Identification of five hub immune genes and characterization of two immune subtypes of osteoarthritis.

Background: Osteoarthritis (OA) is one of the most prevalent chronic diseases, leading to degeneration of joints, chronic pain, and disability in the elderly. Little is known about the role of immune-related genes (IRGs) and immune cells in OA. Method: Hub IRGs of OA were identified by differential expression analysis and filtered by three machine learning strategies, including random forest (RF), least absolute shrinkage and selection operator (LASSO), and support vector machine (SVM). A diagnostic nomogram model was then constructed by using these hub IRGs, with receiver operating characteristic (ROC) curve, decision curve analysis (DCA), and clinical impact curve analysis (CICA) estimating its performance and clinical impact. Hierarchical clustering analysis was then conducted by setting the hub IRGs as input information. Differences in immune cell infiltration and activities of immune pathways were revealed between different immune subtypes. Result: Five hub IRGs of OA were identified, including TNFSF11, SCD1, PGF, EDNRB, and IL1R1. Of them, TNFSF11 and SCD1 contributed the most to the diagnostic nomogram model with area under the curve (AUC) values of 0.904 and 0.864, respectively. Two immune subtypes were characterized. The immune over-activated subtype showed excessively activated cellular immunity with a higher proportion of activated B cells and activated CD8 T cells. The two phenotypes were also seen in two validation cohorts. Conclusion: The present study comprehensively investigated the role of immune genes and immune cells in OA. Five hub IRGs and two immune subtypes were identified. These findings will provide novel insights into the diagnosis and treatment of OA.

FDA Approval Summary: Calaspargase Pegol-mknl For Treatment of Acute Lymphoblastic Leukemia in Children and Young Adults.

On December 20, 2018, the Food and Drug Administration approved calaspargase pegol-mknl (CALASP), an asparagine-specific enzyme, as a component of a multi-agent chemotherapeutic regimen for acute lymphoblastic leukemia (ALL) in pediatric and young adult patients age 1 month to 21 years. Efficacy was determined on the basis of achievement and maintenance of steady-state nadir serum asparaginase activity (NSAA) above 0.1 U/mL when using CALASP, 2,500 U/m2 intravenously, every 3 weeks. In a randomized comparison to pegaspargase (PEGASP) every 2 weeks, treatment with CALASP every 3 weeks had a similar safety profile and no substantial impairment in event-free survival. The pharmacokinetics of CALASP were studied when administered in combination with multiagent chemotherapy in 124 patients with B-cell ALL in Study AALL07P4 and Study DFCI 11-001. The results showed that 123 [99%, 95% confidence interval (CI), 96%-100%] of the 124 patients maintained NSAA >0.1 U/mL at weeks 6, 12, 18, 24, and 30 of post-induction phase. Maintaining adequate NSAA levels is critical to successful treatment of ALL. Herein, we describe the FDA review and approval of CALASP.See related commentary by Lew, p. 325.

U.S. Food and Drug Administration Approval: Neratinib for the Extended Adjuvant Treatment of Early-Stage HER2-Positive Breast Cancer.

On July 17, 2017, the FDA approved neratinib (NERLYNX; Puma Biotechnology, Inc.) for the extended adjuvant treatment of adult patients with early-stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy. Approval was based on data from ExteNET, a randomized, double-blind, placebo-controlled multicenter trial. Women with early-stage HER2-positive breast cancer and within 2 years of completing adjuvant trastuzumab were randomized to neratinib (n = 1,420) or placebo (n = 1,420) for 1 year. The primary endpoint was invasive disease-free survival (iDFS), defined as the time between randomization date to first occurrence of invasive recurrence (local/regional, ipsilateral, or contralateral breast cancer), distant recurrence, or death from any cause, with 2 years and 28 days of follow-up. The trial showed a statistically significant treatment effect favoring neratinib with a stratified HR of 0.66 [95% confidence interval (CI), 0.49-0.90, P = 0.008]. The estimated iDFS rate at 2 years was 94.2% (95% CI, 92.6%-95.4%) in patients treated with neratinib versus 91.9% (95% CI, 90.2%-93.2%) in those receiving placebo. Diarrhea was the most common adverse event (AE), with a 40% incidence of grade 3 or 4 diarrhea, and represents the most common AE leading to treatment discontinuation. Other frequent AEs (>10% incidence) were nausea, abdominal pain, fatigue, vomiting, rash, stomatitis, decreased appetite, and muscle spasms. Other than diarrhea, neratinib is associated with a low incidence of severe AEs; toxicities are generally reversible and manageable with dose interruptions, dose reductions, and/or standard medical care. This article summarizes FDA decision-making and data supporting the neratinib approval. Clin Cancer Res; 24(15); 3486-91. ©2018 AACRSee related commentary by Unni et al., p. 3483.

FDA Approval Summary: TAS-102.

The FDA approved TAS-102 (Lonsurf; Taiho Oncology, Inc.) for the treatment of patients with metastatic colorectal cancer (mCRC) who have been previously treated with fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy; an anti-VEGF biological therapy; and if RAS wild type, an anti-EGFR therapy. In an international, multicenter, double-blinded, placebo-controlled trial (TPU-TAS-102-301, herein referred to as RECOURSE), 800 patients with previously treated mCRC were randomly allocated (2:1) to receive either TAS-102 35 mg/m2 orally twice daily after meals on days 1 through 5 and 8 through 12 of each 28-day cycle (n = 534) or matching placebo (n = 266). The trial demonstrated a statistically significant improvement in overall survival for those randomized to receive TAS-102, with a median survival of 7.1 months in the TAS-102 arm [confidence interval (CI), 6.5-7.8] and 5.3 months in the placebo arm [CI, 4.6-6.0; hazard ratio (HR), 0.68; 95% CI, 0.58-0.81; P < 0.001, stratified log-rank test]. The trial also demonstrated a statistically significant prolongation of progression-free survival (HR, 0.47; 95% CI, 0.40-0.55; P < 0.001). The most common adverse reactions, in order of decreasing frequency, observed in the patients who received TAS-102 were anemia, neutropenia, asthenia/fatigue, nausea, thrombocytopenia, decreased appetite, diarrhea, vomiting, abdominal pain, and pyrexia. Adverse events led to discontinuation of TAS-102 in 3.6% of patients, and 13.7% required a dose reduction. The most common adverse reactions leading to dose reduction were neutropenia, anemia, febrile neutropenia, fatigue, and diarrhea. Clin Cancer Res; 23(12); 2924-7. ©2017 AACR.

U.S. FDA Approval Summary: Nivolumab for Treatment of Unresectable or Metastatic Melanoma Following Progression on Ipilimumab.

On December 22, 2014, the FDA granted accelerated approval to nivolumab (OPDIVO; Bristol-Myers Squibb) for the treatment of patients with unresectable or metastatic melanoma and disease progression following ipilimumab and, if BRAF V600 mutation positive, a BRAF inhibitor. Approval was based on a clinically meaningful, durable objective response rate (ORR) in a non-comparative analysis of 120 patients who received 3 mg/kg of nivolumab intravenously every 2 weeks with at least 6-month follow-up in an ongoing, randomized, open-label, active-controlled clinical trial. The ORR as assessed by a blinded independent review committee per RECIST v1.1 was 31.7% (95% confidence interval, 23.5-40.8). Ongoing responses were observed in 87% of responding patients, ranging from 2.6+ to 10+ months. In 13 patients, the response duration was 6 months or longer. The risks of nivolumab, including clinically significant immune-mediated adverse reactions (imARs), were assessed in 268 patients who received at least one dose of nivolumab. The FDA review considered whether the ORR and durations of responses were reasonably likely to predict clinical benefit, the adequacy of the safety database, and systematic approaches to the identification, description, and patient management for imARs in product labeling. Clin Cancer Res; 23(14); 3484-8. ©2017 AACR.

SAR studies of non-zinc-chelating MMP-13 inhibitors: improving selectivity and metabolic stability.

SAR studies to improve the selectivity and metabolic stability of a class of recently discovered MMP-13 inhibitors are reported. Improved selectivity was achieved by modifying interactions with the S1' pocket. Metabolic stability was improved through reduction of inhibitor lipophilicity. This translated into lower in vivo clearance for the preferred compound.

Dynamic schema for near infrared detection of pressure-induced changes in solid tumors.

Differentiation among malignant tumors, benign tumors, and normal tissue is highly important in the diagnosis and treatment of many malignancies. We have proposed a dynamic schema for noninvasive characterization of pressure-induced changes in solid tumors. Our hypothesis has been that the altered neovascularization processes within cancer-bearing tissues may significantly increase vascular resistance and cause a much slower response of hemoglobin concentration during a dynamic compression stimulus. This hypothesis was tested by the evaluation of data generated from human tumor clinical testing and from animal tumor model testing. In the human tumor clinical testing, a unified diagnostic criterion was derived that integrated the relative characteristics of tumor oxygen, hemoglobin, and hemoglobin dynamics. By applying such a unified criterion, we were able to differentiate benign breast lesions and malignant breast tumors with high sensitivity and specificity within a subset of 14 suspicious breast lesions with similar size and depth characteristics. In the animal testing, a stepped compression load was applied to the subcutaneous tumor deposit on an athymic NU/NU nude mouse model with subcutaneous xenograft BxPC-3 cancer. Characteristic differences were observed between the premortem tumor and the postmortem tumor in terms of pressure-induced tumor structural and functional changes.

Synergistic antipancreatic tumor effect by simultaneously targeting hypoxic cancer cells with HSP90 inhibitor and glycolysis inhibitor.

PURPOSE: We sought to examine the synergistic antipancreatic cancer effect by simultaneously targeting hypoxic cancer cells with heat-shock protein 90 (HSP90) inhibitor and blockade of energy production. EXPERIMENTAL DESIGN: The anticancer effects of an HSP90 inhibitor (geldanamycin) in pancreatic cells were investigated in hypoxia and normoxia. A hexokinase II inhibitor, 3-broma-pyruvate (3BrPA), was evaluated for selective glycolysis inhibition in hypoxia as a sensitizer of HSP90 inhibitor against pancreatic cancer. The HSP90 client protein degradation was monitored by Western blot. The synergistic antitumor effect of geldanamycin and 3BrPA was evaluated in a xenograft pancreatic cancer model and monitored by a noninvasive dynamic contrast-enhanced magnetic resonance imaging. RESULTS: Hypoxia enhanced HIF-1alpha expression by 11-fold in pancreatic cancer cells, and HSP90 inhibitor exhibited a seven- to eightfold higher anticancer effect in hypoxia compared with normoxia via HSP90 client protein degradation. 3BrPA selectively inhibited glycolysis and sensitized geldanamycin against pancreatic cancer cells by 17- to 400-fold through HSP90 client protein degradation. The synergistic anticancer effect of reduced doses of geldanamycin and 3-BrPA was confirmed in xenograft models in vivo by more than 75% tumor growth inhibition. CONCLUSIONS: The combination of HSP90 inhibitors and glycolysis inhibitors provides preferential inhibition of cancer cells in hypoxia through HSP90 client protein degradation and selective glycolysis inhibition. This may provide a new therapeutic regimen to battle chemotherapy-resistant pancreatic cancers, by enhancing the synergistic therapeutic efficacy and reducing dose-limiting toxicity.

Non-invasive MRI tumor imaging and synergistic anticancer effect of HSP90 inhibitor and glycolysis inhibitor in RIP1-Tag2 transgenic pancreatic tumor model.

PURPOSES: To utilize non-invasive MRI imaging for real-time testing the synergistic effects of HSP90 inhibitor and glycolysis inhibitor for pancreatic cancer therapy in spontaneous pancreatic cancer mouse model. MATERIAL AND METHODS: Transgenic RIP1-Tag2 spontaneous pancreatic cancer mice were treated with geldanamycin (GA, 5 mg/kg) and /or 3-Bromo-pyruvate (3-BrPA, 5 mg/kg) from 8 to 12 weeks of age. Non-invasive MRI imaging measured and calculated the total tumor mass and volumes in real-time and compared to ex vivo tumors size. Serum VEGF levels were measured by ELISA. HSP 90 client protein levels (AKT and VEGF) were measured by western blots. RESULTS: RIP-Tag2 transgenic mice developed pancreatic tumors from 8 to 12 weeks of age. Non-invasive MRI imaging detected primary tumors in pancreas and metastasis in intestine and mesenterium with minimal resolution of 20 mm(3). VEGF, AKT, hexokinase II, and Hsp90 were expressed in the pancreatic cancer tissues from RIP1-Tag2 transgenic mice. Combination of GA and 3-BrPA decreased serum VEGF levels by 70% compared to control group. Non-invasive MRI imaging showed that combination of GA and 3-BrPA inhibited pancreatic tumor and metastasis by more than 90% and significantly prolonged life span of RIP1-Tag2 transgenic pancreatic cancer mice. The synergistic effect of geldanamycin and 3-BrPA is through inhibition of two different pathways on HSP90 for its client protein degradation and on HK II for energy metabolism. CONCLUSION: Non-invasive MRI imaging revealed synergistic effects of Hsp90 inhibitors and glycolysis inhibitors, which may provide a new therapeutic option for pancreatic cancer therapy.

A novel Hsp90 inhibitor to disrupt Hsp90/Cdc37 complex against pancreatic cancer cells.

Pancreatic cancer is an aggressive disease with multiple biochemical and genetic alterations. Thus, a single agent to hit one molecular target may not be sufficient to treat this disease. The purpose of this study is to identify a novel Hsp90 inhibitor to disrupt protein-protein interactions of Hsp90 and its cochaperones for down-regulating many oncogenes simultaneously against pancreatic cancer cells. Here, we reported that celastrol disrupted Hsp90-Cdc37 interaction in the superchaperone complex to exhibit antitumor activity in vitro and in vivo. Molecular docking and molecular dynamic simulations showed that celastrol blocked the critical interaction of Glu33 (Hsp90) and Arg167 (Cdc37). Immunoprecipitation confirmed that celastrol (10 micromol/L) disrupted the Hsp90-Cdc37 interaction in the pancreatic cancer cell line Panc-1. In contrast to classic Hsp90 inhibitor (geldanamycin), celastrol (0.1-100 micromol/L) did not interfere with ATP binding to Hsp90. However, celastrol (1-5 micromol/L) induced Hsp90 client protein degradation (Cdk4 and Akt) by 70% to 80% and increased Hsp70 expression by 12-fold. Celastrol induced apoptosis in vitro and significantly inhibited tumor growth in Panc-1 xenografts. Moreover, celastrol (3 mg/kg) effectively suppressed tumor metastasis by more than 80% in RIP1-Tag2 transgenic mouse model with pancreatic islet cell carcinogenesis. The data suggest that celastrol is a novel Hsp90 inhibitor to disrupt Hsp90-Cdc37 interaction against pancreatic cancer cells.

Population pharmacokinetics of humanized monoclonal antibody HuCC49deltaCH2 and murine antibody CC49 in colorectal cancer patients.

To predict the optimal time for surgery after antibody administration, the population pharmacokinetics of (125)I-HuCC49deltaCH2 and (125)I-CC49 were characterized in 55 patients with colorectal cancers. A 2-compartment linear model was used to fit the pharmacokinetic data. Model stability and performance were assessed using a visual predictive check procedure. Different clinical trial designs were evaluated by simulation in combination with Bayesian estimation method to predict the optimal time for surgery. The results showed that HuCC49deltaCH2 had 65% faster clearance from blood circulation and 24% shorter mean residence time than CC49. Population pharmacokinetic analysis identified body weight as the only covariate to explain between-subject variability in clearance, intercompartmental flow rate, and volume of distribution. Model predictions indicated a wide interval for the optimal time of surgery, suggesting that it would be beneficial to individualize the time of surgery for each patient by measurement of antibody disposition. Clinical trial designs with at least 3 measurements of antibody disposition were found to be better than an empirical direct observation method for the optimal prediction of surgery time.

Glucose uptake inhibitor sensitizes cancer cells to daunorubicin and overcomes drug resistance in hypoxia.

PURPOSE: A high-rate glycolysis is a fundamental property of solid tumors and is associated with an over-expression of glucose transporters and glycolytic enzymes. We hypothesize that over-expression of glucose transporters in tumors prevents apoptosis, promotes cancer cell survival, and confers drug resistance. Inhibition of glucose transporter will preferentially sensitize the anticancer effects of chemotherapeutic drugs to overcome drug resistance in hypoxia. METHODS: Glucose transporter expressions were detected in cancer tissues and NCI 60 cancer cells with immunostaining and DNA microarray. Glucose uptake was measured with 3H-2-deoxy-glucose. Cytotoxicity of daunorubicin (DNR) in combination of glucose inhibitor was detected by MTS assay under hypoxic condition. Early stage apoptosis was monitored with Annexin V-FITC staining. RESULTS: Immunostaining showed that GLUT1 was significantly increased in hypoxic regions of the human colon and breast tumors. The expression profiles of all glucose transporters in NCI 60 cancer cells exhibited distinct expression patterns. Phloretin exhibited more than 60% glucose uptake inhibition. Hypoxia conferred two to fivefold higher drug resistance in SW620 and K562 to DNR. Inhibition of glucose uptake by phloretin sensitized cancer cells to DNR for its anticancer activity and apoptosis to overcome drug resistance only under hypoxia. CONCLUSION: Cancer cells heavily rely on glucose transporters for glucose uptake to facilitate a high-rate glycolysis under hypoxia for their survival and drug resistance. Combination of glucose transporter inhibitors and chemotherapeutic drugs may provide a preferential novel therapeutic strategy to overcome drug resistance in hypoxia.

Why is it challenging to predict intestinal drug absorption and oral bioavailability in human using rat model.

PURPOSE: To study the correlation of intestinal absorption for drugs with various absorption routes between human and rat, and to explore the underlying molecular mechanisms for the similarity in drug intestinal absorption and the differences in oral bioavailability between human and rat. MATERIALS AND METHODS: The intestinal permeabilities of 14 drugs and three drug-like compounds with different absorption mechanisms in rat and human jejunum were determined by in situ intestinal perfusion. A total of 48 drugs were selected for oral bioavailability comparison. Expression profiles of transporters and metabolizing enzymes in both rat and human intestines (duodenum and colon) were measured using GeneChip analysis. RESULTS: No correlation (r(2) = 0.29) was found in oral drug bioavailability between rat and human, while a correlation (r(2) = 0.8) was observed for drug intestinal permeability with both carrier-mediated absorption and passive diffusion mechanisms between human and rat small intestine. Moderate correlation (with r(2) > 0.56) was also found for the expression levels of transporters in the duodenum of human and rat, which provides the molecular mechanisms for the similarity and correlation of drug absorption between two species. In contrast, no correlation was found for the expressions of metabolizing enzymes between rat and human intestine, which indicates the difference in drug metabolism and oral bioavailability in two species. Detailed analysis indicates that many transporters (such as PepT1, SGLT-1, GLUT5, MRP2, NT2, and high affinity glutamate transporter) share similar expression levels in both human and rat with regional dependent expression patterns, which have high expression in the small intestine and low expression in the colon. However, discrepancy was also observed for several other transporters (such as MDR1, MRP3, GLUT1, and GLUT3) in both the duodenum and colon of human and rat. In addition, the expressions of metabolizing enzymes (CYP3A4/CYP3A9 and UDPG) showed 12 to 193-fold difference between human and rat intestine with distinct regional dependent expression patterns. CONCLUSIONS: The data indicate that rat and human show similar drug intestinal absorption profiles and similar transporter expression patterns in the small intestine, while the two species exhibit distinct expression levels and patterns for metabolizing enzymes in the intestine. Therefore, a rat model can be used to predict oral drug absorption in the small intestine of human, but not to predict drug metabolism or oral bioavailability in human.

Molecular analysis of expression and function of hFcgammaRIIbl and b2 isoforms in myeloid cells.

The inhibitory receptor FcgammaRIIb becomes tyrosine phosphorylated and associates with the inositol phosphatase SHIP to downregulate phagocytosis. The two splice variants of FcgammaRIIb, b1 and b2, are differentially expressed in hematopoetic cells. Both isoforms of FcgammaRIIb are expressed in human myeloid cells although FcgammaRIIb2 predominates. In murine B cells FcgammaRIIb2 associates with clathrin-coated pits and undergoes endocytosis, whereas FcgammaRIIbl is excluded from the coated pits, indicating that the two isoforms serve partially differing functions. In humans, there are conflicting reports with regard to the ability of FcgammaRIIb2 to become tyrosine phosphorylated, and the functional capacities of the two isoforms are poorly understood. We, and others, have previously reported that the expression of FcgammaRIIb is upregulated in human monocytes by the anti-inflammatory cytokine IL-4. Here, we extend these findings to demonstrate that the IL-4-induced upregulation of FcgammaRIIb is synergistically enhanced by the addition of IL-10, both at the protein and the mRNA level. The upregulated receptors are functional as assessed by their ability to become tyrosine phosphorylated and to downregulate phagocytosis. Interestingly, both b1 and b2 isoforms are upregulated by anti-inflammatory cytokines. Transfection experiments expressing human FcgammaRIIbl or b2 in Raw 264.7 murine macrophage cells revealed that both isoforms are tyrosine phosphorylated and promote SHIP phosphorylation. Finally, both b1 and b2 isoforms of FcgammaRIIb downregulate phagocytosis to a similar extent. Thus we conclude that FcgammaRIIbl and b2 are both functional inhibitory receptors in the phagocytic process.

Syntheses and biological activities of daunorubicin analogs with uncommon sugars.

To study the effects of the sugar structure on the activity of anthracycline against cancer cells, six daunorubicin analogs containing different uncommon sugars were synthesized. Their cytotoxicities were tested against colon cancer cells by MTS assay. The results showed that the aglycon without sugar moiety has 70-100-fold lower activity against cancer cells than daunorubicin derivatives with various uncommon sugars. It suggests that the sugar structure in daunorubicin plays a critical role in determining its anticancer activity. In the compounds with various sugars, the 4'-OH of the sugar is an important determinant for their activity, while the axial-3'-substituent in the sugar interferes with the binding of daunorubicins to DNA. Therefore, 2,6-dideoxy sugars are a better choice for generating biologically active daunorubicin analogs than 6-deoxysugars, 2,3,6-trideoxysugars, or 2,3,4,6-tetradeoxysugars.

Permeability dominates in vivo intestinal absorption of P-gp substrate with high solubility and high permeability.

Three purposes are presented in this study: (1) to study the in vivo regional dependent intestinal absorption of a P-gp substrate with high solubility and high permeability, (2) to study the gene expression difference in the various regions of the intestine, and (3) to study the contributions of P-gp or any other transporters for the absorption of a P-gp substrate. The in vivo permeability of verapamil and propranolol were determined by single-pass in situ intestinal perfusion in rat. The gene expression profiles were measured using Affymetrix GeneChip. Correlation analysis between drug in vivo permeability and expression of 3500 genes was performed with nonparametric bootstrap and ANOVA analysis. The permeability of verapamil and propranolol did not demonstrate regional dependency even though significant differences in gene expression were observed in various regions of the intestine. Verapamil permeability significantly correlates with propranolol permeability in both jejunum and ileum, but did not correlate with the permeability of other hydrophilic compounds (valacyclovir, acyclovir, and phenylalanine). Four different regions (duodenum, jejunum, ileum, and colon) showed distinct gene expression patterns with more than 70-499 genes showing at least 5-fold expression differences. Interestingly, P-gp expression is gradually increased by 6-fold from the duodenum to colon. Despite the distinct gene expression patterns in the various regions of the intestine, verapamil permeability did not correlate with any gene expression from 3500 expressed genes in the intestine. A 2-6-fold P-gp expression difference did not seem to associate verapamil permeability in the various intestinal regions in vivo. These data suggest that P-gp plays a minimal role in the in vivo intestinal absorption process of verapamil with high water solubility and high membrane permeability. The intestinal absorption of verapamil in vivo is primarily dominated by its high permeability. However, it is important to note that the findings in this paper do not undermine the importance of P-gp in oral drug bioavailability, drug disposition from the liver, drug efflux from the blood-brain barrier, and drug-drug interaction.

Pharmacokinetics and clinical evaluation of 125I-radiolabeled humanized CC49 monoclonal antibody (HuCC49deltaC(H)2) in recurrent and metastatic colorectal cancer patients.

INTRODUCTION: CC49 is an antitumor monoclonal antibody that is promising for use in radioimmunoguided surgery (RIGS). However, the murine antibody has been limited by human antimouse antibody (HAMA) response and slow clearance. This study examined the pharmacokinetics and tissue localization of a humanized domain-deleted CC49 antibody (HuCC49DeltaC(H)2 MAb) in humans. METHODS: Twenty-one patients with colorectal carcinoma were given 1 mg intravenous (I.V.) bolus of HuCC49DeltaC(H)2 MAb radiolabeled with 2 mCi (125)I after thyroid blockade. The level of circulating HuCC49DeltaC(H)2 MAb was measured daily as precordial counts using a handheld gamma-detecting probe. Each patient underwent an exploratory laparotomy on postinjection days 3-20. Gamma counts were measured at normal organs, aortic bifurcation (AB), and both clinically evident and occult tumors. RESULTS: Precordial and AB gamma counts showed an excellent linear correlation. HuCC49DeltaC(H)2 MAb followed a two-compartment pharmacokinetic model. Normal organs and AB showed similar exposures to HuCC49DeltaC(H)2 MAb, while HuCC49DeltaC(H)2 MAb favorably distributed into tumors from day 3. Intestinal and metastatic liver lesions showed the highest partition coefficients. All patients showed no HAMA response. DISCUSSION: C(H)2 region deletion of HuCC49DeltaC(H)2 MAb did not alter the pharmacokinetics compared to murine CC49. The favorable partition coefficient K of HuCC49DeltaC(H)2 MAb into tumors supports its use in RIGS.

Synthesis and enzyme-specific activation of carbohydrate-geldanamycin conjugates with potent anticancer activity.

Geldanamycin (GA) is a potent anticancer antibiotic that inhibits Hsp90. Its potential clinical utility is hampered by its severe toxicity. To alleviate this problem, we synthesized a series of carbohydrate-geldanamycin conjugates for enzyme-specific activation to increase tumor selectivity. The conjugation was carried out at the C-17-position of GA. Their anticancer activity was tested in a number of cancer cell lines. The enzyme-specific activation of these conjugates was evaluated with beta-galactosidase and beta-glucosidase. Evidently, glycosylation of C-17-position converted GA to an inactive prodrug before enzyme cleavage. Glucose-GA, as positive control, showed anticancer activity with IC(50) of 70.2-380.9 nM in various cancer cells by beta-glucosidase activation inside of the tumor cells, which was confirmed by 3-fold inhibition using beta-glucosidase specific inhibitor [2,5-dihydroxymethy-3,4-dihydroxypyrrolidine (DMDP)]. Compared to glucose-GA, galactose- and lactose-GA conjugates exhibited much less activity with IC(50) greater than 8000-25 000 nM. However, when galactose- and lactose-GA were incubated with beta-galactosidase in the cells, their anticancer activity was enhanced by 3- to 40-fold. The results suggest that GA can be inactivated by glycosylation of C-17-position and reactivated for anticancer activity by beta-galactosidase. Therefore, galactose-GA can be exploited in antibody-directed enzyme prodrug therapy (ADEPT) with beta-galactosidase for enzyme-specific activation in tumors to increase tumor selectivity.

Lipopolysaccharide-induced macrophage inflammatory response is regulated by SHIP.

LPS stimulates monocytes/macrophages through TLR4, resulting in the activation of a series of signaling events that potentiate the production of inflammatory mediators. Recent reports indicated that the inflammatory response to LPS is diminished by PI3K, through the activation of the serine/threonine kinase Akt. SHIP is an inositol phosphatase that can reverse the activation events initiated by PI3K, including the activation of Akt. However, it is not known whether SHIP is involved in TLR4 signaling. In this study, we demonstrate that LPS stimulation of Raw 264.7 mouse macrophage cells induces the association of SHIP with lipid rafts, along with IL-1R-associated kinase. In addition, SHIP is tyrosine phosphorylated upon LPS stimulation. Transient transfection experiments analyzing the function of SHIP indicated that overexpression of a wild-type SHIP, but not the SHIP Src homology 2 domain-lacking catalytic activity, up-regulates NF-kappaB-dependent gene transcription in response to LPS stimulation. These results suggest that SHIP positively regulates LPS-induced activation of Raw 264.7 cells. To test the validity of these observations in primary macrophages, LPS-induced events were compared in bone marrow macrophages derived from SHIP(+/+) and SHIP(-/-) mice. Results indicated that LPS-induced MAPK phosphorylation is enhanced in SHIP(+/+) cells, whereas Akt phosphorylation is enhanced in SHIP(-/-) cells compared with SHIP(+/+) cells. Finally, LPS-induced TNF-alpha and IL-6 production was significantly lower in SHIP(-/-) bone marrow-derived macrophages. These results are the first to demonstrate a role for SHIP in TLR4 signaling, and propose that SHIP is a positive regulator of LPS-induced inflammation.

The inositol 3-phosphatase PTEN negatively regulates Fc gamma receptor signaling, but supports Toll-like receptor 4 signaling in murine peritoneal macrophages.

Fc gamma R clustering in macrophages activates signaling events that result in phagocytosis. Phagocytosis is accompanied by the generation harmful byproducts such as reactive oxygen radicals and production of inflammatory cytokines, which mandate that the phagocytic process be subject to a tight regulation. The molecular mechanisms involved in this regulation are not fully understood. In this study, we have examined the role of the inositol 3-phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in Fc gamma R-induced macrophage function. We demonstrate that in ex vivo murine peritoneal macrophages that are deficient in PTEN expression, Fc gamma R-induced Akt and extracellular signal-regulated kinase phosphorylation are enhanced. Notably, PTEN(-/-) macrophages showed constitutively high phosphorylation of Akt. However, PTEN did not seem to influence tyrosine phosphorylation events induced by Fc gamma R clustering. Furthermore, PTEN(-/-) macrophages displayed enhanced phagocytic ability. Likewise, Fc gamma R-induced production of TNF-alpha, IL-6, and IL-10 was significantly elevated in PTEN(-/-) macrophages. Surprisingly, LPS-induced TNF-alpha production was down-regulated in PTEN(-/-) macrophages. Analyzing the molecular events leading to PTEN influence on LPS/Toll-like receptor 4 (TLR4) signaling, we found that LPS-induced activation of mitogen-activated protein kinases is suppressed in PTEN(-/-) cells. Previous reports indicated that LPS-induced mitogen-activated protein kinase activation is down-regulated by phosphatidylinositol 3-kinase through the activation of Akt. Our observation that Akt activation is basally enhanced in PTEN(-/-) cells suggests that PTEN supports TLR4-induced inflammatory responses by suppressing the activation of Akt. Thus, we conclude that PTEN is a negative regulator of Fc gamma R signaling, but a positive regulator of TLR4 signaling. These findings are the first to demonstrate a role for PTEN in Fc gamma R- and TLR4-mediated macrophage inflammatory response.