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1.
Sci Adv ; 5(12): eaax9586, 2019 12.
Article in English | MEDLINE | ID: mdl-31897428

ABSTRACT

The neonatal crystallizable fragment receptor (FcRn) functions as an intracellular protection receptor for immunoglobulin G (IgG). Recently, several clinical studies have reported the lowering of circulating monomeric IgG levels through FcRn blockade for the potential treatment of autoimmune diseases. Many autoimmune diseases, however, are derived from the effects of IgG immune complexes (ICs). We generated, characterized, and assessed the effects of SYNT001, a FcRn-blocking monoclonal antibody, in mice, nonhuman primates (NHPs), and humans. SYNT001 decreased all IgG subtypes and IgG ICs in the circulation of humans, as we show in a first-in-human phase 1, single ascending dose study. In addition, IgG IC induction of inflammatory pathways was dependent on FcRn and inhibited by SYNT001. These studies expand the role of FcRn in humans by showing that it controls not only IgG protection from catabolism but also inflammatory pathways associated with IgG ICs involved in a variety of autoimmune diseases.


Subject(s)
Antibodies, Monoclonal, Humanized/pharmacokinetics , Antibodies, Monoclonal/pharmacokinetics , Antigen-Antibody Complex/immunology , Immunity, Humoral/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Receptors, Fc/antagonists & inhibitors , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal, Humanized/adverse effects , Autoantibodies/drug effects , Autoimmune Diseases/drug therapy , Cohort Studies , Double-Blind Method , Female , Healthy Volunteers , Histocompatibility Antigens Class I , Humans , Macaca fascicularis , Male , Mice , Protein Binding
2.
Eur J Orthop Surg Traumatol ; 26(4): 423-7, 2016 May.
Article in English | MEDLINE | ID: mdl-27026091

ABSTRACT

BACKGROUND: The minimally invasive direct anterior approach (MDAA) has been reported to be useful in total hip arthroplasty. The benefits of this approach may be useful for the treatment of femoral neck fractures. Aim of this study is to compare MDAA and postero-lateral approach (PLA) in patients treated with hip hemiarthroplasty for femoral neck fractures. MATERIALS AND METHODS: Between 2013 and 2014, 109 patients underwent bipolar hip hemiarthroplasty for femoral neck fracture: 88 female and 21 male with a mean age of 88 years old. PLA was performed in 54 cases and MDAA in 55 cases. RESULTS: The mean surgery time was significantly lower in MDAA group (P = 0.001). The hemoglobin loss was significantly lower in MDAA group (P = 0.02). The mean postoperative pain was significantly lower in the MDAA group (P = 0.001). The mean hospitalization period was 2 days lower in the MDAA group but with no significant difference between the two groups (P = 0.09). Hip dislocation was higher in PLA cases (7.4 %) than in MDAA cases (1.8 %). Periprosthetic fracture occurred only in one case of PLA. Great trochanter fracture occurred in 1 MDAA cases, while no cases were observed in the PLA group. CONCLUSIONS: Minimally invasive direct anterior approach for hip hemiarthroplasty in elderly people with femoral neck fracture provided significant benefit in the early postoperative period when compared to the postero-lateral approach in terms of surgery time, hemoglobin loss, postoperative pain, time of recovery and dislocation rate. LEVEL OF EVIDENCE: Therapeutic study, level IV (case series).


Subject(s)
Arthroplasty, Replacement, Hip/methods , Femoral Neck Fractures/surgery , Hemiarthroplasty/methods , Aged , Aged, 80 and over , Blood Loss, Surgical/statistics & numerical data , Female , Humans , Length of Stay/statistics & numerical data , Male , Operative Time , Pain, Postoperative/etiology , Prosthesis Failure
3.
J Thromb Haemost ; 11(1): 132-41, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23205847

ABSTRACT

BACKGROUND: Hemophilia A results from a deficiency in factor VIII activity. Current treatment regimens require frequent dosing, owing to the short half-life of FVIII. A recombinant FVIII-Fc fusion protein (rFVIIIFc) was molecularly engineered to increase the half-life of FVIII, by 1.5-2-fold, in several preclinical animal models and humans. OBJECTIVE: To perform a biochemical and functional in vitro characterization of rFVIIIFc, with existing FVIII products as comparators. METHODS: rFVIIIFc was examined by utilizing a series of structural and analytic assays, including mass spectrometry following lysyl endopeptidase or thrombin digestion. rFVIIIFc activity was determined in both one-stage clotting (activated partial thromboplastin time) and chromogenic activity assays, in the context of the FXase complex with purified components, and in both in vitro and ex vivo rotational thromboelastometry (ROTEM) assays performed in whole blood. RESULTS: rFVIIIFc contained the predicted primary structure and post-translational modifications, with an FVIII moiety that was similar to other recombinant FVIII products. The von Willebrand factor-binding and specific activity of rFVIIIFc were also found to be similar to those of other recombinant FVIII molecules. Both chromogenic and one-stage assays of rFVIIIFc gave similar results. Ex vivo ROTEM studies demonstrated that circulating rFVIIIFc activity was prolonged in mice with hemophilia A in comparison with B-domain-deleted or full-length FVIII. Clot parameters at early time points were similar to those for FVIII, whereas rFVIIIFc showed prolonged improvement of clot formation. CONCLUSIONS: rFVIIIFc maintains normal FVIII interactions with other proteins necessary for its activity, with prolonged in vivo activity, owing to fusion with the Fc region of IgG(1) .


Subject(s)
Blood Coagulation/drug effects , Coagulants/pharmacology , Factor VIII/pharmacology , Hemophilia A/drug therapy , Immunoglobulin Fc Fragments/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cloning, Molecular , Coagulants/chemistry , Coagulants/pharmacokinetics , Cysteine Endopeptidases/metabolism , Disease Models, Animal , Factor VIII/chemistry , Factor VIII/genetics , Factor VIII/pharmacokinetics , Half-Life , Hemophilia A/blood , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/genetics , Male , Mass Spectrometry , Mice , Neoplasm Proteins/metabolism , Partial Thromboplastin Time , Peptide Mapping/methods , Protein C/metabolism , Protein Conformation , Protein Engineering , Protein Stability , Recombinant Fusion Proteins/pharmacokinetics , Recombinant Fusion Proteins/pharmacology , Structure-Activity Relationship , Thrombelastography , von Willebrand Factor/metabolism
5.
Hum Reprod ; 20(7): 1805-13, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15817590

ABSTRACT

BACKGROUND: The alpha and beta subunits of FSH were fused to the Fc domain of IgG1 either in a single chain or a heterodimer format. These molecules were absorbed through the epithelium in lung and intestine by neonatal Fc receptor (FcRn)-mediated transcytosis. METHODS AND RESULTS: Single chain and heterodimer FSH-Fc were made recombinantly in Chinese hamster ovary cells. Treatment of rats with a single s.c. dose of single chain or heterodimer FSH-Fc resulted in greater stimulation of ovarian weight (20.8+/-3.9 and 26.9+/-6.1 mg respectively) compared to those receiving vehicle (12.1+/-1.0 mg) or an equimolar dose of recombinant human FSH (14.3+/-1.7 mg). Both FSH-Fc fusion proteins were absorbed after oral dosing of newborn rats with long terminal half-lives of approximately 60 h, and pulmonary delivery in four cynomolgus monkeys produced maximum serum concentrations between 69 and 131 ng/ml with long terminal half-lives between 55 and 210 h. Serum inhibin levels increased after pulmonary dosing with single chain FSH-Fc (1.3- and 1.4-fold) and heterodimer FSH-Fc (5.9- and 7.1-fold) and remained elevated for >12 days after treatment with heterodimer FSH-Fc. CONCLUSIONS: We have shown that FSH-Fc fusion proteins have increased stability in blood and improved bioactivity in vivo, and that heterodimer FSH-Fc is more active in rats and monkeys than single chain FSH-Fc. These data suggest that Fc fusion proteins offer the potential for oral and pulmonary delivery of FSH.


Subject(s)
Follicle Stimulating Hormone/administration & dosage , Immunoglobulin Fc Fragments/administration & dosage , Administration, Oral , Aerosols , Animals , Animals, Newborn , Base Sequence , Biological Transport, Active , CHO Cells , Cricetinae , DNA, Complementary/genetics , Female , Follicle Stimulating Hormone/pharmacokinetics , Humans , Immunoglobulin Fc Fragments/metabolism , Macaca fascicularis , Male , Organ Size/drug effects , Ovary/anatomy & histology , Ovary/drug effects , Rats , Receptors, Fc/metabolism , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/pharmacokinetics , Testis/anatomy & histology , Testis/drug effects
6.
Nucleosides Nucleotides Nucleic Acids ; 20(4-7): 1067-78, 2001.
Article in English | MEDLINE | ID: mdl-11562960

ABSTRACT

Cyclin-dependent kinases (CDKs) belong to a class of enzymes that control the ability of a cell to enter into and proceed through the cell division cycle. Using purine as a scaffold, we have synthesized a number of nanomolar inhibitors of CDK-2/cyclin E. In this report, the synthesis of a series of piperidine-substituted purine analogs will be presented, as well as some of their in vitro and in vivo biological effects.


Subject(s)
Adenine/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , CDC2-CDC28 Kinases , Cyclin-Dependent Kinases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Piperidines/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Purines/chemical synthesis , Purines/pharmacology , Adenine/analogs & derivatives , Adenine/chemical synthesis , Animals , Cyclin-Dependent Kinase 2 , Drug Design , Drug Screening Assays, Antitumor , HT29 Cells/drug effects , Humans , Mice , Mice, Nude , Piperidines/chemical synthesis , Structure-Activity Relationship , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
7.
J Med Chem ; 44(4): 524-30, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11170642

ABSTRACT

Cyclin-dependent kinases (CDKs) are regulatory proteins of the eukaryotic cell cycle. They act after association with different cyclins, the concentrations of which vary throughout the progression of the cell cycle. As central mediators of cell growth, CDKs are potential targets for inhibitory molecules that would allow disruption of the cell cycle in order to evoke an antiproliferative effect and may therefore be useful as cancer therapeutics. We synthesized several inhibitory 2,6,9-trisubstituted purine derivatives and solved the crystal structure of one of these compounds, H717, in complex with human CDK2 at 2.6 A resolution. The orientation of the C2-p-diaminocyclohexyl portion of the inhibitor is strikingly different from those of similar moieties in other related inhibitor complexes. The N9-cyclopentyl ring fully occupies a space in the enzyme which is otherwise empty, while the C6-N-aminobenzyl substituent points out of the ATP-binding site. The structure provides a basis for the further development of more potent inhibitory drugs.


Subject(s)
Adenine/chemistry , CDC2-CDC28 Kinases , Cyclin-Dependent Kinases/chemistry , Enzyme Inhibitors/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/chemistry , Adenine/analogs & derivatives , Crystallography, X-Ray , Cyclin-Dependent Kinase 2 , Humans , Models, Molecular , Molecular Structure
8.
Anticancer Res ; 20(4): 2289-96, 2000.
Article in English | MEDLINE | ID: mdl-10953287

ABSTRACT

BACKGROUND: Three approaches to doxorubicin therapy are directly compared: free doxorubicin, liposomal doxorubicin and beta-glucuronidase-activated prodrug (HMR 1826). MATERIALS AND METHODS: The optimal dose of HMR 1826 was determined to be 200 mg/kg once a week and subsequent studies were carried out comparing HMR 1826 at 200 mg/kg 1x/wk, liposomal doxorubicin (Doxil) at 9 mg/kg 1x/wk and free doxorubicin at 7 mg/kg 1x/wk in seven different human tumor xenograft models. RESULTS: All three forms of doxorubicin inhibited tumor growth with similar efficacy in each of the tumor models with the exception of MDA-MB-231 tumor xenografts, which were resistant to free doxorubicin but sensitive to Doxil and HMR 1826. Overall less weight loss was observed with HMR 1826 treatment. CONCLUSIONS: The efficacy of HMR 1826 is equal to or better than that of doxorubicin and Doxil at a safe dose and schedule, indicating that the beta-glucuronidase activated prodrug approach is safe and effective.


Subject(s)
Antibiotics, Antineoplastic/therapeutic use , Doxorubicin/analogs & derivatives , Doxorubicin/therapeutic use , Glucuronates/therapeutic use , Neoplasms, Experimental/drug therapy , Prodrugs/therapeutic use , Animals , Doxorubicin/administration & dosage , Drug Administration Schedule , Drug Carriers , Female , Humans , Liposomes/administration & dosage , Male , Mice , Mice, Nude , Neoplasm Transplantation
9.
Biochem Pharmacol ; 55(6): 841-51, 1998 Mar 15.
Article in English | MEDLINE | ID: mdl-9586957

ABSTRACT

Six hundred triphenylethylenes were assayed for antiproliferative activity against MCF-7, LY2, and MDA-MB-231 breast cancer cells using sulforhodamine B dye to measure proliferation. Here we report on just 63 of the compounds, mostly clomiphene analogs, with substitutions on the alpha' or beta ring, at the vinyl position or in the side chain, of which 23 were active, as defined by antiproliferation IC50 values < or =1 microM. Activity profiles showed that 23 and 11 analogs were active toward MCF-7 and LY2, respectively, but none were active against MDA-MB-231. The IC50 values of tamoxifen were 2.0 microM against MCF-7 and 7.5 microM against LY2 and MDA-MB-231. Estradiol reversed antiproliferative activities of several E isomers but not their Z isomer counterparts. Clomiphene side chain analogs 46 [(E)-1-butanamine, 4-[4-(2-chloro-1,2-diphenylethenyl) phenoxy]-N,N-diethyl-dihydrogen citrate (MDL 103,323)] and 57 [(E)-N-[p-(2-chloro-1,2-diphenylvinyl) phenyl]-N,N-diethylethylenediamine dihydrogen citrate (MDL 101,986)] were 4- to 5-fold more effective than tamoxifen. Methylene additions up to (-CH2-)12 in the clomiphene side chain showed that analog 46 [(-CH2-)4 side chain] had maximal antiproliferative activity, binding affinity, and inhibition of transcription of an estrogen response element luciferase construct in transfected MCF-7 cells. Intraperitoneal administration of 46 or 57 inhibited progression of MCF-7 breast tumor xenografts in nude mice with ED50 values of <0.02 mg/mouse/day. Both analogs may hold promise for treating ER positive breast cancer and are of interest for further development.


Subject(s)
Antineoplastic Agents/pharmacology , Clomiphene/analogs & derivatives , Mammary Neoplasms, Experimental/drug therapy , Animals , Cell Division/drug effects , Clomiphene/pharmacology , Estradiol/pharmacology , Humans , Mice , Mice, Nude , Structure-Activity Relationship , Transplantation, Heterologous , Tumor Cells, Cultured
10.
Oncol Res ; 9(10): 543-52, 1997.
Article in English | MEDLINE | ID: mdl-9507532

ABSTRACT

The effect of the antitumor drug MDL 101,731 [(E)-2'-deoxy-2'-(fluoromethylene)cytidine] on tumor growth and on steady-state vascular endothelial growth factor (VEGF) mRNA levels in MDA-MB-231, PC-3, MCF-7, and HT-29 human tumor xenografts grown in nude mice was examined, using quantitative in situ hybridization. MDL 101,731 caused regression of MDA-MB-231 and PC-3 tumor xenografts, but only inhibition of growth (without regression) of MCF-7 xenografts. The drug caused inhibition of growth of HT-29 xenografts at low doses, and regression at high doses. When treatment with MDL 101,731 led to tumor regression, VEGF mRNA levels were decreased. When treatment led only to inhibition of growth, there was no significant change in VEGF mRNA. Further examination of the tumor xenografts revealed that elevated VEGF mRNA was associated with hypoxic zones surrounding areas of necrosis in the tumors, and that the drop in VEGF mRNA observed in tumors from mice treated with MDL 101,731 correlated with a loss of zones of necrosis. In contrast, treatment with cisplatin led to either an increase (PC-3) or no change (MDA-MB-231) in VEGF mRNA levels, and no loss of necrotic zones. Quantitative analysis of changes in VEGF mRNA levels was supported by immunohistochemical analysis of VEGF protein in the same tumor specimens. In vitro, MDL 101,731 was a potent inhibitor of VEGF secretion in cells exposed to hypoxia, whereas there was no effect of cisplatin on VEGF secretion by three of the four cell lines tested. These findings suggest that inhibition of VEGF expression by MDL 101,731 may distinguish this compound from other classes of cytotoxic agents, such as cisplatin.


Subject(s)
Antineoplastic Agents/therapeutic use , Deoxycytidine/analogs & derivatives , Endothelial Growth Factors/genetics , Lymphokines/genetics , Neoplasms, Experimental/drug therapy , RNA, Messenger/biosynthesis , Animals , Deoxycytidine/therapeutic use , Enzyme-Linked Immunosorbent Assay , Humans , Immunohistochemistry , Mice , Mice, Nude , Neoplasms, Experimental/metabolism , Transplantation, Heterologous , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
11.
Anticancer Res ; 16(5A): 2553-7, 1996.
Article in English | MEDLINE | ID: mdl-8917350

ABSTRACT

MDL 103,323, an enclomiphene analog, was tested for binding to the estrogen receptor, inhibition of human tumor xenografts and prevention of carcinogen-induced mammary tumors. MDL 103,323 had 5-6 fold greater affinity for the human estrogen receptor than did either tamoxifen or enclomiphene. Consistent with enhanced binding affinity, MDL 103,323 was more potent against MCF-7 cell proliferation and MCF-7 xenografts in nude mice were inhibited almost completely by MDL 103,323 doses > or = 0.02 mg/mouse/day (ED50 l 0.01 mg/mouse/day). N-methylnitrosourea induced rat mammary carcinomas were inhibited by > or = 50% at 0.003 mg/kg daily and by 90% at 0.1 mg/kg/day. The antitumor potency and efficacy of MDL 103,323 are striking and further evaluation of the compound for potential clinical utility is warranted.


Subject(s)
Antineoplastic Agents, Hormonal/therapeutic use , Breast Neoplasms/drug therapy , Clomiphene/analogs & derivatives , Clomiphene/therapeutic use , Enclomiphene , Animals , Antineoplastic Agents, Hormonal/metabolism , Clomiphene/metabolism , Drug Screening Assays, Antitumor , Female , Humans , Mammary Neoplasms, Experimental/chemically induced , Mammary Neoplasms, Experimental/prevention & control , Mice , Mice, Nude , Rats , Rats, Sprague-Dawley , Receptors, Estrogen/metabolism , Transplantation, Heterologous , Tumor Cells, Cultured
12.
J Steroid Biochem Mol Biol ; 58(1): 21-30, 1996 Apr.
Article in English | MEDLINE | ID: mdl-8809183

ABSTRACT

Steroidal antiestrogens appear to have at least two major modes of action in breast cancer cells, direct antagonism of estrogen binding to its receptor and depletion of estrogen receptors (ER) due to inhibition of dimerization of the receptor and a resultant destabilization of the receptor protein. In a search for other classes of compounds which would act as dimerization inhibitors, a novel substituted indole (8-{2-[1-(4-chlorobenzoyl)-5-hydroxy-2-methyl-1H-indol-3-yl]-acetylamino} octanoic acid butyl-methyl amide, MDL 101,906) was synthesized. Binding of the ER to its consensus response element (ERE) was apparently decreased in nuclear extracts from MCF-7 human breast cancer cell treated with MDL 101,906. This decreased binding was found to be due to depletion of ER based on direct measurement of ER using an enzyme-linked immunoassay. Other transcription factors were apparently unaffected by MDL 101,906 treatment. Whereas depletion of ER with a steroidal antiestrogen was almost complete after 3 h of treatment of MCF-7 cells, the effect of MDL 101,906 took significantly longer to occur, suggesting a fundamental difference in the mechanisms of action of the two drugs. This was also evident in the lack of binding of MDL 101,906 to the hormone binding domain of ER. MDL 101,906 treatment also caused depletion of ER mRNA in MCF-7 cells. Depletion of ER mRNA was noted by 3 h of drug treatment and was apparently almost complete after 24 h of treatment. Depletion of ER from MCF-7 cells led to a dose-dependent decrease in the expression of luciferase by an ERE-driven luciferase reporter gene assay system. The mechanism of MDL 101,906 appears to be unique and additional studies with this chemical class seem to be warranted to assess the potential for therapeutic utility.


Subject(s)
Breast Neoplasms/chemistry , Estrogen Antagonists/pharmacology , Indoles/pharmacology , Receptors, Estrogen/analysis , Base Sequence , Binding, Competitive , Breast Neoplasms/metabolism , Cell Extracts , Cell Nucleus , DNA/metabolism , Dose-Response Relationship, Drug , Estradiol/analogs & derivatives , Estradiol/metabolism , Estradiol/pharmacology , Estrogen Antagonists/chemical synthesis , Gene Expression , Genes, Reporter/genetics , Humans , Indoles/chemical synthesis , Molecular Sequence Data , Polyunsaturated Alkamides , RNA, Messenger/analysis , RNA, Neoplasm/analysis , Receptors, Estradiol/metabolism , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Time Factors , Transcription Factors/metabolism , Tumor Cells, Cultured
13.
Cell Growth Differ ; 7(3): 351-9, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8838865

ABSTRACT

We have isolated a variant of the MCF-7 human breast tumor that is characterized by a hormone-independent, yet hormone-responsive, phenotype. This tumor, designated MCF-WES, was derived from MCF-7 tumor cells implanted in the mammary fat pad of a nude mouse in the absence of estradiol supplementation. MCF-WES tumors remain responsive to estradiol; however, unlike the parental MCF-7 tumors, they are stimulated to grow by tamoxifen. Additionally, MCF-WES cells are resistant to the pure steroidal antiestrogen, ICI 182,780. To our knowledge, a tumor with this combination of properties has not yet been described. Nuclear estrogen receptor (ER) levels in MCF-WES cells were 10% of those for MCF-7 under steroid-depleted conditions. MCF-WES tumor ER levels were 32% of those in MCF-7 tumors. Similarly, in vivo expression of ER mRNA for MCF-WES was 20% of levels determined for MCF-7. Further characterization of MCF-WES cells showed that they have increased levels of AP-1 DNA-binding activity. The marked increase in AP-1 binding activity may act to bypass the hormone dependence that is a characteristic of MCF-7 cells. It is also probable that the increase in AP-1 binding activity is responsible for the finding that MCF-WES cells secrete greater quantities of metalloproteinase activity in comparison to parental MCF-7 cells, suggesting progression to a more invasive, malignant phenotype. More complete characterization of this new cell line will help elucidate hormone-independent breast cancer and possibly identify targets for therapy.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , DNA, Neoplasm/metabolism , Estradiol/analogs & derivatives , Estrogen Antagonists/pharmacology , Transcription Factor AP-1/metabolism , Animals , Cell Division , DNA, Neoplasm/analysis , Drug Resistance, Neoplasm , Estradiol/pharmacology , Fulvestrant , Gelatinases/metabolism , Humans , Mice , Mice, Nude , Phenotype , Protein Binding , RNA, Messenger/analysis , Receptors, Estrogen/analysis , Receptors, Estrogen/genetics , Receptors, Progesterone/analysis , Tamoxifen/pharmacology , Tumor Cells, Cultured
14.
Exp Cell Res ; 222(1): 54-60, 1996 Jan 10.
Article in English | MEDLINE | ID: mdl-8549673

ABSTRACT

MDL 101,731, (E)2'-fluoromethylene-2'-deoxycytidine, is an irreversible inhibitor of ribonucleotide diphosphate reductase and causes regression of human tumors in nude mouse models. Messenger RNA levels for testosterone-repressed prostatic message-2 (TRPM-2), a transcript that increases in human tumor xenografts undergoing programmed cell death, were analyzed by in situ hybridization. Xenografts derived from a human prostate tumor cell line (PC-3) regressed following treatment with MDL 101,731 and the relative levels of TRPM-2 mRNA increased up to threefold in drug-treated animals. Apoptosis in the tumor xenografts was further indicated by in situ labeling of DNA strand breaks by incorporation of biotinylated-dUTP with terminal deoxynucleotidyl transferase. In vitro, PC-3 cells incubated with MDL 101,731 showed evidence of apoptosis based on flow cytometry and DNA laddering. These data support the hypothesis that MDL 101,731 stimulates programmed cell death in regressing PC-3 xenografts.


Subject(s)
Apoptosis/drug effects , Deoxycytidine/analogs & derivatives , Glycoproteins/genetics , Molecular Chaperones , Neoplasm Proteins/genetics , Prostatic Neoplasms/pathology , Ribonucleoside Diphosphate Reductase/antagonists & inhibitors , Animals , Biomarkers, Tumor , Clusterin , Deoxycytidine/pharmacology , Enzyme Inhibitors , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , In Situ Hybridization , Male , Mice , Mice, Nude , Neoplasm Transplantation , Prostatic Neoplasms/metabolism , RNA, Complementary , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/biosynthesis , Transplantation, Heterologous , Tumor Cells, Cultured
15.
Cancer Res ; 56(2): 359-61, 1996 Jan 15.
Article in English | MEDLINE | ID: mdl-8542592

ABSTRACT

We examined the effects of MDL101731, a novel ribonucleoside reductase inhibitor, against human glioblastomas and neuroblastoma, both in vitro and in xenograft models, to determine its activity against malignant brain tumors. MDL101731 produced a concentration-dependent inhibition of both glioblastoma cell lines (HS683 and J889H) and neuroblastoma (SK-N-MC) in nanomolar concentrations (IC50, 30-90 nM). s.c. xenografts of human glioblastoma (D54) in athymic mice increased to five times their initial volume at a median of 7.4 days in control animals, while tumor regression occurred in 12 of 12 animals treated with MDL101731 (100 mg/kg, i.p., two times/week) during 22 days of treatment (P < 0.0001). Intracerebral implants of D54 carried a median survival of 20 days in control animals, whereas animals receiving MDL101731 (100 mg/kg, i.p., two times/week, days 10-35) had a median survival of 46.5 days (P < 0.0001). Intracerebral xenografts of SK-N-MC in athymic mice resulted in a median survival of 23 days in control animals and 26 days in animals treated with carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea 20 mg/kg/week, i.v. x 2; difference not significant). There was 90% survival in animals treated with MDL101731 (200 mg/kg, i.v., two times/week, days 7-35) up to 90 days after implant. These studies indicate that MDL101731 has potent antiproliferative activity against human malignant brain tumors.


Subject(s)
Antineoplastic Agents/pharmacology , Brain Neoplasms/drug therapy , Deoxycytidine/analogs & derivatives , Enzyme Inhibitors/pharmacology , Glioblastoma/drug therapy , Ribonucleoside Diphosphate Reductase/antagonists & inhibitors , Animals , Brain Neoplasms/enzymology , Deoxycytidine/pharmacology , Glioblastoma/enzymology , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Transplantation, Heterologous
16.
Anticancer Res ; 15(4): 1179-82, 1995.
Article in English | MEDLINE | ID: mdl-7653997

ABSTRACT

Daily oral or intravenous administration of the ribonucleoside diphosphate reductase inhibitor, (E)-2'-deoxy-2'-(fluoromethylene)cytidine (MDL 101,731), to nude mice caused rapid regression of colon and prostate xenografts. Studies were performed to optimize dosing schedule and route of administration. MDL 101,731 was tested against colon (HT-29) and prostate (PC-3) xenografts using twice weekly oral and intravenous administration. PC-3 tumors regressed almost completely with doses of 20 mg/kg. HT-29 xenografts regressed during intravenous administration of 100 mg/kg MDL 101,731, whereas oral administration was less effective. Based on these data it seems that MDL 101,731 is effective when administered intravenously, twice weekly and is an excellent candidate for clinical development against solid tumors.


Subject(s)
Colonic Neoplasms/drug therapy , Deoxycytidine/analogs & derivatives , Prostatic Neoplasms/drug therapy , Ribonucleotide Reductases/antagonists & inhibitors , Animals , Deoxycytidine/administration & dosage , Deoxycytidine/therapeutic use , Humans , Male , Mice , Mice, Nude , Neoplasm Transplantation , Transplantation, Heterologous , Tumor Cells, Cultured
17.
FASEB J ; 9(2): 279-83, 1995 Feb.
Article in English | MEDLINE | ID: mdl-7781931

ABSTRACT

In situ methodologies allow qualitative and semi-quantitative analysis of spatial gene expression in whole organisms or tissues. We have applied quantitative autoradiography to in situ hybridizations of sections from human breast tumor xenografts to measure mRNA levels for ornithine decarboxylase, estrogen receptor, transforming growth factor alpha, and glyceraldehyde-3-phosphate dehydrogenase. Comparisons of control and tamoxifen-treated animals show significant decreases in MCF-7 tumor estrogen receptor mRNA levels in the drug-treated animals. Combining quantitative autoradiography with in situ hybridization allows measurement of absolute rather than relative mRNA levels for genes of interest, and to monitor effector-induced changes in these mRNAs in vivo.


Subject(s)
Autoradiography/methods , Breast Neoplasms/metabolism , In Situ Hybridization/methods , Ornithine Decarboxylase/biosynthesis , RNA, Messenger/analysis , Receptors, Estrogen/biosynthesis , Animals , Breast Neoplasms/pathology , Carbon Radioisotopes , Cell Line , DNA Probes , Gene Expression/drug effects , Humans , Mice , Mice, Nude , Molecular Sequence Data , Tamoxifen/pharmacology , Transforming Growth Factor alpha/biosynthesis , Transplantation, Heterologous , Tritium , Tumor Cells, Cultured
18.
Biochem Biophys Res Commun ; 204(1): 264-72, 1994 Oct 14.
Article in English | MEDLINE | ID: mdl-7945369

ABSTRACT

A correlation between changes in protein kinase C (PKC) activity and tumor metastasis has been reported previously with several murine tumor cell lines. Treatment of a human metastatic melanoma cell line, M24met, with phorbol ester, phorbol-12-myristate-13-acetate (PMA), followed by injection into the tail vein of scid mice doubled pulmonary metastasis. Adhesion of M24met cells exposed to PMA, was enhanced to collagens I and IV, but not to laminin or fibronectin, suggesting a change in specific adhesion receptors on the tumor cells. Treatment of M24met cells with PMA did not affect de novo synthesis of integrin subunits (alpha 2, alpha 3, beta 1) known to form collagen receptors. However, PMA stimulated the phosphorylation of integrin subunits alpha 3 and beta 1 on serine. Therefore, PMA effects on metastasis and cell adhesion may occur through PKC-mediated phosphorylation of integrins.


Subject(s)
Cell Adhesion , Integrins/metabolism , Lung Neoplasms/secondary , Melanoma/pathology , Melanoma/physiopathology , Protein Kinase C/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Amino Acids/analysis , Animals , Cell Line , Collagen , Fibronectins , Humans , Laminin , Lung Neoplasms/pathology , Melanoma/enzymology , Mice , Mice, SCID , Phosphorylation , Transplantation, Heterologous , Tumor Cells, Cultured
19.
Cancer Res ; 54(6): 1485-90, 1994 Mar 15.
Article in English | MEDLINE | ID: mdl-8137252

ABSTRACT

(E)-2'-Deoxy-2'-(fluoromethylene)cytidine (MDL 101,731) is a mechanism-based inhibitor of ribonucleoside diphosphate reductase (J. Stubbe, personal communication), an enzyme involved in DNA synthesis and therefore a potential target for cancer chemotherapy. In the present report, we show that MDL 101,731 inhibits the proliferation of several human breast cancer cell lines, including the estrogen-dependent cell line, MCF-7, and the estrogen-independent cell lines MDA-MB-231, MDA-MB-468, and MDA-MB-435 in vitro at nanomolar concentrations (50% inhibitory concentration, 15-26 nM). Administration of MDL 101,731 caused marked regression of tumors which formed after s.c. inoculation of all four of the cell lines in athymic (nude) mice. MDA-MB-231 tumors were found to be most sensitive to MDL 101,731 with a 90-100% cure rate at doses of MDL 101,731 between 2 and 20 mg/kg, given as once daily i.p. injections, 5 days/week for as little as 3 weeks. Almost complete cessation of MDA-MB-231 tumor growth was obtained with a dose of 0.5 mg/kg MDL 101,731 following the same dosing regimen. MDA-MB-468, MDA-MB-435, and MCF-7 tumors were not as sensitive as MDA-MB-231, but tumor regression of 50, 65, and 80%, respectively, was obtained after 5-6 weeks of treatment. The effects of MDL 101,731 on spontaneous metastasis of MDA-MB-435 cells from the mammary fat pad to the lung was also examined, and it was found that the number of lung metastases was significantly decreased if mice received MDL 101,731 while the primary tumors were growing and after primary tumors were surgically excised. Additionally, preliminary evidence raises the possibility that MDL 101,731 may induce apoptosis in MDA-MB-231 tumors. Our data suggest that the use of MDL 101,731 for the treatment of breast cancer and possibly other solid tumors should be pursued.


Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/enzymology , Deoxycytidine/analogs & derivatives , Neoplasms, Hormone-Dependent/drug therapy , Neoplasms, Hormone-Dependent/enzymology , Ribonucleoside Diphosphate Reductase/antagonists & inhibitors , Animals , Breast Neoplasms/pathology , Cell Death/drug effects , Cell Death/physiology , Cell Division/drug effects , Deoxycytidine/pharmacology , Estrogens , Female , Humans , Mice , Mice, Nude , Neoplasm Metastasis , Neoplasm Transplantation , Neoplasms, Hormone-Dependent/pathology , Transplantation, Heterologous , Tumor Cells, Cultured/drug effects
20.
Biochem J ; 293 ( Pt 1): 263-7, 1993 Jul 01.
Article in English | MEDLINE | ID: mdl-7687129

ABSTRACT

Integrin binding to proteins often involves recognition of domains containing the arginine-glycine-aspartate (RGD) motif. Different binding affinities and specificities of the integrin-ligand protein interactions involve additional protein domains. The n.m.r. structure of the snake-venom protein echistatin suggested that the C-terminal portion of the molecule might be important, in addition to the RGD domain, in binding to the integrin glycoprotein IIbIIIa (GPIIbIIIa) [Saudek, Atkinson and Pelton (1991) Biochem. 30, 7369-7372]. The synthetic C-terminal peptide, echistatin-(40-49), PRNPHKGPAT, (1) inhibited binding of GPIIbIIIa to immobilized echistatin (IC50 3-6 mM), but did not inhibit binding of GPIIbIIIa to immobilized fibrinogen (up to 5 mM peptide), (2) activated GPIIbIIIa binding to fibronectin and vitronectin, usual ligands for the activated integrin, (3) activated binding of GPIIbIIIa to collagen type I and type IV, proteins not usually regarded as ligands for the integrin, and (4) stimulated 125I-fibrinogen binding by human platelets. These findings argue for an interaction of this non-RGD domain in echistatin with GPIIbIIIa, leading to activation of the integrin and extension of the ligand specificity to include immobilized collagen.


Subject(s)
Blood Platelets/drug effects , Peptides , Platelet Aggregation Inhibitors/pharmacology , Platelet Membrane Glycoproteins/metabolism , Viper Venoms/pharmacology , Amino Acid Sequence , Binding Sites , Blood Platelets/metabolism , Collagen/metabolism , Enzyme-Linked Immunosorbent Assay , Fibrinogen/metabolism , Fibronectins/metabolism , Glycoproteins/metabolism , Humans , Intercellular Signaling Peptides and Proteins , Molecular Sequence Data , Vitronectin
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