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1.
Biochem Pharmacol ; 57(8): 917-25, 1999 Apr 15.
Article in English | MEDLINE | ID: mdl-10086326

ABSTRACT

It has been shown previously that 4-anilino quinazolines compete with the ability of ATP to bind the epidermal growth factor receptor (EGF-R), inhibit EGF-stimulated autophosphorylation of tyrosine residues in EGF-R, and block EGF-mediated growth. Since millimolar concentrations of ATP in cells could reduce the efficacy of 4-anilino quinazolines in cells and the activity of these compounds would not be sustained once they were removed from the body, we reasoned that irreversible inhibitors of EGF-R might improve the activity of this series of compounds in animals. Molecular modeling of the EGF-R kinase domain was used to design irreversible inhibitors. We herein describe one such inhibitor: N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]2-butynamide, known as CL-387,785. This compound covalently bound to EGF-R. It also specifically inhibited kinase activity of the protein (IC50 = 370+/-120 pM), blocked EGF-stimulated autophosphorylation of the receptor in cells (ic50 approximately 5 nM), inhibited cell proliferation (IC50 = 31-125 nM) primarily in a cytostatic manner in cell lines that overexpress EGF-R or c-erbB-2, and profoundly blocked the growth of a tumor that overexpresses EGF-R in nude mice (when given orally at 80 mg/kg/day for 10 days, daily). We conclude that CL-387,785 is useful for studying the interaction of small molecules with EGF-R and may have clinical utility.


Subject(s)
Enzyme Inhibitors/pharmacology , ErbB Receptors/antagonists & inhibitors , Quinazolines/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Cycle/drug effects , Cell Division/drug effects , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/metabolism , ErbB Receptors/metabolism , Female , Mice , Mice, Nude , Models, Molecular , Neoplasm Transplantation , Neoplasms, Experimental/drug therapy , Phosphorylation/drug effects , Quinazolines/chemical synthesis , Tumor Cells, Cultured
2.
Anal Chem ; 69(14): 2716-26, 1997 Jul 15.
Article in English | MEDLINE | ID: mdl-9230680

ABSTRACT

Calicheamicin derivatives (MW approximately 1500) and monoclonal antibodies (MoAbs) conjugated to calicheamicin derivatives (MW approximately 150,000) were analyzed by UV-MALDI/MS, IR-MALDI/MS, and ESI/MS. These materials are potent anticancer agents. Calicheamicin derivatives and conjugates rapidly degrade upon UV irradiation but are relatively stable during IR irradiation and under ESI conditions. A unique feature of IR-MALDI/MS is a 2 times enhancement in resolution relative to UV-MALDI/MS for masses above approximately 50,000 Da resulting in a molecular ion envelope containing a series of partially resolved peaks of the calicheamicin-MoAb conjugates. The mass shift difference between the peak maxima corresponded to the mass change due to the covalent addition of calicheamicin derivatives to the monoclonal antibody. The distribution of the calicheamicin derivatives in the monoclonal antibodies was computed by deconvoluting the partially resolved peak envelope. A unique feature of the ESI mass spectra, under unit resolution conditions, is that the distribution of the carbohydrates can be well resolved for pure MoAbs and can be only partially resolved for conjugated MoAbs. Average loading values for calicheamicia derivatives when conjugated to MoAbs were computed from UV-MALDI/MS, IR-MALDI/MS, and ESI/MS data and the results compared with the average loading values obtained by UV absorption spectrometry. Very low average loading values were computed from UV-MALDI/MS data due to the degradation of the conjugated calicheamicin derivatives during the UV irradiation process. The IR-MALDI/MS average loading values, obtained with glycerol as the matrix, were consistent with the UV absorption spectrometry values for conjugates having hydrolytically stable linkers, but not when the linker contained a hydrolytically labile hydrazone. ESI/MS average loading values were generally lower than the corresponding values obtained by IR-MALDI/MS. The average loading values and distributions obtained using IR-MALDI/MS were more reliable than the corresponding ESI/MS values because the partially resolved, singly and doubly charged peaks in the IR-MALDI spectra can be mathematically deconvoluted, while the overlapping, highly multiply charged peaks of the electrospray spectra can only be partially deconvoluted.


Subject(s)
Aminoglycosides , Anti-Bacterial Agents/chemistry , Antibiotics, Antineoplastic/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Anti-Bacterial Agents/analysis , Antibiotics, Antineoplastic/analysis , Antibodies, Monoclonal/chemistry , Carbohydrate Sequence , Drug Delivery Systems , Infrared Rays , Mass Spectrometry , Molecular Sequence Data , Molecular Structure , Ultraviolet Rays
3.
Am J Surg Pathol ; 16(9): 868-76, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1384377

ABSTRACT

Adenomyoepitheliomas of the breast have been considered to have limited metastatic potential; axillary node metastasis has been reported, but there has been no report of distant metastasis. We report six cases, including two malignant adenomyoepitheliomas, one of which metastasized to the lung and brain. Patient age ranged from 26 to 63 years (mean 46). Primary tumors were solitary and measured 0.9-3.5 cm (mean 1.7). Five of six tumors presented as palpable masses. Two patients treated by local resection have no evidence of disease at 5 and 18 months' follow-up. Two patients treated by local resection had recurrences, one at 48 the other at 60 months. The fifth patient had a spindle-cell type adenomyoepithelioma diagnosed as malignant because of high mitotic rate and cytologic atypicality of the myoepithelial component. This patient was treated by mastectomy and has no evidence of disease at 18 months. The sixth patient, initially treated by local excision, had six local recurrences over 52 months treated by reexcisions, mastectomies, and radiation. A lung metastasis was resected at 54 months and brain metastases were identified at 60 months with death occurring at 64 months. Both malignant adenomyoepitheliomas had high mitotic rates [11-14/10 high-power fields (HPF)] diffusely throughout the tumors and foci of cytologically malignant cells. The malignant adenomyoepithelioma that metastasized had an infiltrative growth pattern that increased with successive local recurrences. The four other tumors had only isolated areas of mitotic activity (maximum 1-9/10 HPF) and minimal cytologic atypia. Immunohistochemistry performed on five of six cases confirmed dual epithelial/myoepithelial cell populations in all tumors examined, including the metastasis. Electron microscopic examination of the malignant adenomyoepithelioma that metastasized also confirmed dual epithelial/myoepithelial cell populations in a local recurrence and the lung metastasis. We conclude that there is a spectrum of behavior for breast adenomyoepitheliomas with potential for local recurrence and, rarely, distant metastasis.


Subject(s)
Breast Neoplasms/pathology , Myoepithelioma/pathology , Adult , Brain Neoplasms/secondary , Breast Neoplasms/chemistry , Breast Neoplasms/ultrastructure , Carcinoembryonic Antigen/analysis , Female , Humans , Immunohistochemistry , Keratins/analysis , Lung Neoplasms/secondary , Membrane Glycoproteins/analysis , Microscopy, Electron , Middle Aged , Mitotic Index , Mucin-1 , Myoepithelioma/chemistry , Myoepithelioma/ultrastructure , Neoplasm Metastasis/pathology , Recurrence
4.
Anal Chem ; 63(21): 2470-81, 1991 Nov 01.
Article in English | MEDLINE | ID: mdl-1763805

ABSTRACT

The chemically averaged molecular weights of a variety of native and conjugated monoclonal antibodies, approximately 150,000, were measured by matrix-assisted UV-laser desorption/ionization mass spectrometry. The average mass of the carbohydrate present in a monoclonal antibody was estimated from the difference between the measured mass of the monoclonal antibody and the mass of the protein present in the monoclonal antibody computed from the amino acid translation of the DNA sequence. The loading of chelators and anticancer drugs conjugated to a monoclonal antibody was quantitated from the difference in the measured masses for the conjugated and untreated monoclonal antibody relative to the expected mass change upon conjugation of 1 mol of chelator or drug. The loading results obtained by mass spectrometry were consistent in most cases with measurements obtained by radioactivity trace assay or UV spectrometry. Similar matrix-assisted UV-laser desorption/ionization mass spectrometric studies were also made after reducing untreated and conjugated monoclonal antibodies with dithiothreitol to determine the distribution of carbohydrate and chelator between the light and heavy chains of the molecules. Matrix-assisted UV-laser desorption/ionization mass spectra were used to compute loading values for covalently bound drugs and proteins, while the loading values obtained by use of gel-filtration HPLC and UV spectrometry cannot distinguish between covalently and noncovalently bound drugs and proteins.


Subject(s)
Antibodies, Monoclonal/analysis , Carbohydrates/analysis , Chelating Agents/analysis , Pharmaceutical Preparations/analysis , Lasers , Mass Spectrometry , Spectrophotometry, Ultraviolet
5.
Ann Intern Med ; 110(10): 847, 1989 May 15.
Article in English | MEDLINE | ID: mdl-2712470

Subject(s)
Reference Values , Humans
6.
Crit Rev Biotechnol ; 6(3): 253-71, 1987.
Article in English | MEDLINE | ID: mdl-3146436

ABSTRACT

Plasminogen activators (PAs) are proteases that convert plasminogen to plasmin. Plasmin, in turn, is a protease that can lyse a fibrin clot and, therefore, PAs have a primary role in fibrinolysis. Two PAs, urokinase (UK) and streptokinase (SK), have been available for therapeutic use for years. Unfortunately, both can cause systemic fibrinogenolysis and other side effects which have limited their use. Interest has focused on a different enzyme, tissue plasminogen activator (t-PA), which will cause specific clot lysis without systemic problems. The gene for t-PA has been cloned and many biotechnology firms are preparing to produce t-PA for therapeutic use. The properties and potential for therapy of t-PA are reviewed and compared to new forms of other activators, such as pro-urokinase. How the interactions of PAs and inhibitors may affect the use of PAs is also discussed.


Subject(s)
Plasminogen Activators/therapeutic use , Amino Acid Sequence , Animals , Humans , Molecular Sequence Data , Myocardial Infarction/drug therapy , Thrombosis/drug therapy , Tissue Plasminogen Activator/therapeutic use , Urokinase-Type Plasminogen Activator/therapeutic use
7.
Science ; 224(4649): 610-2, 1984 May 11.
Article in English | MEDLINE | ID: mdl-6546810

ABSTRACT

Isopenicillin N synthetase (cyclase) has been purified to homogeneity from Cephalosporium acremonium strain C-10. The enzyme has a molecular weight of 40,000 to 42,000 and yields a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was purified in 10 percent yield by a combination of protamine sulfate and ammonium sulfate precipitations, gel filtration, and ion-exchange high-performance liquid chromatography. The purified enzyme can be stabilized with sucrose and stored at -20 degrees C for several weeks without any loss in activity.


Subject(s)
Enzymes/isolation & purification , Oxidoreductases , Penicillins/biosynthesis , Acremonium/enzymology , Chromatography, Gel , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Enzymes/metabolism
8.
Ann Clin Lab Sci ; 10(3): 204-8, 1980.
Article in English | MEDLINE | ID: mdl-6994579

ABSTRACT

The familial nonhemolytic hyperbilirubinemias include the syndromes of Gilbert, Crigler-Najjar, Dubin-Johnson and Rotor. Gilbert's syndrome is probably very common in occult form, and patients come to clinical attention partially owing to subtle coincidental hemolysis. The biochemical defect may lie not in microsomal glucuronyl transferase but rather in the plasma membrane enzyme which transglucuronidates bilirubin monoglucuronide to diglucuronide. Patients with Crigler-Najjar type I, a severe disease, exhibit virtual absence of glucuronyltransferase. Type II is milder and appears related to Gilbert's syndrome. Dubin-Johnson's syndrome and Rotor's syndrome, the conjugated hyperbilirubinemias, are separate entities. The former is a block in hepatic excretion, while the defect in the latter lies at least partially in uptake of bilirubin.


Subject(s)
Hyperbilirubinemia, Hereditary/diagnosis , Crigler-Najjar Syndrome/diagnosis , Crigler-Najjar Syndrome/etiology , Gilbert Disease/diagnosis , Gilbert Disease/etiology , Humans , Hyperbilirubinemia, Hereditary/etiology , Jaundice, Chronic Idiopathic/diagnosis , Jaundice, Chronic Idiopathic/etiology
10.
Ann Clin Lab Sci ; 9(4): 268-74, 1979.
Article in English | MEDLINE | ID: mdl-485090

ABSTRACT

Malignant tumours of nonendocrine tissues may produce ectopic hormones. The most likely mechanism is depression of genes which code for hormones. Ectopic hormones are invariably peptides, and each is identical to some peptide product of an endocrine gland. However, the majority of ectopic hormones occur as biologically inactive precursors or subunits and therefore remain occult unless they are specifically sought. When appropriate assays are made for such inactive forms, it is found that ectopic production of hormone-like peptides occurs frequently. Clinical syndromes result only in the relatively rare patients in whom a biologically active form is synthesized in large quantities. Laboratory research in this area improves our understanding of genetic control mechanisms in neoplasia. Ectopic hormones may be of limited use in diagnosis of cancer, especially when multiple markers are measured simultaneously.


Subject(s)
Hormones/metabolism , Neoplasms/metabolism , Chorionic Gonadotropin/metabolism , Female , Humans , Neoplasms/genetics , Placental Lactogen/metabolism , Pregnancy
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