Comparison of the impact of different anionic salts on the acid-base status and calcium metabolism in non-lactating, non-pregnant dairy cows.

Eleven mature, non-lactating, non-pregnant, Holstein-Friesian crossbred cows were given, via a ruminal cannula, 2000mEq of one of three chloride salts, four sulfate salts, two combinations of anionic salts (AS), sodium chloride (as neutral salt), or water as control. The salts and controls were assigned in an 11x11 Latin square and the cows were randomly distributed. All of the AS induced a metabolic acidosis that resulted in a small reduction of blood pH, base excess, and bicarbonate (P<0.001), and notable changes in urinary pH, net acid base excretion (P<0.001), and urinary calcium excretion (P<0.001). Only calcium chloride had a significantly greater impact on acid-base status (ABS) than the sulfate salts. The effect of other chloride salts did not differ from calcium sulfate. There was no indication that chloride salts in general have a greater impact than sulfate salts on the ABS.

Conditional up-regulation of MHC class I in skeletal muscle leads to self-sustaining autoimmune myositis and myositis-specific autoantibodies.

In the human inflammatory myopathies (polymyositis and dermatomyositis), the early, widespread appearance of MHC class I on the surface of muscle cells and the occurrence of certain myositis-specific autoantibodies are striking features. We have used a controllable muscle-specific promoter system to up-regulate MHC class I in the skeletal muscles of young mice. These mice develop clinical, biochemical, histological, and immunological features very similar to human myositis. The disease is inflammatory, limited to skeletal muscles, self-sustaining, more severe in females, and often accompanied by autoantibodies, including, in some mice, autoantibodies to histidyl-tRNA synthetase, the most common specificity found in the spontaneous human disease, anti-Jo-1. This model suggests that an autoimmune disease may unfold in a highly specific pattern as the consequence of an apparently nonspecific event-the sustained up-regulation of MHC class I in a tissue-and that the specificity of the autoantibodies derives not from the specificity of the stimulus, but from the context, location, and probably the duration of the stimulus. This model further suggests that the presumed order of events as an autoimmune disease develops needs to be reconsidered.

The inhibition of apoptosis in myositis and in normal muscle cells.

The mechanism of injury and death of muscle cells in the inflammatory myopathies (dermatomyositis, polymyositis, and inclusion body myositis) remains obscure. We and others have not detected apoptosis in the muscle biopsies from patients with myositis despite clear evidence of cell damage and loss. We provide evidence in this study that Fas ligand (FasL) as well as Fas is present on muscle cells and inflammatory cells in myositis biopsies: Fas is present on most muscle cells and lymphocytes, and FasL is present on degenerating muscle cells and many infiltrating mononuclear cells. The expression of both Fas and FasL in the inflamed tissue makes the absence of apoptosis more striking. To address the mechanisms of this resistance to classical apoptosis in muscle cells, we have investigated the expression of the antiapoptotic molecule FLICE (Fas-associated death domain-like IL-1-converting enzyme)-inhibitory protein (FLIP) in muscle biopsies of myositis patients and in cultured human skeletal muscle cells. Using laser capture microscopy, we have shown that FLIP is expressed in the muscle fibers and on infiltrating lymphocytes of myositis biopsies. Furthermore, we have shown that FLIP, but not Bcl-2, is expressed in cultured human skeletal muscle cells stimulated with proinflammatory cytokines, and inhibition of FLIP with antisense oligonucleotides promotes significant cleavage of poly(ADP-ribose) polymerase autoantigen, a sensitive indicator of apoptosis. These studies strongly suggest that the resistance of muscle to Fas-mediated apoptosis is due to the expression of FLIP in muscle cells in the inflammatory environment in myositis.

Costimulatory markers in muscle of patients with idiopathic inflammatory myopathies and in cultured muscle cells.

In an attempt to understand the mechanisms of cell injury in the inflammatory myopathies, we analyzed the expression of costimulatory molecules, CTLA4, CD28, CD86, CD40, and CD154 as well as HLA class I, HLA class II, and ICAM-I in normal muscle and in muscle biopsies from patients with polymyositis (PM) or dermatomyositis (DM). By immunohistochemical staining, DM and PM biopsies showed the presence of CTLA4, CD28, CD86, and CD40 on inflammatory cells. More strikingly, however, low levels of CTLA4 and CD28 were observed on muscle cells. The expression of CTLA4 and CD28 on nonlymphoid cells has not been previously reported. These unexpected findings were confirmed in cultured normal human myoblasts: various proinflammatory cytokines induced the expression of CTLA4 and CD28 on normal human muscle cells. The sequences of the cDNAs were found to be identical to the sequences for these molecules in T cells. The data suggest a novel complexity in the network of cellular interactions between the infiltrated immune cells and the muscle cells in which the normal relationship between infiltrating inflammatory cells and target tissue is under a previously unrecognized set of controls.

A variety of cytokines and immunologically relevant surface molecules are expressed by normal human skeletal muscle cells under proinflammatory stimuli.

Muscle is an attractive target for gene therapy and for immunization with DNA vaccines and is also the target of immunological injury in myositis. It is important therefore to understand the immunologic capabilities of muscle cells themselves. In this study, we show that proinflammatory stimuli induce the expression of other cytokines such as IL-6, transforming growth factor-beta (TGF-beta), and granulocyte-macrophage colony-stimulating factor (GM-CSF) by muscle cells themselves, as well as the up-regulation of human leucocyte antigen (HLA) class I, class II and intercellular adhesion molecule-1 (ICAM-1). Thus, muscle cells have an inherent ability to express and respond to a variety of cytokines and chemokines. The levels of HLA class I, class II and ICAM-1 in inflamed muscle may be affected by the secreted products of the stimulation.

Synthesis and antitumor evaluation of selected 5,6-disubstituted 1(2)H-indazole-4,7-diones.

A series of novel aziridinyl-substituted 1(2)H-indazole-4,7-diones and related 1(2)H-indazole-4,7-diones was synthesized and tested against Ehrlich ascites carcinoma growth in male CF1 mice. Ten of the test compounds, including two aziridinyl-substituted 1(2)H-indazole-4,7-diones, were found to be significantly active (inhibition of tumor growth greater than 80%) in the Ehrlich ascites carcinoma screen. Several structure-activity relationships were indicated for antitumor activity in this screen. An aziridinyl-substituted derivative, 5-aziridinyl-6-chloro-1H-indazole-4,7-dione (8a), also exhibited significant activity against the growth of P-388 lymphocytic leukemia cells in male BDF1 mice (% T/C = 145; % T/C greater than 125 is considered significant).

Effects of 3-chloromethylthiochromone-1,1-dioxide on nucleic acid, protein, and aerobic and anaerobic metabolism of Ehrlich ascites tumor cells.

3-Chloromethylthiochromone-1,1-dioxide was observed to be a potent inhibitor of Ehrlich ascites carcinoma growth and a moderate inhibitor of P-388 lymphocytic leukemia growth at 10 mg/kg/day. Preliminary in vitro studies showed that the agents significantly inhibited RNA and DNA synthesis in Ehrlich ascites cells. In vivo studies after dosing on Days 6, 7, and 8 demonstrated the same reductions in nucleic acid synthesis and a moderate reduction in protein synthesis. The primary site of nucleic acid synthesis, which was blocked by 3-chloromethylthiochromone, was at orotidine monophosphate decarboxylase in the primidine pathway. Other enzymes, in anaerobic and aerobic glycolysis, which were blocked include hexokinase, phosphofructokinase, succinic and alpha-ketoglutarate dehydrogenases, as well as States 3 and 4 of oxidative phosphorylation.

Synthesis and antitumor testing of 3-methenylthiochroman-4-one-1,1-dioxide.

Treatment of thiochroman-4-one-1,1-dioxide (II) with paraformaldehyde and dimethylamine hydrochloride in isopropyl alcohol at reflux afforded directly in 80% yield the dimeric dihydropyran (IV), corresponding to the dimerization of the target compound 3-methenyl-thiochroman-4-one-1,1-dioxide (III). Neither the monomer III nor the expected Mannich base, 3-dimethylaminomethylthiochroman-4-one-1,1-dioxide, were isolated under conditions of the reaction. The monomer III could be prepared in 55% yield by sublimation of the dimer IV at 230-250 degrees; however, redimerization slowly occurred at room temperature. The dimer IV was also prepared by the use of paraformaldehyde and N-methylanilinium trifluoroacetate. The monomer III was found to be marginally active at 10 mg/kg/day versus Ehrlich ascites tumor growth in mice.

Synthesis and antitumor activity of a series of sulfone analogues of 1,4-naphthoquinone.

A series of novel substituted thiochromones and thiochroman-4-ones was synthesized. Compounds were designed as analogues of naphthoquinone and as potential "bioreductive alkylating agents" and were tested for antitumor activity. The lead compound, 3-(chloromethyl)thiochromone 1,1-dioxide (4), inhibited Ehrlich ascites tumor growth by 100% in CF1 male mice at 10 (mg/kg)/day ip. Similarly, 18 of the 29 related compounds demonstrated good activity in this tumor screen. Few definitive structure-activity correlations were evident regarding the nature of the 3-substituent. However, the 2,3 double bond and a sulfone or sulfoxide were required for activity. Four of the compounds synthesized showed marginal but significant activity against P-388 lymphocytic leukemia.

Antifertility activity of N-protected glycine activated esters.

A series of N-protected glycine activated esters was found to have antifertility activity in mice when administered intravaginally. The N-carbobenzoxyglycine vinyl ester and N-carbobenzoxyglycine 1,2-dibromoethyl ester analogs possessed the best activity in inhibiting pregnancy but were much less active when administered intraperitoneally. The acrosin enzymatic activity of sperm also was inhibited by these N-protected glycine activated esters, as measured by N-alpha-benzoyl-L-arginine ethyl ester and azocasein hydrolysis. The ability to inhibit N-alpha-benzoyl-L-arginine ethyl ester hydrolysis, a trypsin-like activity, appeared to have a positive correlation with antifertility activity when the agents were administered intravaginally.

PIP: A series of glycine esters were tested for antifertility activity in mice. N-protected glycine, when administered intravaginally had antifertility activity in mice both in vitro and in vivo. Greatest antifertility activity was found with 2 glycine esters, N-carbobenzoxyglycine vinyl ester, and N-carbobenzoxyglycine 1,2-dibromoethyl ester analogs; 0% pregnancy occurred when administered intravaginally. Their inhibiting activity was much less when administered intraperitoneally. In vitro, the acrosin enzymatic activity of sperm was inhibited by these N-protected glycine-activated esters, as determined by measuring N-alpha-benzoyl-L-arginine ethyl ester and azocasein hydrolysis. The ability to inhibit the arginine ethyl ester hydrolysis, which is a trypsin-like activity, seemed to be the positive correlate with antifertility activity when agents were administered intravaginally.

Effects of cis-malonato-diammino platinum (II) on P-388 lymphocytic leukemia cell metabolism.

cis-Malonato-diammino platinum(II) significantly inhibited P-388 lymphocytic leukemia cell proliferation at 10 mg/kg/day. Incorporation studies showed that DNA synthesis was inhibited following in vivo drug therapy. The major inhibitory effects appeared to be on thymidine kinase and dihydrofolate reductase activities and on overall purine synthesis, with marginal effects on DNA polymerase and ribonucleotide reductase activities. In addition to the DNA inhibition, a marked increase in cyclic adenosine 3',5'-monophosphate levels was noted, which correlated with a rapid decrease in histone phosphorylation. Other minor effects of the drug included significant reduction of proteolytic activity, suppression of States 4 and 3 respiration, and an increase in adenosine triphosphatase and acid phosphatase activities of P-388 cells.

Antitumor agents: diazomethyl ketone and chloromethyl ketone analogues prepared from N-tosyl amino acids.

Diazomethyl ketone and chloromethyl ketone analogues prepared from N-tosyl amino acids have been synthesized and tested for antitumor activity in Ehrlich ascites carcinoma and P-388 lymphocytic leukemia screens in mice. The N-tosyl chloromethyl ketone analogues prepared from glycine, L-alanine, beta-alanine, L-valine, and 6-(N-tosyl-amino)caproic acid were the most potent antineoplastic agents in the Ehrlich ascites carcinoma screen. The N-tosyl diazomethyl ketone analogues synthesized from glycine, L-leucine, and L-proline were the most active of this series in the Ehrlich ascites screen, along with 5-keto-1-tosyl-2-(diazoacetyl)pyrrolidine and the diazomethyl ketone analogues prepared from 6-(N-tosylamino)caproic acid. In the P-388 lymphocytic leukemia screen, the N-tosyl chloromethyl ketone prepared from glycine and the compound 5-keto-1-tosyl-2-(diazoacetyl)pyrrolidine were the most active.

Antitumor and antiinflammatory agents: N-benzoyl-protected cyanomethyl esters of amino acids.

A series of N-protected cyanomethyl esters of various amino acids was synthesized and tested for antineoplastic and antiinflammatory activity in rodents. Utilizing the L-phenylalanine cyanomethyl ester and varying the N-protecting moiety demonstrated that the N-tosyl and the N-Cbz analogues were the most active against Ehrlich ascites cell proliferation. The N-(carbobenzyloxy)- and N-benzoyl-L-phenylalanine cyanomethyl esters were the most active against carrageenan-induced inflammation. In the N-benzoyl series of cyanomethyl esters, L-alanine, DL-valine, and L-leucine amino acid analogues were the most active against Ehrlich ascites cell proliferation. The glycine and L-alanine analogues possessed the best inhibitor activity in the antiinflammatory screen. The cyanomethyl esters also demonstrated immunosuppressive activity and the ability to suppress the writhing reflex which is associated with inflammatory pain. However, no antipyretic or narcotic analgesic activity was demonstrated by these agents.

Development of radioimmunoassay for guanethidine.

A radioimmunoassay was developed for measuring plasma concentrations of the antihypertensive agent guanethidine at the nanogram level. Guanethidine was conjugated covalently to human serum albumin by two procedures, and the degree of conjugation was determined using tracer amounts of 3H-guanethidine. Immunization of sheep ethidine, as determined in competitive binding studies using 3H-guanethidine and a dextran-coated charcoal technique for the separation of free and antibody-bound drug. The major human metabolities, an N -oxide and a ring-opened derivative, were not cross-reactive in antibody binding studies. Constituents of human plasma or serum do not appear to interfere with the assay. Preliminary results from immunoassay of plasma samples from patients receiving guanethidine indicate potential use for assessing dosage regimens and studying pharmacokinetics.

Antibody specificity studies for reserpine, its metabolites, and synthetic reserpine congeners: radioimmunoassay.

Progress in the development of radioimmunoassay techniques for reserpine and related compounds is reported. A conjugate of reserpine with human serum albumin was prepared, involving linkage at the indole nitrogen atom of reserpine. Injection of the purified conjugate into sheep elicited antibodies of high titer, which bound reserpine selectively. Tritiated reserpine was employed in the procedure, and dextran-coated charcoal was utilized to separate free and bound forms of the drug. Antibodies exhibited a selectivity for reserpine and did not cross-react significantly with major human metabolites. Cross-reactivity of antibodies with other reserpine derivatives (i.e., syrosingopine, deserpidine, and rescinnamine) also was investigated. A stable tritiated or radioiodinated reserpine derivative of high specific activity is being sought to improve assay sensitivity for use in bioequivalence and bioavailability studies. In the absence of any extraction or concentration procedures, at least a 10-fold increase in immunoassay sensitivity would be required to follow reserpine levels in humans given normal doses of the drug. The methods show promise also for the assay of reserpine derivatives such as deserpidine, which exhibits cross-reactivity to reserpine antibodies.

Antifertility and antiproteolytic activity of activated N-carbobenzoxy amino acid esters.

N-Carbobenzoxy L-phenylalanine, glycine, L-leucine, and L-proline derivatives, their vinyl esters, and their 1,2-dibromoethyl esters were tested for antifertility activity in mice. Intraperitoneal administration reduced the pregnancy percentage and the number of fetuses per litter. Intravaginal administration reduced the pregnancy percentage significantly, with N-carbobenzoxyglycine vinly ester, N-carbobenzoxyglycine-1,2-dibromoethyl ester, N-carbobenzoxyl-L-leucine-1,2-dibromoethyl ester, and N-carbobenzoxy-L-proline-1,2-dibromoethyl ester producing 100% inhibition at 10 mg/kg/day. Sperm enzyme hydrolysis of the nonspecific substrate azocasein was inhibited significantly by certain N-carbobenzoxy amino acid esters in vitro. Specific substrate N-benzoyl-L-arginine ethyl ester hydrolysis was also inhibited. Compounds that inhibited N-benzoyl-L-arginine ethyl ester hydrolysis also demonstrated in vivo intravaginal antifertility activity.

Antineoplastic agents III: effects of dibromoethyl and vinyl esters of N-benzyloxycarbonyl-l-phenylalanine on Ehrlich ascites tumor cell metabolism.

Evidence is presented that N-benzyloxycarbonyl-L-phenylalanine vinyl ester and 1,2-dibromoethyl ester are inhibitors of Walker 256 carcinosarcoma and Ehrlich ascites carcinoma tumor growth. The major effects of these two agents on Ehrlich ascites cell metabolism were the inhibition of deoxyribonucleic acid and protein synthesis and the alteration of cellular regulatory processes controlling cytokinetics. Deoxynucleotide (purine) kinase enzymes appeared to be the focal site for inhibition of deoxyribonucleic acid synthesis with marginal inhibition of thymidylate synthetase activity. Cyclic adenosine monophosphate levels were elevated by drug treatment whereas chromatin protein phosphorylation, cell respiration, and lysosomal activities were inhibited. N-Benzyloxycarbonyl-L-phenylalanine 1,2-dibromoethyl ester was a latent in vitro chymotrypsin inhibitor. Some preliminary evidence suggests that these activated esters may inhibit cellular enzymatic activity by alkylating imidazole and lysine residues of proteins.

Antineoplastic agents. 1. N-Protected vinyl, 1,2-dihaloethyl, and cyanomethyl esters of phenylalanine.

A series of N-protected vinyl, 1,2-dihaloethyl, and cyanomethyl esters of phenylalanine was synthesized and these compounds were evaluated for antitumor activity against the growth of Ehrlich ascites carcinoma in CF1 male mice (33 mg/kg/day), Walker 256 carcinosarcoma in Sprague-Dawley male rats (2.5 mg/kg/day), and P388 lymphocytic leukemia in DBA/2 mice (20 mg/kg/day). Structure-activity relationships were evaluated and acute toxicity studies (LD50 determinations) in male CF1 mice were also carried out on selected compounds. Carbobenzoxy-L0phenylalanine vinyl ester (5), N-carbobenzoxy-L-phenylalanine 1,2-dibromoethyl ester (12), and N-carbobenzoxy-L-phenylalanine cyanomethyl ester (8) were found to be very potent inhibitors of Ehrlich ascites tumor growth at nontoxic doses cited above. Compounds 5 and 12 also tripled survival time in the Walker 256 system. LD50 values for compounds 5, 12, and 8 were greater than 2000 mg/kg (greater than 6.15 mmol/kg), 74 mg/kg (0.15 mmol/kg), and 150 mg/kg (0.44 mmol/kg), respectively.

Antineoplastic agents. 2. Structure-activity studies on N-protected vinyl, 1,2-dibromoethyl, and cyanomethyl esters of several amino acids.

Previously reported work on N-protected activated esters of phenylalanine has been extended to include N-protected vinyl, dibromoethyl, and cyanomethyl esters of several other amino acids. These compounds have been synthesized and evaluated in Ehrlich ascites carcinoma, Walker 256 carcinosarcoma, and and P388 lymphocytic leukemia tests. Among compounds tested were derivatives of tyrosine, tryptophan, glycine, leucine, proline, aspartic acid, glutamic acid, 4-aminobutyric acid, and 6-aminocaproic acid. Compounds of greatest potential interest from this study are N-carbobenzoxyglycine 1,2-dibromoethyl ester and N-carbobenzoxy-L-leucine 1,2-dibromoethyl ester. Both compounds were highly active in Ehrlich ascites test systems (33 mg/kg/day). The glycine derivative was also active in the Walker 256 test (2.5 mg/kg/day. Values for LD50's in mice were 148 mg/kg (0.37 mmol/kg) and 225 mg/kg (0.50 mmol/kg) for glycine and leucine derivatives, respectively; therefore, these compounds do not appear to be toxic at effective dose levels.