The novel fungal CYP51 inhibitor VT-1598 is efficacious alone and in combination with liposomal amphotericin B in a murine model of cryptococcal meningitis.

Objectives: Annual global deaths from cryptococcal meningitis (CM) are estimated at 180 000 and mortality is as high as 30%, even with optimal therapy. VT-1598 is a novel fungal CYP51 inhibitor with potent intrinsic antifungal activity against Cryptococcus. We report here VT-1598's in vivo antifungal activity in a murine model of CM. Methods: Single-dose plasma and brain pharmacokinetics in mice and MIC for Cryptococcus neoformans H99 were determined prior to efficacy studies. Short-course monotherapy and combination doses were explored with the endpoint of brain fungal burden. A survival study was also conducted using monotherapy treatment with fungal burden measured after a 6 day drug washout. Results: Oral doses of VT-1598 had good plasma and brain exposure and resulted in significant (P < 0.0001) and dose-dependent reductions in brain fungal burden, reaching a 6 log10 reduction. Unlike either positive drug control (fluconazole or liposomal amphotericin B), both mid and high doses of VT-1598 reduced fungal burden to below levels measured at the start of treatment. When VT-1598 was dosed in the survival study, no VT-1598-treated animal succumbed to the infection. Whereas fluconazole showed a 2.5 log10 increase in fungal burden after the 6 day washout, the VT-1598 mid- and high-dose animals showed almost no regrowth (<0.5 log10). In a separate fungal burden study using suboptimal doses of VT-1598 and liposomal amphotericin B to probe for combination effects, each combination had a positive effect relative to corresponding monotherapies. Conclusions: These pre-clinical in vivo data strongly support clinical investigation of VT-1598 as a novel therapy for this lethal infection.

Incidence and outcomes of ventral hernia repair after robotic retropubic prostatectomy: A retrospective cohort of 570 consecutive cases.

BACKGROUND: Robotic retropubic prostatectomy (RRP) has become one of the most commonly performed robotic procedures in the United States. Ventral hernia (VH) has been increasingly recognized as an important complication after laparoscopic procedures, in general. However, data related to VH after robotic procedures is relatively scarce, especially after RRP. With increasing popularity of RRP, the purpose of this study was to look at the incidence of VH and outcomes of ventral hernia repair (VHR) after RRP. METHODS: All patients who underwent RRP at a single institution between January 2012 and June 2014 were studied retrospectively using electronic medical records. RESULTS: A total of 570 patients underwent RRP, of which 33 (5.8%) developed VH during the study period. Fourteen (42%) patients were obese and five (15%) had diabetes. One patient (3%) had a surgical site infection after RRP and two (6%) patients were on immunomodulators/steroids. Median duration to develop VH after RRP was 12 (1-25) months. Out of the 33 patients with VH, ten (33%) underwent VHR; five laparoscopic and five open. Median size of hernia defect and mesh used was 25 (1-144) cm2 and 181 (15-285) cm2, respectively. Median length of hospital stay and follow up was 0 (0-4) days and 12 (1-14) months, respectively. One patient who had initial VHR done at an outside institution had a recurrence. Thirty-two (97%) patients were alive at their last follow up. One patient died secondary to progression of prostate cancer. There was no significant 30 day morbidity (surgical site infection, fascial dehiscence, pneumonia, acute kidney injury, myocardial infarction). Of patients who decided non-operative management of VH (n = 23, 67%), none developed a complication requiring emergent surgical intervention. CONCLUSION: The incidence of VH after RRP is likely underreported in prior studies. Repair, either laparoscopic or open, is safe and effective in experienced hands. Patients who decide on watchful waiting can be followed with minimal risk of incarceration/strangulation. Further studies are needed to analyze the extraction techniques after RRP and correlate with incidence of VH.

Challenging a surgical dictum: results from a 10-year experience on the safety of open inguinal herniorrhaphy in patients on chronic warfarin therapy.

BACKGROUND: Previous reports document the safety of open inguinal herniorrhaphy in patients on chronic warfarin therapy; however, the practice remains controversial. This study is a 10-year update of our experience. METHODS: A retrospective review of 1,839 consecutive patients undergoing open inguinal hernia repair was conducted from 2000 to 2010. All patients on chronic warfarin therapy were included. Three groups: continuation (CW), discontinuation (DW) and case-matched control (C) not on warfarin therapy were compared for operative details and postoperative complications. RESULTS: One hundred and fifty-eight patients were on chronic warfarin therapy. Of these, 40 patients (25%) continued on warfarin during the perioperative period (CW). Average preoperative international normalized ratio (INR) was 2.15 ± 0.76 for CW and 1.38 ± 0.42 for DW, p < 0.001. Mean operative times were equivalent between all three groups (88 min CW vs. 85 min DW vs. 79 min C, p = 0.518). Although CW patients experienced higher incidences of both hematoma and urinary retention overall, no statistically significant differences in complication rates were seen between the three groups (hematoma = 10 vs. 8% DW vs. 5% C, p = 0.703; urinary retention = 15 vs. 10% DW vs. 8% C, p = 0.541). Comparing patients by INR, there were no statistically different postoperative complication rates, particularly for hematoma (8% INR <2 vs. 9.5% INR = 2-3 vs. 20% INR >3, p = 0.65). CONCLUSION: Maintenance of warfarin therapy during the perioperative period for open inguinal herniorrhaphy results in equivalent operative times and postoperative complications as discontinuation.

A specific inhibitor of heart cytosolic 5'-nucleotidase I attenuates hydrolysis of adenosine 5'-monophosphate in primary rat myocytes.

ATP breakdown was triggered in primary rat myocytes in the presence of coformycin to force the catabolism of AMP through hydrolysis to adenosine. Selective inhibitors of the cytosolic 5'-nucleotidase I (c-N-I) from myocardium were used to measure the intracellular contribution of this enzyme to AMP hydrolysis under these conditions. The selective inhibitor 5-ethynyl-2',3'-dideoxyuridine inhibited the hydrolysis of AMP to adenosine in a concentration-dependent manner with an IC50 value of 20 microM. Maximal inhibition prevented 76% of the conversion of AMP to adenosine, indicating that under these conditions the majority of AMP hydrolysis in rat myocytes occurs through this enzyme. When ATP breakdown was triggered in the presence of thymidine 5'-phosphonate, a more potent inhibitor of the purified cytosolic 5'-nucleotidase, less inhibition of AMP hydrolysis occurred and only after prolonged preincubation of the myocytes with the inhibitor. These data demonstrate that the selective nucleoside inhibitors of c-N-I can effectively block the hydrolysis of AMP inside myocytes. Thus, these inhibitors may be useful tools in identifying the role of c-N-I during ATP catabolism in whole tissue and animal experiments.

Nucleotide and nucleoside analogues as inhibitors of cytosolic 5'-nucleotidase I from heart.

Substrate and product specificity studies were used to develop inhibitors of the cytosolic 5'-nucleotidase I (c-N-I) from myocardium. As measured by Vmax/Km, c-N-I preferred pyrimidine 2'-deoxyribonucleotides as substrates with thymidine monophosphate (TMP) being the most efficient. In product inhibition studies, thymidine inhibited noncompetitively and inorganic phosphate inhibited competitively, consistent with an ordered release of nucleoside prior to phosphate. Mirroring nucleotide substrate specificities, pyrimidine nucleosides were more potent product inhibitors than purine nucleosides. Thus, pyrimidine nucleotide and nucleoside analogues were developed as inhibitors. Phosphonate analogues of TMP were synthesized by a novel method. The most potent was the 5'-phosphonate of 3'-deoxythymidine (ddT) (apparent Ki value of 63 nM). In addition, pyrimidine nucleoside analogues were inhibitors with 5-ethynyl-2',3'-dideoxyuridine being the most potent (apparent Ki value of 3.7 microM). The most potent nucleotide and nucleoside inhibitor were both greater than 1000-fold more potent inhibiting c-N-I than the cytosolic 5'-nucleotidase II. The nucleoside analogue was also greater than 1000-fold more potent against c-N-I than the membrane ecto-5'-nucleotidase (e-N). Because the phosphonate analogues measurably inhibited e-N (apparent Ki values of 6-12 microM), the selectivity of the phosphonates for c-N-I versus e-N was less (40-200-fold). Because of the high selectivity for c-N-I versus both of the other 5'-nucleotidases, the nucleoside inhibitors of c-N-I may be useful biochemical tools in discerning the role that c-N-I plays in generating adenosine within myocardium.

Unique intracellular activation of the potent anti-human immunodeficiency virus agent 1592U89.

The anabolism of 1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, a selective inhibitor of human immunodeficiency virus (HIV), was characterized in human T-lymphoblastoid CD4+ CEM cells. 1592U89 was ultimately anabolized to the triphosphate (TP) of the guanine analog (-)-carbovir (CBV), a potent inhibitor of HIV reverse transcriptase. However, less than 2% of intracellular 1592U89 was converted to CBV, an amount insufficient to account for the CBV-TP levels observed. 1592U89 was anabolized to its 5'-monophosphate (MP) by the recently characterized enzyme adenosine phosphotransferase, but neither its diphosphate (DP) nor its TP was detected. The MP, DP, and TP of CBV were found in cells incubated with either 1592U89 or CBV, with CBV-TP being the major phosphorylated species. We confirmed that CBV is phosphorylated by 5'-nucleotidase and that mycophenolic acid increased the formation of CBV-TP from CBV 75-fold. However, mycophenolic acid did not stimulate 1592U89 anabolism to CBV-TP. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not inhibit CBV-TP formation from CBV or 1592U89, whereas the adenylate deaminase inhibitor 2'-deoxycoformycin selectively inhibited 1592U89 anabolism to CBV-TP and reversed the antiviral activity of 1592U89. 1592U89-MP was not a substrate for adenylate deaminase but was a substrate for a distinct cytosolic deaminase that was inhibited by 2'-deoxycoformycin-5'-MP. Thus, 1592U89 is phosphorylated by adenosine phosphotransferase to 1592U89-MP, which is converted by a novel cytosolic enzyme to CBV-MP. CBV-MP is then further phosphorylated to CBV-TP by cellular kinases. This unique activation pathway enables 1592U89 to overcome the pharmacokinetic and toxicological deficiencies of CBV while maintaining potent and selective anti-HIV activity.

Phosphorylation of carbovir enantiomers by cellular enzymes determines the stereoselectivity of antiviral activity.

Two enantiomers of carbovir, a carbocyclic analog of 2',3'-dideoxyguanosine, were compared with respect to their phosphorylation and the phosphorylation of their nucleotides by mammalian enzymes. 5'-Nucleotidase catalyzed the phosphorylation of (-)-carbovir, which is active against HIV (human immunodeficiency virus), but did not phosphorylate (+)-carbovir. (-)-Carbovir monophosphate was 7,000 times more efficient as a substrate for GMP kinase than was (+)-carbovir monophosphate. Pyruvate kinase, phosphoglycerate kinase, and creatine kinase phosphorylated both enantiomers of carbovir diphosphate at similar rates. Nucleoside-diphosphate kinase preferentially phosphorylated the (-)-enantiomer. Both enantiomers of carbovir triphosphate were substrates and alternative substrate inhibitors of HIV reverse transcriptase. Thus, the contrasting HIV-inhibitory activities of carbovir enantiomers were due to differential phosphorylation by cellular enzymes and not due to enantioselectivity of HIV reverse transcriptase.

A novel human phosphotransferase highly specific for adenosine.

A novel nucleoside phosphotransferase, referred to as adenosine phosphotransferase (Ado Ptase), was partially purified 1230-fold from human placenta. This enzyme differed from other known nucleoside phosphotransferases in its substrate specificity. Using AMP as the phosphate donor, it readily phosphorylated Ado. Changes in the sugar moiety were tolerated. dAdo and ddAdo were phosphate acceptors and dAMP was a donor. No other nucleotide or nucleoside common in nature displayed appreciable activity as donor or acceptor substrate, respectively. In the absence of nucleoside, the enzyme catalyzed the hydrolysis of AMP, typical of other nucleoside phosphotransferases. However, in the presence of Ado, little, if any, hydrolysis occurred. Ado Ptase had an absolute requirement for a metal cation, with Mg2+ and, to a lesser extent, Mn2+ fulfilling this requisite. The apparent Km for Ado was 0.2 mM. However, the donor AMP displayed cooperativity in both transfer and hydrolytic reactions. This cooperativity was eliminated by nucleotides, 2,3-diphosphoglycerate, and inorganic phosphate. ADP and 2,3-diphosphoglycerate were especially potent. In the presence of these effectors, the apparent Km for AMP was 3.0 mM in the transfer reaction and 4.0 mM in the hydrolytic reaction. Kinetic data suggest that there are two nucleotide binding sites on Ado Ptase, one for the donor, the other for an effector. AMP appeared to bind to both sites. Although this novel enzyme might play a role in the anabolism of nucleoside analogues, the normal physiological role of this nucleoside phosphotransferase is not understood.

A 39-year-old man with acute hemolytic crisis secondary to intravenous injection of hydrogen peroxide.

The diagnosis of cancer or its recurrence is often emotionally devastating for patients and those close to them. People may have a great deal of difficulty accepting the diagnosis and may seek others' opinions about the best course of treatment. When a physician projects that there is no hope, or if a cure is no longer probable, the patient and those close to him or her may feel helpless and hopeless. Given the resultant psychologic turmoil, the patient may be led to try unproven methods. Such methods, often referred to as "cancer quackery," represent a person's attempt to reassert personal control in response to these feelings of helplessness and hopelessness. Cancer quackery involves about $2 billion each year in the United States alone. One study demonstrated that approximately 39% of the pediatric outpatients studied had either tried, considered, or received recommendations for unproven methods of cancer treatment. Laetrile and faith healing were the most frequent methods used. One of the most recent of the unproven methods of cancer treatment that have been shown to cause life-threatening complications is IV injection of hydrogen peroxide. The scientific rationale behind this procedure is still unclear, but the side effects are clearly life threatening.

Effects of multiple replacements at a single position on the folding and stability of dihydrofolate reductase from Escherichia coli.

We have made multiple replacements (alanine, arginine, cysteine, histidine, isoleucine, serine, tyrosine) of valine-75 in dihydrofolate reductase from Escherichia coli to examine the relative importance to protein folding of the position that is substituted and the specific character of the amino acid replacement. Valine-75 is part of the eight-stranded beta sheet that forms the structural core of the protein. The isopropyl side chain participates in van der Waals interactions with a number of nonpolar residues, helping to establish a large hydrophobic cluster. Equilibrium studies showed that arginine, histidine, isoleucine, serine, and tyrosine destabilize the protein by 1.9-2.8 kcal mol-1. Alanine and cysteine substitutions have little or no effect. Contrary to other recent studies of the effect of multiple replacements at a hydrophobic site, there is no observed correlation between the changes of the free energy of folding and the changes of the free energy of transfer for the individual amino acids from water to an organic solvent when they are inserted into this site. The effects observed in kinetic studies are both consistent with and extend the equilibrium results; these data indicate that position 75 participates in a rate-limiting step of folding. Some of the equilibrium and kinetic properties of the tyrosine-75 mutant deviated significantly from those of wild-type protein and the other mutants at position 75. (1) The tyrosine variant displayed a complex banding pattern when analyzed by native gel electrophoresis; the wild-type protein and all other mutants at position 75 migrated as single, discrete bands. (2) Comparison of the difference ultraviolet and circular dichroism transition curves showed that a third species is populated at equilibrium; the wild-type protein and all other mutants at position 75 follow a two-state model involving only native and unfolded forms. (3) A third kinetic phase appeared in the unfolding reaction; the wild-type protein and all other mutants at position 75 only showed two kinetic phases in unfolding. Properties 1 and 3 suggest that the tyrosine mutation significantly alters the distribution of native conformers in the protein. These effects on the equilibrium and kinetic data readily display an overriding pattern: residues that would require hydrogen bonding or lead to an expansion of the tightly packed hydrophobic environment in which valine-75 resides destabilize the protein and alter relaxation times of kinetic phases in a consistent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

An immunohistological study of CD4+ lymphocyte subsets within inflammatory lesions with special reference to rheumatoid arthritis and inflammatory bowel disease.

The monoclonal antibodies (mAbs) WR16, UCHL1 and WR19 identify subsets of CD4+ lymphocytes that have been functionally characterized as suppressor inducer cells or helper inducer cells. These were applied as components of a panel of lymphocyte-specific mAbs for the phenotypic analysis of lymphocyte populations within biopsies taken from rheumatoid synovial membrane and normal and inflamed gut. The phenotype of peripheral blood lymphocytes from patients with rheumatoid arthritis were also compared to normal controls. The rheumatoid synovium was characterized immunohistologically by a lymphocytic infiltrate composed predominantly of CD4+ lymphocytes and a CD4:CD8 ratio of 2.4. The CD4+ population was composed of UCHL1+ cells to the exclusion of WR16+ cells. This finding was confirmed by double immunofluorescence staining using directly conjugated Leu-3a and WR16. The UCHL1+/WR16-/CD4+ phenotype was maintained in the synovial biopsies regardless of whether the patient had commenced treatment with disease modifying drugs. The absence of WR16+ cells within the rheumatoid synovium was shown to be a localized phenomenon as there was a slight elevation of circulating WR16+ lymphocytes in the peripheral blood of rheumatoids whilst the levels of UCHL1+ and WR19+ lymphocytes remained unchanged. As no appropriate normal control tissue is available for comparison to the rheumatoid synovium we also examined the lymphocytes present within Crohn's disease-involved bowel biopsies and compared them to normal gut tissue lymphocytes using WR16 and UCHL1 mAbs. The CD3+ lymphocytes present within normal tissue comprised a mixture of WR16+ and UCHL1+ cells. In contrast the CD3+ lymphocytes within Crohn's involved tissue were exclusively UCHL1+ as previously observed in the rheumatoid synovium. These data indicate that the CD4+ lymphocyte infiltrate present within inflammatory lesions of presumed distinct aetiology exhibit a localized selective loss of cells with the CD45R+/CD4+ suppressor inducer phenotype. This may be a consequence of the selective extravasation of CD4+ helper induced cells or more likely, in view of the previously documented loss of the p220 molecule identified by CD45R mAbs upon T-cell activation, the result of CD4+ T-cell activation at sites of inflammation.

Dopamine receptor agonist activity of some 5-(2-aminoethyl)carbostyril derivatives.

The potency of beta-adrenoreceptor agonists, e.g., isoproterenol, is strikingly increased by substitution of the meta catecholic hydroxyl group with the NH group of a carbostyril system. To explore the possibility that comparable potency enhancement might occur upon similar modification of the catechol ring of dopamine, a series of 5-(2-aminoethyl)carbostyril derivatives was prepared and examined for D-1 and D-2 dopamine receptor-stimulating activity. Only the parent compound, 5-(2-aminoethyl)-8-hydroxycarbostyril (2), produced measurable activation of dopamine-sensitive adenylate cyclase (29% at a concentration of 10 microM). Some of the compounds, however, did produce significant activity in tests, namely displacement of [3H]spiroperidol binding from bovine pituitary homogenate and an isolated perfused rabbit ear artery preparation, that measure interaction with D-2 receptors. Potency of the carbostyrils was enhanced by 8-hydroxylation and by appropriate substitution of the amino group of the ethylamine side chain. The most potent member of the series was 8-hydroxy-5-[2-[[2-(4-hydroxyphenyl)ethyl]-n-propylamino]ethyl] carbostyril (16b). This compound was about 3 times more effective than dopamine in the D-2 receptor tests. Clearly, the results of this study indicate that potency of dopamine receptor agonists is not increased by carbostyril replacement of the m-hydroxyl as is noted with the beta-adrenergic receptor agonists.

Limited proteolysis of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania tropica.

The structure and activity of the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from the protozoan parasite Leishmania tropica were examined by limited proteolysis with five different endopeptidases. Each reaction resulted in a rapid, time-dependent loss of TS activity and no effect on DHFR activity. The proteolytic products were examined by NaDodSO4/PAGE; each digestion produced a fragment of apparent Mr approximately 35,000, and three of the five digestions generated a fragment of Mr approximately 20,000. Attempts to separate the fragments under nondenaturing conditions failed, suggesting that the proteolyzed protein remains a dimer with the gross structure of the subunits more or less undisturbed. In contrast, kinetic data indicate that some aspects of higher-order structure in the native protein are affected by proteolysis. The fragments (Mr 36,600 and 20,000) generated by Staphylococcus aureus V8 protease were subjected to sequence analysis. Whereas neither the native protein nor the Mr 36,600 fragment yielded an NH2-terminal amino acid, we obtained the sequence of the first 28 amino acids of the Mr 20,000 fragment. This sequence bore strong homology with sequences situated within TS of human, Lactobacillus casei, Escherichia coli, and bacteriophage T4. These and other data indicate that the TS-DHFR polypeptide consists of a DHFR sequence at the blocked NH2-terminal and a TS sequence at the COOH-terminal end of the protein. The region that is the target of the five proteases corresponds to a highly variable region within the sequences of the other four TSs. We suggest that an insertion occurs within the TS-DHFR sequence, positioned on the surface of the protein and quite vulnerable to the action of endopeptidases.

Purification and characterization of the bifunctional thymidylate synthetase-dihydrofolate reductase from methotrexate-resistant Leishmania tropica.

Thymidylate synthetase (TS) and dihydrofolate reductase (DHFR) in Leishmania tropica exist as a bifunctional protein. By use of a methotrexate-resistant strain, which overproduces the bifunctional enzyme, the protein was purified 80-fold to apparent homogeneity in two steps. The native protein has an apparent molecular weight of 110 000 and consists of two subunits with identical size and charge. Available data indicate that each of the subunits possesses TS and DHFR. The TS of the bifunctional protein forms a covalent 5-fluoro-2'-deoxyuridylate (FdUMP)-(+/-)-5,10-methylenetetrahydrofolate-enzyme complex in which 2 mol of FdUMP is bound per mole of enzyme. In contrast, titration of DHFR with methotrexate indicated that only 1 mol of the inhibitor is bound per mole of dimeric enzyme. Both TS and DHFR activities of the bifunctional enzyme were inactivated by the sulfhydryl reagent N-ethylmaleimide. Substrates of the individual enzymes afforded protection against inactivation, indicating that each enzyme requires at least one cysteine for catalytic activity. Kinetic evidence indicates that most, if not all, of the 7,8-dihydrofolate produced by TS is channeled to DHFR faster than it is released into the medium. Although the mechanism of channeling is unknown, the possibility that the two enzymes share a common folate binding site has been ruled out.