 100 µg/ml. After intravenous injection, pCAT was degraded at a significantly faster rate than that observed in the whole blood, suggesting that pCAT in vivo was also degraded in other compartments. Following intravenous injection of [32P] pCAT, radioactivity was rapidly eliminated from the plasma due to extensive uptake by the liver. Hepatic accumulation occurred preferentially in the non-parenchymal cells. The hepatic uptake of radioactivity derived from [32P] pCAT was inhibited by preceding administration of polyanions such as polyinosinic acid, dextran sulfate, maleylated and succinylated bovine serum albumin but not by polycytidylic acid. These findings indicate that pCAT is taken up by the liver via scavenger receptors on the non-parenchymal cells. Pharmacokinetic analysis revealed that the apparent hepatic uptake clearance was fairly close to the liver plasma flow. Conclusions. These findings provide useful information for the development of delivery systems for in vivo gene therapy.

The Insulin-like Growth Factor I Receptor Protects Tumor Cells from Apoptosis in Vivo

Abstract: The role of the insulin-like growth factor I receptor (IGF-IR) in programmed cell death has been investigated in vivo in a biodiffusion chamber, where the extent of cell death could be determined quantitatively. We found that a decrease in the number of IGF-IRs causes massive apoptosis in vivo in several transplantable tumors, either from humans or rodents. Conversely, an overexpressed IGF-IR protects cells from apoptosis in vivo. We also show that the same conditions that in vitro cause only partial growth arrest with a minimum of cell death, induce in vivo almost complete cell death. We conclude that the IGF-IR activated by its ligands plays a very important protective role in programmed cell death, and that its protective action is even more striking in vivo than in vitro.

Imaging the expression of transfected genes in vivo.

Abstract: Imaging the expression of successful gene transduction has been demonstrated in vivo for the first time by using an appropriate combination of “marker gene” and “marker substrate” in an experimental animal model The herpes simplex virus 1 thymidine kinase ( HSV1-tk ) gene was selected as an example of a marker gene, and the recombinant STK retrovirus containing HSV1-tk was used to transduce RG2 glioma cells in vitro and in vivo RG2TK + cell lines expressing the HSV1-tk gene and three potential marker substrates for the HSV1-TK enzyme were evaluated Radiolabeled 5-iodo-2′-fluoro-2′deoxy-1-β-d-arabinofuranosyluracil (FIAU) was shown to be a substantially better marker substrate for the HSV1-TK enzyme than 5-iodo-2′-deoxyuridine or ganciclovir The magnitude of FIAU accumulation in different RG2TK + clones corresponded to their sensitivity to ganciclovir and to the level of HSV1-tk mRNA expression Imaging the expression of HSV1-tk in transduced RG2 tumor cells was demonstrated in animals using quantitative autoradiography; 2-[ 14 C]FIAU accumulation was shown to be high in RG2TK + brain tumors growing in one hemisphere and very low in nontransduced RG2 tumors in the contralateral hemisphere Transduction of RG2 tumor cells with the HSV-tk gene in vivo resulted in tumors which accumuiated FIAU to high levels and produced clearly defined images Given the level of FIAU accumulation in the transduced tumors, it is likely that a clinically applicable method for imaging HSV1-tk gene expression can be implemented using existing clinical imaging techniques

Sphingosomes for enhanced drug delivery

Abstract: Liposomal formulations having extended circulation time (in vivo and increased drug retention are comprised of sphingomyelin and cholesterol and have an acidic intraliposomal pH. The formulations have enhanced stability and thus are used in methods which provide improved drug delivery and more effective treatment. The delivery of alkaloid drugs, particularly swainsonine, vincristine and vinblastine, is significantly improved.

KRN7000, a novel immunomodulator, and its antitumor activities.

Abstract: KRN7000, a compound with a novel alpha-galactosylceramide structure, showed potent tumor growth inhibitory activities in B16-bearing mice and markedly stimulated lymphocytic proliferation in allogeneic mixed leukocyte reaction, suggesting that KRN7000 is a biological response modifier. To confirm this suggestion, we examined the abilities of KRN7000 to enhance in vitro and in vivo natural killer (NK) activities, and found that KRN7000 enhanced in vitro and in vivo NK activities, and its potency on in vivo NK activity was much stronger than those of Poly I:C, which is a positive control, or the representative BRMs OK432 and Lentinan. In addition, since spleen cells intravenously treated with KRN7000 also showed potent cytotoxic activities against B16 and EL-4 cells, we evaluated the capability of KRN7000 to prolong the survival period of mice intravenously inoculated with B16 cells or mice intraperitoneally inoculated with EL-4 cells. In these models, KRN7000 (100 micrograms/kg) significantly prolonged the lifespan of mice, and its potency was stronger than that of Mitomycin C, a typical chemotherapeutic agent, in two models. These results demonstrate that KRN7000 would be a useful agent for cancer therapy.

Effect of genistein on in vitro and in vivo models of cancer.

Abstract: In two-thirds of studies on the effect of genistein-containing soy materials in animal models of cancer, the risk of cancer (incidence, latency or tumor number) was significantly reduced. In addition, purified genistein delayed mammary tumor appearance in association with increased cell differentiation in mammary tissue in rats treated with 7, 12-dimethylbenz[a]anthracene when administered neonatally, inhibited phorbol ester-induced H2O2 production in a model of skin cancer, and inhibited aberrant crypt formation in a model of colonic cancer. In in vitro models, genistein inhibited the proliferation of human tumor cell lines in culture with a wide variation in IC50 values (2.6-79 mumol/L, or 1-30 micrograms/mL). In only a few cases was the IC50 below 13.2 mumol/L (5 micrograms/mL), the presumed upper limit for the serum genistein concentration in those on a high soy diet. In future studies, greater emphasis should be placed on the effect of genistein on nontransformed, normal cell lines from the tissues where cancer can occur rather than established tumor cell lines. Similarly, the effect of genistein on the progression and/or promotion of cancer may be more clearly examined using nontransformed cell lines transfected with specific oncogenes thought to be activated during oncogenesis.

Free insulin-like growth factors in human obesity

Abstract: It is well established that spontaneous and stimulated growth hormone (GH) secretion is diminished in human obesity. In contrast to classic GH deficiency, obesity is not associated with hypopituitary levels of circulating total (extractable) insulin-like growth factor-I (IGF-I) and reduced somatic growth. Thus, the riddle of “normal growth without GH” in obese children and the mechanisms behind the GH suppression have remained unsolved. Insulin reduces hepatic production of IGF-binding protein-1 (IGFBP-1), an in vitro inhibitor of IGF bioactivity, and it has been suggested that the obesity-related hyperinsulinemia may increase free (bioactive) IGF in vivo by reducing the concentration of IGFBP-1. We have recently developed a method that during near in vivo conditions isolates the free, unbound fractions of IGF-1 and IGF-II in human serum. Using this method, we have determined overnight fasting serum levels of free IGFs in obese subjects and compared the results with levels of total (extractable) IGFs, IGFBPs, GH, and insulin. The study included 92 healthy subjects (56 males and 36 females) allocated to three age-matched groups depending on body mass index (BMI): 31 controls (BMI ≤ 25), 33 subjects with moderate obesity (25 < BMI < 30), and 28 subjects with severe obesity (BMI ≥ 30). Fasting serum insulin correlated positively (r = .61, P < .0001) with BMI and was significantly elevated in moderate and severe obesity (P < .05). In contrast, levels of serum GH and IGFBP-1 were suppressed in both obese groups (P < .05), and the latter inversely correlated (r = - .60, P < .001) with BMI. Serum free IGF-I was 470 ± 50 ng/L (mean ± SEM) in controls, and was elevated in moderate obesity by 47% (690 ± 90 ng/L, P < .05) and in severe obesity by 72% (810 ± 90 ng/L, P < .05), whereas levels of total IGF-I were unaltered. In addition, serum free IGF-I was inversely correlated with IGFBP-1 (r = - .47, P < .001). Serum IGFBP-3 and total IGF-II were both increased (P < .05) in obese subjects, whereas serum free IGF-II was unaltered. All phenomena were more pronounced in males than in females. We conclude that in obesity, the concentration of free IGF-I in fasting serum is increased. This is likely a result of decreased circulating IGFBP-1, again caused by hyperinsulinemia. Elevated serum free IGF-I may, by feedback, explain the low levels of GH and may be responsible for the normal growth without GH in obese children. Increased levels of IGFBP-3 and normal levels of serum total IGF-I support the interpretation that obese subjects are hypersensitive to the actions of GH.

Neutralization of IL-10 increases survival in a murine model of Klebsiella pneumonia

Abstract: Effective host defense against bacterial infection is dependent upon the vigorous recruitment and activation of neutrophils and macrophages. We hypothesized that IL-10 is produced in the setting of bacterial pneumonia, and this cytokine may attenuate host defense by inhibiting the expression of important activating and chemotactic cytokines. CD-1 mice were challenged with either 30 microliters of saline or saline containing 10(3) CFUs of Klebsiella pneumoniae intratracheally (i.t.) and lungs were harvested at 8, 24, and 48 h. The i.t. inoculation with K. pneumoniae resulted in a 13-, 14-, and 8-fold increase in lung homogenate TNF, macrophage inflammatory protein-2 (MIP-2), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) levels, respectively, as compared with control animals. In addition, we observed an increase in IL-10 mRNA and protein levels in lung homogenates, maximal at 48 h postinoculation. To establish the biologic relevance of IL-10 in Klebsiella pneumonia, we passively immunized CD-1 mice with 0.5 ml of rabbit anti-murine IL-10 serum or preimmune serum i.p. 2 h before i.t. administration of K. pneumoniae. Treatment of animals with anti-IL-10 serum resulted in increased levels of TNF, MIP-2, and MIP-1 alpha, respectively, within lung homogenates at 24 and 48 h, as compared with preimmune-treated animals. Furthermore, neutralization of IL-10 resulted in a significant decrease in K. pneumoniae CFU in both lung homogenates and plasma harvested at 48 h, as well as a significant increase in survival in these animals. Our studies indicate that 1) IL-10 is produced during Klebsiella pneumonia; and 2) inhibition of IL-10 bioactivity in vivo results in enhanced bacterial clearance, increased expression of proinflammatory cytokines, and prolonged survival.

Docetaxel (Taxotere): a review of preclinical and clinical experience. Part I: Preclinical experience.

Abstract: Docetaxel is a taxoid which is currently in phase II/III clinical trials in Europe, the US and Japan. It was found to promote tubulin assembly in microtubules and to inhibit their depolymerization. In vitro, the docetaxel concentrations required to reduce murine and human cell survival by 50% ranged from 4 to 35 ng/ml and the cytotoxic effects were greater on proliferating than on non-proliferating cells. It was also found to be cytotoxic on fresh human tumor biopsies. In vivo, the drug was found to be schedule independent. A total of 13/14 murine transplantable tumors were found very sensitive to i.v. docetaxel and complete regressions of advanced stage tumors were obtained. Activity was also observed in 15/16 human tumor xenografts in nude mice at an advanced stage. In combination studies, synergism was observed in vivo with 5-fluorouracil, cyclophosphamide and etoposide. Pharmacokinetic evaluation revealed linear pharmacokinetics in tumor-bearing mice. There was a good tumor retention with a 22 h elimination half-life. Plasma protein binding ranged from 76 to 89%. Preclinical toxicology evaluation of docetaxel included single-dose toxicity in rats, mice and dogs, 5-day toxicity in mice and dogs, intermittent-dose toxicity in rats, dogs and monkeys, genetic and reproductive toxicity, as well as investigation of the irritation and sensitization potential. The principal toxicities were hematopoietic (all species), gastrointestinal (dog, monkey) and neuromotor (mice). Dogs appeared to be the most sensitive species. The clinical entry dose of 5 mg/m2 was based on one-third of the 'toxic dose low' in dogs (15 mg/m2).

The antiproliferative activity of c-myb and c-myc antisense oligonucleotides in smooth muscle cells is caused by a nonantisense mechanism.

Abstract: Smooth muscle cell (SMC) proliferation is thought to play a major role in vascular restenosis after angioplasty and is a serious complication of the procedure. Developing antisense (AS) oligonucleotides as therapeutics is attractive because of the potentially high specificity of binding to their targets, and several investigators have reported inhibition of SMC proliferation in vitro and in vivo by using AS strategies. We report here the results of our experiments on vascular SMCs using AS oligonucleotides directed toward c-myb and c-myc. We found that significant inhibition of SMC proliferation occurred with these specific AS sequences but that this inhibition was clearly not via a hybridization-dependent AS mechanism. Rather, inhibition was due to the presence of four contiguous guanosine residues in the oligonucleotide sequence. This was demonstrated in vitro in primary cultures of SMCs and in arteries ex vivo. The ex vivo model developed here provides a rapid and effective system in which to screen potential oligonucleotide drugs for restenosis. We have further explored the sequence requirements of this non-AS effect and determined that phosphorothioate oligonucleotides containing at least two sets of three or four consecutive guanosine residues inhibit SMC proliferation in vitro and ex vivo. These results suggest that previous AS data obtained using these and similar, contiguous guanosine-containing AS sequences be reevaluated and that there may be an additional class of nucleic acid compounds that have potential as antirestenosis therapeutics.

Induction of acute phase gene expression by brain irradiation

Abstract: Purpose: To investigate the in vivo acute phase molecular response of the brain to ionizing radiation Methods and Materials: C3Hf/Sed/Kam mice were given midrain or whole-body irradiation Cerebral expression of interleukins (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6), interferon (IFN-γ), tumor necrosis factors (TNF-α and TNK-β), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthetase (iNOS), von Willebrand factor (vWF), α1-antichymotrypsin (EB22/53), and glial fibrillary acidic protein (GFAP) was measured at various times after various radiation doses by ribonuclease (RNase) protection assay The effects of dexamethasone or pentoxifylline treatment of mice on radiation-induced gene expression were also examined Results: Levels of TNF-α, IL-1β, ICAM-1, EB22/53, and to alesser extent IL-1α and GFAP, messenger RNA were increased in the brain after irradiation, whether the dose was delivered to the whole body or only to the midbrain Responses were radiation dose dependent, but were not found below 7 Gy; the exception being ICAM-1, which was increased by doses as low as 2 Gy Most responses were rapid, peaking within 4–8, but antichymotrypsin and GFAP responses were delayed and still elevated at 24 h, by which time the others had subsides Pretreatment of mice with dexamethasone or pentoxifylline suppressed radiation-induced gene expression, either partially or completely Dexamethasone was more inhibitory than pentoxifylline at the doses chosen Conclusions: The initial response of thebrain to irradiation involves expression of inflammatory gene products, which are probably responsible for clinically observed early symptoms of brain radiotherapy This mechanism explains the beneficial effects of the clincial use of steroids in such circumtances