Target interaction profiling of midostaurin and its metabolites in neoplastic mast cells predicts distinct effects on activation and growth.

Proteomic-based drug testing is an emerging approach to establish the clinical value and anti-neoplastic potential of multikinase inhibitors. The multikinase inhibitor midostaurin (PKC412) is a promising new agent used to treat patients with advanced systemic mastocytosis (SM). We examined the target interaction profiles and the mast cell (MC)-targeting effects of two pharmacologically relevant midostaurin metabolites, CGP52421 and CGP62221. All three compounds, midostaurin and the two metabolites, suppressed IgE-dependent histamine secretion in basophils and MC with reasonable IC(50) values. Midostaurin and CGP62221 also produced growth inhibition and dephosphorylation of KIT in the MC leukemia cell line HMC-1.2, whereas the second metabolite, CGP52421, which accumulates in vivo, showed no substantial effects. Chemical proteomic profiling and drug competition experiments revealed that midostaurin interacts with KIT and several additional kinase targets. The key downstream regulator FES was recognized by midostaurin and CGP62221, but not by CGP52421 in MC lysates, whereas the IgE receptor downstream target SYK was recognized by both metabolites. Together, our data show that the clinically relevant midostaurin metabolite CGP52421 inhibits IgE-dependent histamine release, but is a weak inhibitor of MC proliferation, which may have clinical implications and may explain why mediator-related symptoms improve in SM patients even when disease progression occurs.

Short stay after cleft palate surgery.

Although algorithms for the repair of soft and hard palatal clefts continue to be debated, the appropriate length of postoperative stay has not yet been defined. Recent reports of cleft palate repair advocate a 2- to 5-day hospitalization. The plastic surgery service at St. Joseph Hospital frequently uses same-day admission with 23-hour observation postoperatively, with no increase in complications from the reported 2- to 5-day stay. The authors inspected the records for all the cleft palate patients undergoing cleft repair at St. Joseph Hospital Cleft Clinic from August of 1988 through June of 1998. After excluding syndromic patients and secondary or revision surgical cases, 79 patients remained in the study. These 79 patients underwent 104 procedures; all procedures were performed by a single surgeon (E.D.C.) with resident assistance. Short-term morbidity, length of stay, and operation performed were studied. All patients were admitted the day of surgery. Mean age at the time of operation was 13.2 months, with a range of 6 months to 20 years. The length of operation averaged 1 hour and 37 minutes; 94 percent of patients stayed 24 hours or less postoperatively, and 97 percent stayed 36 hours or less. The longest stay was 72 hours, which was related to delay in resuming adequate oral intake. The overall complication rate was 3.8 percent for this cohort, which included two partial palatal dehiscences and two small fistulas. No blood transfusions were needed, and no infections were noted postoperatively. No patients required readmission postoperatively for bleeding, respiratory compromise, or inadequate oral intake. The authors do not advocate a 1-night stay for all cleft palate cases. However, they do think it is safe for a healthy group of patients undergoing routine cleft palate surgery. The decision to discharge a patient early must always be left to the treating physician.

Markers of tumor angiogenesis and proteolysis independently define high- and low-risk subsets of node-negative breast cancer patients.

PURPOSE: To compare the prognostic impact of tumor angiogenesis factors (vascular endothelial growth factor [VEGF], angiogenin, and basic fibroblast growth factor [bFGF]), tumor proteolysis factors (urokinase-type plasminogen activator [uPA] and plasminogen activator inhibitor-1 [PAI-1]), and conventional tumor markers (stage, grade, and steroid receptors) in early breast cancer. PATIENTS AND METHODS: In the primary clinical study, tumor angiogenesis and other factors were detected in frozen biopsies from 305 primary breast tumors. VEGF expression was assessed by chemiluminescence immunosorbent assay (ICMA); angiogenin, bFGF, uPA, and PAI-1 by enzyme-linked immunosorbent assay (ELISA); and steroid receptors (estrogen receptor [ER] and progesterone receptor [PgR]) by enzyme immunoassay (EIA). In the validating clinical study, another set of 190 node-negative primary breast tumor samples were collected at a separate institution. RESULTS: Univariate analysis of the primary study showed that VEGF levels were positively correlated with recurrence (P < .001). Angiogenin levels were positively correlated with disease relapse (P < .005) for the overall collective group, but not within the node-negative subset. No significant correlations were found between tumor bFGF levels and patient survival. In multivariate regression analysis, the only independent predictors of relapse-free survival (RFS) were VEGF, uPA, and lymph node status. In the validation set, the distribution of VEGF and uPA values were similar to those in the primary study; low expression of both VEGF and uPA identified patients with a < or = 20% likelihood of recurrence within 7 years. CONCLUSION: Separate primary and validating clinical studies concur that tumor VEGF level is the most important prognostic parameter among several markers of tumor angiogenesis and proteolysis.

Novel spirodihydrobenzofuranlactams as antagonists of endothelin and as inhibitors of HIV-1 protease produced by Stachybotrys Sp. I. Fermentation, isolation and biological activity.

Six novel spirodihydrobenzofuranlactams I - VI (1 - 6) and a related spirodihydrobenzofuranalcohol, the previously described natural compound L-671,776 (7), were isolated from cultures of two different Stachybotrys species. These secondary metabolites showed antagonistic effects in the endothelin receptor binding assay and inhibited HIV-1 protease. Both biological activities are novel for L-671,776 (7). The pseudosymmetric spirodihydrobenzofuranlactam VI (6) is the most potent representative of this class of compounds exhibiting IC50 values of 1.5 microM in the ET-A receptor binding assay and 11 microM in the HIV-1 protease inhibition assay.

Pharmacokinetics of a novel HIV-1 protease inhibitor incorporated into biodegradable or enteric nanoparticles following intravenous and oral administration to mice.

CGP 57813 is a peptidomimetic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease. This lipophilic compound was successfully entrapped into poly(D,L-lactic acid) (PLA) and pH sensitive methacrylic acid copolymers nanoparticle. The intravenous administration to mice of PLA nanoparticles loaded with CGP 57813 resulted in a 2-fold increase of the area under the plasma concentration-time curve, compared to a control solution. An increase in the elimination half-life (from 13 to 61 min) and in the apparent volume of distribution (1.7-3.6 L/kg) was observed for the nanoparticle incorporated compound vs control solution. Following oral administration, only nanoparticles made of the methacrylic acid copolymer soluble at low pH provided sufficient plasma levels of CGP 57813. In vitro, these nanoparticles dissolved completely within 5 min at pH 5.8. PLA nanoparticles, which are insoluble in the gastrointestinal tract, did not provide significant plasma concentrations of CGP 57813. From these observations, one can conclude that the passage of intact PLA nanoparticles across the gastrointestinal mucosa appears to be very low.

Assessment of differential cytokine effects on angiogenesis using an in vivo model of cutaneous wound repair.

Angiogenesis, or new blood vessel formation, has been a subject of intense investigation in recent years. A major obstacle in this research has been the selection of an appropriate in vivo model with which comparisons to humans can be made as well as a reliable quantitative method. Using the porcine excisional wound healing model, we report a new and simple technique for obtaining objective assessments of the microvascular compartment. Factor VIII immunostaining of histological specimens was utilized for specific identification of endothelium devoid of background interference. This technique was coupled with morphometric analysis to quantitate the differential effects of tumor necrosis factor alpha (TNF alpha), transforming growth factor beta (TGF beta), basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1), and epidermal growth factor (EGF) within healing porcine wounds. All cytokines stimulated angiogenesis, with low dose TNF alpha and bFGF treatments exhibiting the most profound effects at 7 days postwounding. With increasing levels of TNF alpha (1 ng, 10 ng, 100 ng, and 2.5 micrograms), a step-wise decrease in microvascular area was noted. Although no significant dose responsive differences in angiogenesis were noted following bFGF treatments, a profound increase in capillary area was shown. Significant yet less dramatic increases were noted in capillary area following treatment with EGF or IGF-1. Comparison of the angiogenic effects of TGF beta at 7 and 10 days postwounding showed a significant decrease in the microvasculature as wounds matured. Our data are consistent with previous qualitative in vitro and in vivo reports, thereby confirming the validity of this new model. The data furthermore provide the first quantitative evidence of differential angiogenic responses to cytokines within a clinically relevant model of cutaneous wound repair.

The G-C specific DNA binding drug, mithramycin, selectively inhibits transcription of the C-MYC and C-HA-RAS genes in regenerating liver.

Expression of the c-myc and c-Ha-ras protooncogenes is dramatically increased in regenerating rat liver as an early response to partial hepatectomy. Nuclear runon transcription studies confirm that the increased c-myc and c-Ha-ras mRNA levels in regenerating livers reflect transcriptional activation of these genes. Mithramycin, a G-C specific DNA binding drug, prevents the increased transcriptional activity of c-myc and c-Ha-ras genes after hepatectomy but does not alter the transcriptional activity of the beta-actin gene. Continuous exposure of rats to mithramycin after hepatectomy prevents the increase in both c-myc and c-Ha-ras expression and blocks the increased cellular proliferation characteristic of regeneration. The delayed increase in c-myc and c-Ha-ras gene expression is associated with a delay in cellular proliferation. The inhibition of c-myc and c-Ha-ras transcription by mithramycin, the delay in cellular proliferation, and the ability of mithramycin to prevent protein binding to the c-myc promoter, suggest that the increased expression of these genes is a necessary component of liver regeneration.

3-Alkanoyl-5-hydroxymethyl tetronic acid homologues and resistomycin: new inhibitors of HIV-1 protease. I. Fermentation, isolation and biological activity.

In the course of a screening program for HIV-1 protease inhibiting activity, six new homologues of 3-alkanoyl-5-hydroxymethyl tetronic acids (1 approximately 6) and the known natural product resistomycin (7) were isolated from cultures of the Actinomycete strain DSM 7357. The substituted tetronic acids belong to a recently described structural class of secondary metabolites. The HIV-1 activity of resistomycin (7) has not been reported before.

CGP 53437, an orally bioavailable inhibitor of human immunodeficiency virus type 1 protease with potent antiviral activity.

CGP 53437 is a peptidomimetic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease containing a hydroxyethylene isostere. The compound inhibited recombinant HIV-1 protease with a Ki of 0.2 nM. The inhibition constant versus human cathepsin D and human cathepsin E was 4 nM. Human pepsin and gastricsin were inhibited with Kis of 8 and 500 nM, respectively, and human renin was inhibited with a Ki of 190 microM. The replication of HIV-1/LAV, HIV-1/Z-84, and HIV-1/pLAI was inhibited with a 90% effective dose of 0.1 microM in acutely infected MT-2 cells. The 50% cytotoxic dose was 100 microM. Similar antiviral activity was observed when the compound was added up to 10 h after infection. At the effective concentration, processing of Gag precursor protein p55 was greatly reduced, confirming an action on the late stage of the virus life cycle, as expected. The efficacy of the inhibitor was also demonstrated by using primary human peripheral blood lymphocytes infected with the HIV-1/LAV strain, low-passage clinical isolates obtained from HIV-1-seropositive individuals (including a zidovudine-resistant strain), and HIV-2/ROD. In these cells, CGP 53437 delayed the onset of HIV replication in a dose-dependent fashion (substantial effects with concentrations of > or = 0.1 microM) as long as the inhibitor was maintained in the culture. CGP 53437 was orally bioavailable in mice. Concentrations in plasma 10-fold in excess of the in vitro antiviral 90% effective dose could be sustained for several hours after oral application of 120 mg/kg. Therefore, CGP 53437 has the potential to be a therapeutically useful anti-HIV agent for the treatment of AIDS.

Cloning, expression and purification of a recombinant poly-histidine-linked HIV-1 protease.

The gene coding for the HIV-1 protease was cloned in an Escherichia coli expression vector adding three-histidine codons to the amino and carboxy terminus of the protease sequence. Expression of the protease from this construct led to the accumulation of high amounts of insoluble histidine-linked protease entrapped in inclusion bodies. The histidine-linked protease could be efficiently released from purified inclusion bodies with 6 M guanidine hydrochloride and further purified by metal chelate affinity chromatography. The refolded protease cleaved synthetic peptide substrates and the viral polyprotein p55 with the same specificity as the wild type protease. It displays a specific activity of 4.4 mumol/min/mg.

Benzopyranones and benzothiopyranones: a class of tyrosine protein kinase inhibitors with selectivity for the v-abl kinase.

Abelson murine leukemia virus is an acutely transforming replication-defective virus which encodes a transforming protein with tyrosine-specific protein kinase activity. A variety of benzopyranone and benzothiopyranone derivatives have been identified which selectively inhibit the v-abl tyrosine protein kinase with 50% inhibitory concentrations ranging from 1 to 30 microM. The most active derivative inhibited v-abl with a Ki value of 0.9 microM. Active derivatives showed selectivity for the v-abl tyrosine protein kinase relative to the epidermal growth factor receptor tyrosine protein kinase (50% inhibitory concentration greater than 100 microM). Protein kinase C and protein kinase A, two members of the serine/threonine protein kinase family, were not inhibited by benzopyranones or benzothiopyranones (50% inhibitory concentration greater than 100 microM). Kinetically, a representative derivative (compound 2) showed competitivity with respect to ATP and noncompetitive behavior with respect to the exogenous peptide substrate. Autophosphorylation of p120v-abl and recombinant p70v-abl tyrosine protein kinases were also inhibited by benzopyranones and benzothiopyranones in vitro. When tested in Abelson murine leukemia virus-transformed BALB/c cell, active benzopyranone and benzothiopyranone derivatives inhibited tyrosine phosphorylation of cellular proteins by the v-abl tyrosine protein kinase.

Sulfonylbenzoyl-nitrostyrenes: potential bisubstrate type inhibitors of the EGF-receptor tyrosine protein kinase.

The synthesis and biological activities of a series of sulfonylbenzoyl-nitrostyrene derivatives, a novel class of selective bisubstrate type inhibitors of the EGF-receptor tyrosine protein kinase, are described. The most potent derivatives inhibited the EGF-R tyrosine kinase, using angiotensin II as exogenous substrate, with IC50 values of less than or equal to 1 microM. No inhibition of the v-abl tyrosine kinase or the serine/threonine kinases PKC and PK-A was observed. In addition, active derivatives (compounds 5 and 12) effectively blocked the autophosphorylation of the EGF-R in vitro. Starting from the acids 5, 7, and 9, a series of esters, amides, and peptides was synthesized with the aim of increasing cellular penetration. Amides 14-18 showed potent antiproliferative effects using the EGF-dependent Balb/MK mouse epidermal keratinocyte cell line. Additionally, with the amide 14 inhibition of EGF-R autophosphorylation was demonstrated in the A431 cell line. CAMM studies using a computer-generated model for the transition state of the gamma-phosphoryl transfer from ATP to a tyrosine moiety and fitting experiments using the highly potent derivative 7 (IC50 value = 54 nM) support the hypothesis that the sulfonylbenzoyl group mimics a diphosphate moiety in the transition state. These results demonstrate that the rational design of tyrosine kinase inhibitors, using the inhibitory nitrostyrene moiety as a tyrosine mimic together with the sulfonylbenzoyl moiety as a diphosphate mimic, leads to highly potent and selective multisubstrate type inhibitors.

Thiazolidine-diones. Biochemical and biological activity of a novel class of tyrosine protein kinase inhibitors.

Various derivatives of thiazolidine-diones have been identified as tyrosine protein kinase inhibitors. The epidermal growth factor (EGF) receptor kinase and c-src kinase were inhibited in vitro with IC50 values in the range of 1-7 microM. The v-abl tyrosine protein kinase was not inhibited by thiazolidine-diones. Inhibition was found to be specific for tyrosine protein kinases. Inhibition of serine/threonine protein kinases was not observed. The active derivatives were shown to inhibit EGF-induced receptor autophosphorylation, either in vitro or in intact cells, and were also found to inhibit growth of the EGF-dependent BALB/MK and A431 cell lines (IC50 1-3 microM). Growth of the interleukin-3-dependent myeloid cell line FDC-P1 was inhibited with equal efficiency. Thus, in these cell lines, members of the c-src kinase family are also potential targets for inhibition by the compounds.

Stimulation of renal and hepatic c-myc and c-Ha-ras expression by unilateral nephrectomy.

Unilateral nephrectomy induces compensatory hypertrophy of the contralateral kidney in rats, resulting in a 25% weight increase in 14 days. We have demonstrated that expression of the c-myc and c-Ha-ras protooncogenes is increased more than ten-fold in the contralateral kidney within 4 to 8 hr following unilateral nephrectomy in rats. The increased expression of these genes is analogous to the increased expression of c-myc and c-Ha-ras that occurs early in liver regeneration, preceding the first increase in DNA synthesis by at least 20 hr. In order to define the tissue specificity of the signals for compensatory renal hypertrophy, we also determined the early protooncogene response and the proliferative response in the liver of rats following unilateral nephrectomy. The expression of c-myc and c-Ha-ras was also increased (five- to ten-fold) in the livers of these animals. DNA synthesis was stimulated in the contralateral kidney at 26-30 hr following nephrectomy as measured by 3H thymidine incorporation, indicating a hyperplastic response to unilateral nephrectomy. However, there was no increase in DNA synthesis in the liver despite the dramatic increase in c-myc and c-Ha-ras expression. Our data suggest that the early increase in protooncogene expression in response to unilateral nephrectomy is stimulated by circulating signals that are not tissue-specific. Increased protooncogene expression in both kidney and liver following unilateral nephrectomy is an early response to the regenerative stimulus, but later signals must provide the tissue specificity necessary for regeneration and cellular proliferation.

Stimulation of protooncogene expression by partial hepatectomy is not tissue-specific.

We have characterized the early protooncogene response and the later cell proliferative response in the kidneys and livers of normal rats cross-circulated with partially hepatectomized animals. Increase c-myc and C-Ha-ras expression was observed in the kidneys of totally hepatectomized rats, as well as those of their cross-circulated partners. This indicates that the initial response to hepatectomy is not organ-specific, although the later DNA synthetic response of the kidney is only approximately one-tenth that of regenerating liver. Expression of c-myc and c-Ha-ras is dramatically increased in the livers of both hepatectomized and nonhepatectomized, parabiotic (cross-circulated) rats within 1 hr of partial hepatectomy, confirming the presence of a circulating factor which stimulates protooncogene expression early in regeneration. DNA synthesis was also stimulated in the livers of the cross-circulated animals between 20 and 26 hr following hepatectomy, but only to a level one-eighth that of the livers of hepatectomized animals. Normal rats cross-circulated with totally hepatectomized animals also demonstrated an early increase in hepatic c-myc and c-Ha-ras expression, indicating that regeneration must be stimulated by an extrahepatic signal. Our data suggest that the early increase in protooncogene expression is a non-organ-specific response to partial hepatectomy which does not insure subsequent cellular proliferation. The organ specificity of liver regeneration must involve an event separate from the early stimulation of protooncogene expression.