Discovery of spirohydantoins as selective, orally bioavailable inhibitors of p300/CBP histone acetyltransferases.

p300 and CREB-binding protein (CBP) are essential for a multitude of cellular processes. Dysregulation of p300/CBP histone acetyltransferase activity is linked to a broad spectrum of human diseases including cancers. A novel drug-like spirohydantoin (21) has been discovered as a selective orally bioavailable inhibitor of p300/CBP histone acetyltransferase. Lead compound 21 is more potent than the first-in-class lead A-485 in both enzymatic and cellular assays and lacks the off-target inhibition of dopamine and serotonin transporters, that was observed with A-485.

Dose-independent genotoxic response in A549 cell line exposed to fungicide Iprodione.

The fungicide Iprodione is widely applied in vegetables and raises concern for human health. The A549 human lung carcinoma cell line is a suitable model for assessing the toxicological effects of drugs. The goal of this work was to evaluate the genotoxicity and oxidative stress in the A549 cell line exposed to sublethal concentrations from 3 to 100 µg/mL Iprodione considering LC50 = 243.4 µg/mL Iprodione, as determined by the MTT assay. Generalized Linear Mixed Models (GLMM) were performed to determine the association between the responses NDI, MNim and MNib and the explanatory variables. Iprodione and solvent were relativized to the control whereas the concentration was included as numeric variable. ANOVA was used for the comparison of treatments. The coefficients of linear association between the explanatory variables and NDI, and the coefficients of logistic association between explanatory variables and MNim were not significant. However, these coefficients showed significant association with MNib only for Iprodione treatment but not for Iprodione concentration, indicating lack of dose-response relationship. Genotoxicity risk assessment indicated that the increase in Iprodione concentrations increased slightly the probability of belonging to the genotoxic category. ANOVA showed significant differences in MNib, and non-significant differences in NDI and MNim among treatments. The oxidative stress analysis performed at 3, 12, and 25 µg/mL Iprodione showed a significant and linear increase in SOD, and a significant and linear decrease in GSH and GST. The Dunnett test was significant for GSH at 12 and SOD at 25 µg/mL.

Development of a prodrug of hydantoin based TACE inhibitor.

Our research on hydantoin based TNF-α converting enzyme (TACE) inhibitors led to fused bi-heteroaryl hydantoin series that demonstrate sub-nanomolar potency (Ki) as well as excellent activity in human whole blood (hWBA). However, lead compound 2 posed some formulation challenges which prevented it for further development. A prodrug approach was investigated to address this issue. The pivalate prodrug 3 can be formulated as stable neutral form and demonstrated improved DMPK properties when compared with parent compound.

Revisiting the SAR of the Antischistosomal Aryl Hydantoin (Ro 13-3978).

The aryl hydantoin 1 (Ro 13-3978) was identified in the early 1980s as a promising antischistosomal lead compound. However, this series of aryl hydantoins produced antiandrogenic side effects in the host, a not unexpected outcome given their close structural similarity to the antiandrogenic drug nilutamide. Building on the known SAR of this compound series, we now describe a number of analogs of 1 designed to maximize structural diversity guided by incorporation of substructures and functional groups known to diminish ligand-androgen receptor interactions. These analogs had calculated polar surface area (PSA), measured LogD7.4, aqueous kinetic solubility, and estimated plasma protein binding values in ranges predictive of good ADME profiles. The principal SAR insight was that the hydantoin core of 1 is required for high antischistosomal activity. We identified several compounds with high antischistosomal efficacy that were less antiandrogenic than 1. These data provide direction for the ongoing optimization of antischistosomal hydantoins.

Formaldehyde-releasers in cosmetics: relationship to formaldehyde contact allergy. Part 2. Patch test relationship to formaldehyde contact allergy, experimental provocation tests, amount of formaldehyde released, and assessment of risk to consumers allergic to formaldehyde.

This is the second part of an article on formaldehyde-releasers in cosmetics. The patch test relationship between the releasers in cosmetics to formaldehyde contact allergy is reviewed and it is assessed whether products preserved with formaldehyde-releasers may contain enough free formaldehyde to pose a threat to individuals with contact allergy to formaldehyde. There is a clear relationship between positive patch test reactions to formaldehyde-releasers and formaldehyde contact allergy: 15% of all reactions to 2-bromo-2-nitropropane-1,3-diol and 40-60% of the reactions to the other releasers are caused by a reaction to the formaldehyde in the test material. There is only fragmented data on the amount of free formaldehyde in cosmetics preserved with formaldehyde donors. However, all releasers (with the exception of 2-bromo-2-nitropropane-1,3-diol, for which adequate data are lacking) can, in the right circumstances of concentration and product composition, release >200 p.p.m. formaldehyde, which may result in allergic contact dermatitis. Whether this is actually the case in any particular product cannot be determined from the ingredient labelling. Therefore, we recommend advising patients allergic to formaldehyde to avoid leave-on cosmetics preserved with quaternium-15, diazolidinyl urea, DMDM hydantoin, or imidazolidinyl urea, acknowledging that many would tolerate some products.

Synthesis and in vitro cytotoxic evaluation of novel diazaspiro bicyclo hydantoin derivatives in human leukemia cells: a SAR study.

To study the structure activity relationship (SAR) on the cytotoxic activity and probe the structural requirement for the potent antitumor activity, a series of novel diazaspiro bicyclo hydantoin derivatives were designed and synthesized. Their structures were confirmed by (1)H NMR, LCMS and IR analyses. The antiproliferative effect of these compounds were determined against human leukemia, K562 (chronic myelogenous leukemia) and CEM (T-cell leukemia) cells using trypan blue and MTT assay, and the SAR associated with the position of N-terminal substituents in diazaspiro bicyclo hydantoin have also been discussed. It has been observed that these compounds displayed strong, moderate and weak cytotoxic activities. Interestingly, compounds having electron withdrawing groups at third and fourth position of the phenyl ring displayed selectively cytotoxic activities to both the cell lines tested with IC(50) value lower than 50 muM. In addition, the cytotoxic activities of the compounds 7(a-o) bearing the substituents at N-3 position of diazaspiro bicyclo hydantoin increases in the order alkene > ester > ether and plays an important role in determining their antitumor activities. The position and number of substituents in benzyl group attached to N-8 of diazaspiro bicyclo hydantoin nucleus interacted selectively with specific targets leading to the difference of biochemical and pharmacological effects.

Discovery of potent, orally-active, and muscle-selective androgen receptor modulators based on an N-aryl-hydroxybicyclohydantoin scaffold.

A novel, N-aryl-bicyclohydantoin selective androgen receptor modulator scaffold was discovered through structure-guided modifications of androgen receptor antagonists. A prototype compound (7R,7aS)-10b from this series is a potent and highly tissue-selective agonist of the androgen receptor. After oral dosing in a rat atrophied levator ani muscle model, (7R,7aS)-10b demonstrated efficacy at restoring levator ani muscle mass to that of intact controls and exhibited >50-fold selectivity for muscle over prostate.

Structure-activity relationship study of novel necroptosis inhibitors.

Necroptosis is a regulated caspase-independent cell death mechanism that results in morphological features resembling necrosis. It can be induced in a FADD-deficient variant of human Jurkat T cells treated with TNF-alpha. 5-(1H-Indol-3-ylmethyl)-2-thiohydantoins and 5-(1H-indol-3-ylmethyl)hydantoins were found to be potent necroptosis inhibitors (called necrostatins). A SAR study revealed that several positions of the indole were intolerant of substitution, while small substituents at the 7-position resulted in increased inhibitory activity. The hydantoin ring was also quite sensitive to structural modifications. A representative member of this compound class demonstrated moderate pharmacokinetic characteristics and readily entered the central nervous system upon intravenous administration.

Possible involvement of p38 in mechanisms underlying acceleration of proliferation by 15-deoxy-Delta(12,14)-prostaglandin J2 and the precursors in leukemia cell line THP-1.

15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ2), which is a ligand for peroxisome proliferator-activated receptor gamma (PPARgamma), induced apoptosis of several human tumors including gastric, lung, colon, prostate, and breast. However, the role of PPARgamma signals in other types of cancer cells (e.g., leukemia) except solid cancer cells is still unclear. The aim of this study is to evaluate the ability of 15dPGJ2 to modify the proliferation of the human leukemia cell line THP-1. 15dPGJ2 at 5 microM stimulated the proliferation in THP-1 at 24 to 72 h after incubation. In contrast, 15dPGJ2 at concentrations above 10 microM inhibited the proliferation through the induction of apoptosis. PGD2, PGJ2, and Delta12-PGJ2 (DeltaPGJ2), precursors of 15dPGJ2, had similar proliferative effects at lower concentrations, whereas they induced apoptosis at high concentrations. 15dPGJ2 and three precursors failed to induce the differentiation in THP-1 as assessed by using the differentiation marker CD11b. FACScan analysis revealed that PGD2 at 5 microM, PGJ2 at 1 microM, DeltaPGJ2 at 1 microM and 15dPGJ2 at 5 microM all accelerated cell cycle progression in THP-1. Immunoblotting analysis revealed that PGD2 at 5 microM and 15dPGJ2 at 5 microM inhibited the expression of phospho-p38, phospho-MKK3/MKK6, and phospho-ATF-2, and the expression of Cdk inhibitors including p18, p21, and p27 in THP-1. In contrast, PGJ2 at 1 microM and DeltaPGJ2 at 1 microM did not affect their expressions. These results suggest that 15dPGJ2 and PGD2 may, through inactivation of the p38 mitogen-activated protein kinase pathway, inhibit the expression of Cdk inhibitors, leading to acceleration of the THP-1 proliferation.

Action of prostanoids on the emetic reflex of Suncus murinus (the house musk shrew).

Several prostanoids were investigated for a potential to induce emesis in Suncus murinus. The TP receptor agonist 11alpha,9alpha-epoxymethano-15S-hydroxyprosta-5Z,13E-dienoic acid (U46619) induced emesis at doses as low as 3 microg/kg, i.p. but the DP receptor agonist 5-(6-Carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl) hydantoin (BW245C) was approximately 1000 times less potent. The emetic action of U46619 (300 microg/kg, i.p.) was antagonized significantly by the TP receptor antagonist, vapiprost (P<0.05). EP (prostaglandin E(2), 17-phenyl-omega-trinor prostaglandin E(2), misoprostol and sulprostone), FP (prostaglandin F(2alpha) and fluprostenol) and IP (iloprost and cicaprost) receptor agonists failed to induce consistent emesis at doses up to 300-1000 microg/kg, i.p. Fluprostenol reduced nicotine (5 mg/kg, s.c.)-but not copper sulphate (120 mg/kg, intragastric)-induced emesis; the other inconsistently emetic prostanoids were inactive to modify drug-induced emesis. The results indicate an involvement of TP and possibly DP and FP receptors in the emetic reflex of S. murinus.

Capillary gas chromatographic-electron-capture assay for the aldose reductase inhibitor imirestat in lens and plasma.

A sensitive and selective gas chromatographic-electron-capture assay was developed for the determination of the aldose reductase inhibitor imirestat in lens and plasma. The method involves solid-phase extraction of drug and internal standard from the plasma specimen or lens sample homogenate using "Baker"-10 SPE extraction columns followed by derivatization with pentafluorobenzyl bromide and further purification. Derivatives of drug and internal standard were separated on a fused-silica capillary column and analyzed using a 63Ni electron-capture detector. The limit of detection was 2.5 ng per lens or ml of plasma. The method was used to evaluate the pharmacokinetics of imirestat in human subjects and to quantitate imirestat in animal lens tissue following topical ocular administration.

The prevention of biochemical changes in lens, retina, and nerve of galactosemic dogs by the aldose reductase inhibitor AL01576.

Normal eight month old beagle dogs were fed a diet of 30% galactose for a period of two weeks. One group of dogs was untreated while three other groups were orally dosed with 0.25, 1.0, and 5.0 mg/kg of the aldose reductase inhibitor (ARI), AL01576. No visible changes were observed in the lens but glutathione (GSH) and inositol were depleted while dulcitol was elevated. These biochemical changes closely parallel those found in the (two week) streptozotocin induced diabetic rat. In contrast with the diabetic rat model, retina and nerve inositol was not found to differ from normal in spite of significant dulcitol accumulation. Plasma AL01576 was found to be inversely correlated with lens dulcitol concentration. When plasma AL0P1576 concentration was greater than 1 microgram/mL (5 mg/kg), there was a 95% reduction in dulcitol concentration (relative to untreated), while concentrations of 0.2 to 0.2 mg/mL (1 mg/kg) of AL01576 resulted in a dulcitol reduction of at least 70%. Retina and nerve dulcitol concentrations of galactosemic dogs were similarly diminished by AL01576 treatment. The dog model exhibits a biochemical profile of change and responsiveness to ARI therapy similar to that observed in hyperglycemic rats. Changes in retina morphology in diabetic and galactosemic dogs has been shown to closely resemble those occurring in human diabetics; these early biochemical events may also parallel.

Importancia de la dosificación plasmática de fármacos antiepilépticos en el tratamiento de la epilepsia: estudio de 65 pacientes / Significance of plasmic dosification of antiepileptic drugs in the management of epilepsia: study of 65 cases

Se presentan los resultados del primer trabajo cubano sobre dosificación de antiepilépticos en plasma y tratamiento de la epilepsia, de carácter interdisciplinario y en colaboración por 4 instituciones. Se estudiaron 65 adultos que padecían crisis epilépticas parciales, tonicoclónicas generalizadas o ambas, con más de 1 año, por lo menos, de observación en la Consulta Especial de Epilepsia. Se utilizó la cromatografía líquida de alta resolución para la dosificación plasmática de 3 drogas: defenilhidantoína (DFH) carbamazepina (CBZ) y fenobarbital (FB). El 58,4 % de los pacientes tenían un control total de sus crisis al momento de la determinación; el 32,3 un control parcial y el 9,2 no estaba controlado. La relación entre el control clínico y las cifras de antiepilépticos en plasma fue adecuada. Los pacientes no contolados tenían una concentración promedio más baja (entre 11,72 y 13,87 *g/mL) que los controlados (24,5 *g/mL para la DFH. Las cifras de DFH por debajo de 15 *g/mL se comportaron como subterapéuticas en nuestra serie. La concentración media de FB fue de 13,87 *g/mL y la de CBZ de 5,50. La DFH sola, la CBZ sola y la DFH asociada al FB fueron igualmente efectivas para el control de la crisis. Las dosis terapéuticas de DFH se encontraron en 4,8 mg por kg de peso corporal, y las de CBZ en 10,9. SE presentan casos demostrativos y se comentan los avances que han representado estas técnicas para el tratamiento de la epilepsia, por lo que se sugiere la introducción de las mismas en nuestro país al nivel provincial por la organización de salud cubana

Drug cytotoxicity at elevated temperature. In vitro study on the U-87MG glioma cell line.

The malignant glioma cell line U-87MG was used for 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), aziridinylbenzoquinone (AZQ), cis-diaminodichloroplatinum (II) (cis-DDP), and spirohydantoin mustard (SHM) treatments at 37 degrees and 42 degrees C. With the exception of SHM, all drugs killed a greater proportion of cells at the higher temperature, as assessed by the colony-formation assay. Drug-dose enhancement ratios were 1.6, 2.8, 2, and 1:1 for BCNU, AZQ, cis-DDP, and SHM, respectively. Because methods to heat discrete volumes of brain are now available, we conclude that hyperthermic increase of BCNU, AZQ, and cis-DDP cytotoxicity might have therapeutic application for malignant gliomas.

Phase I trial of spiromustine (NSC 172112) and evaluation of toxicity and schedule in a murine model.

Phase I evaluation of spiromustine was performed using an every-3-week schedule and a weekly X 3 schedule. Neurotoxicity was the dose-limiting toxicity presenting as alterations in cortical integrative functions (orientation, language, coordination), leading to a decrease in the level of consciousness. Traditional criteria for grading neurotoxicity poorly characterized these toxicities. The maximum tolerated dose was 6 mg/m2 every 3 weeks and 3 mg/m2 weekly X 3. Concurrent murine studies confirmed spiromustine as a schedule independent drug with toxicity correlating with peak plasma levels. Physostigmine had little effect on decreasing neurotoxicity in the murine model. The solvating agent used was not responsible for the neurotoxicity. Injection of spiromustine on a split-dose schedule decreased the acute neurological toxicity in mice and allowed a larger total dosage to be delivered (compared to single bolus dosage). Based on these results a split-dose schedule is suggested for future clinical trials.

A phase I trial of spirohydantoin mustard (NSC 172112) in patients with advanced cancer.

Spirohydantoin mustard (spiromustine, NSC 172112) is a classical bifunctional alkylating agent synthesized in an effort to develop antitumor agents effective against CNS tumors. The rationale was to combine the reactive moiety of an active antitumor agent with the hydantoin part of the molecule, which might serve as a carrier to cross the blood brain barrier. Thirty-eight patients with refractory solid tumors received spiromustine as part of a phase I trial at the Johns Hopkins Oncology Center. Three schedules were investigated: intravenously (IV) daily for three consecutive days, IV every other day for 3 days, and IV on a weekly basis for three doses, all cycled every 28 days. Hematologic toxicity was infrequently seen. Mild to moderate nausea and vomiting occurred on all schedules. The dose limiting toxicity was CNS toxicity characterized by mydriasis, xerostomia, lethargy, confusion, and hallucinations. This CNS toxicity was dose related, cumulative, and reversible. IV physostigmine appeared to diminish the neurotoxicity if administered before spiromustine and at frequent intervals following the drug. The maximum tolerated dose of spiromustine (without concomitant physostigmine) on the three times a week schedule is 6 mg/m2. With physostigmine pretreatment, 8 mg/m2 can be administered. The three times daily and every other day for three days schedules are not recommended for further study due to the severity of neurotoxicity. It is recommended that 6 mg/m2 be used as the starting dose for any phase II studies using the three times weekly schedule, and that physostigmine be used as needed to minimize neurotoxicity. Dose escalation above this level can be considered when individual tolerance has been established. Phase II trials to investigate the activity of this agent against primary and metastatic CNS malignancies appear indicated on the basis of three transient radiographic responses in refractory malignancies metastatic to the CNS.

Spiromustine and intracarotid artery cisplatin in the treatment of glioblastoma multiforme.

Glioblastoma multiforme is a highly malignant and rapid-growing primary brain tumor. It constitutes one-fourth of all intracranial tumors and about half of all gliomas. Survival rate following conventional treatment is only 12 to 18 months. At the National Institutes of Health, two promising therapies are currently undergoing clinical trials. Spirohydantoin mustard (spiromustine) is a combination of a nitrogen mustard and a derivative of phenytoin, an anticonvulsant drug that rapidly penetrates the blood-brain barrier and localizes in brain tumors. Intracarotid administration of cis-diamminedichloroplatinum (cisplatin) increases drug delivery to the tumor and, through hemodialysis, systemic exposure is reduced. Nursing management of patients receiving these two agents requires precise planning and implementation of an individualized plan of care to ensure a successful chemotherapeutic regimen.

The effect of treatment with single and split doses of spirohydantoin mustard on the growth delay of 9L rat brain tumor multicellular spheroids.

The effects of single and split doses of spirohydantoin mustard on the growth delay and cell survival of 9L rat brain tumor multicellular spheroids have been investigated. Treatment with 10-25 microM concentrations of spirohydantoin mustard inhibited the growth of spheroids. Growth delay increased rapidly when assayed during the first 6-10 days after treatment, after which a decrease in delay was observed. Plots of the values for the inflection points from growth curves vs drug concentration were linear, and growth delay correlated well with cell survival. There was less growth delay caused by treating spheroids with two 10 microM concentrations of spirohydantoin mustard than with a single 20 microM concentration. When a 2 or 4 hr time interval was allowed between treatment with 10 microM concentrations of spirohydantoin mustard the delay observed was greater than that obtained with the split dose control. When the resolution of the growth delay assay is considered, the split dose effect can be explained by the existence of a 5 microM threshold before any growth delay is observed. Nonetheless, the finding that spheroids treated on the split dose protocol recover from drug damage may have implications for the design of clinical protocols.

[New developments in chemotherapy for experimental brain metastasis].

Numerous animal models of brain tumor have been developed for screening new chemotherapeutic agents effective for brain tumors. However, most of these models have problem (s) of mechanical disruption of the blood-brain barrier by tumor inoculation and/or discrepancy in histologic nature and site of growth between inoculated tumors and human tumors. With the purpose of establishing a better model for the evaluation of antitumor activity, we have developed rat models of leptomeningeal tumors by intralateral-ventricle inoculation of myelogenous leukemia cells (DBLA-6) or AH130 ascites hepatoma cells. It was proved that in the two models there was no disruption of blood-brain barrier function by tumor inoculation and that the histologic pattern of DBLA-6 model tumor closely resembled the diffuse meningeal involvement of leukemic cells seen in the human disease. Using these models, several antitumor agents have been tested and ACNU, CCNU and procarbazine were found to be highly effective when given intravenously or intraperitoneally, and the intralateral-ventricle administration of methotrexate was also found to show a moderate antitumor activity. Furthermore it was found that intraperitoneal administration of spirohydantoin mustard, and intralateral-ventricle administration of 5-fluorodeoxyuridine were highly effective against the AH130 model.

Effect of a hydantoin prostaglandin analogue, BW245C, during oral dosing in man.

Following an open pilot study, the effects of repeated oral doses of BW245C, a hydantoin prostaglandin analogue, were studied in man. Six healthy volunteers received 150 micrograms BW245C or placebo 6-hourly for 5 days according to a double blind randomised balanced design with 7 days interval between treatments. Measurements of headache, facial flushing, heart rate, blood pressure, systolic time intervals, ECG, platelet aggregation responses to ADP and of subjective effects were made before and 1 and 3 h after the first dose of BW245C/placebo on days 1, 3 and 5 of dosing. BW245C produced significantly (p less than 0.05) higher headache scores than placebo on days 3 and 5; facial flushing, nasal stuffiness and abdominal discomfort were more frequent on BW245C than placebo. Heart rate, derived from the ECG, was significantly (p less than 0.05) higher and pre-ejection period significantly (p less than 0.05) shorter on BW245C at 1 h after dosing on each day. Left ventricular ejection time index, QS2 index, PR interval, QRS duration and T wave height were unchanged. Heart rate, counted at the radial pulse, and systolic and diastolic blood pressure, all measured lying and standing, were similar for BW245C and placebo. Platelet aggregation responses were not significantly different between the two treatments. The results indicate that repeated oral doses of BW245C, sufficient to cause moderately uncomfortable subjective effects, do not inhibit platelet aggregation.