Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways.

Skeletal muscle is composed of multinucleated fibres, formed after the differentiation and fusion of myoblast precursors. Skeletal muscle atrophy and hypertrophy refer to changes in the diameter of these pre-existing muscle fibres. The prevention of atrophy would provide an obvious clinical benefit; insulin-like growth factor 1 (IGF-1) is a promising anti-atrophy agent because of its ability to promote hypertrophy. However, the signalling pathways by which IGF-1 promotes hypertrophy remain unclear, with roles suggested for both the calcineurin/NFAT (nuclear factor of activated T cells) pathway and the PtdIns-3-OH kinase (PI(3)K)/Akt pathway. Here we employ a battery of approaches to examine these pathways during the hypertrophic response of cultured myotubes to IGF-1. We report that Akt promotes hypertrophy by activating downstream signalling pathways previously implicated in activating protein synthesis: the pathways downstream of mammalian target of rapamycin (mTOR) and the pathway activated by phosphorylating and thereby inhibiting glycogen synthase kinase 3 (GSK3). In contrast, in addition to demonstrating that calcineurin does not mediate IGF-1-induced hypertrophy, we show that IGF-1 unexpectedly acts via Akt to antagonize calcineurin signalling during myotube hypertrophy.

Differentiation stage-specific inhibition of the Raf-MEK-ERK pathway by Akt.

Extracellular signals often result in simultaneous activation of both the Raf-MEK-ERK and PI3K-Akt pathways (where ERK is extracellular-regulated kinase, MEK is mitogen-activated protein kinase or ERK kinase, and PI3K is phosphatidylinositol 3-kinase). However, these two signaling pathways were shown to exert opposing effects on muscle cell hypertrophy. Furthermore, the PI3K-Akt pathway was shown to inhibit the Raf-MEK-ERK pathway; this cross-regulation depended on the differentiation state of the cell: Akt activation inhibited the Raf-MEK-ERK pathway in differentiated myotubes, but not in their myoblast precursors. The stage-specific inhibitory action of Akt correlated with its stage-specific ability to form a complex with Raf, suggesting the existence of differentially expressed mediators of an inhibitory Akt-Raf complex.

Identification of pharmacokinetically stable 3, 10-dibromo-8-chlorobenzocycloheptapyridine farnesyl protein transferase inhibitors with potent enzyme and cellular activities.

Farnesyl protein transferase (FPT) is a promising target for the development of cancer chemotherapeutics because it is responsible for the farnesylation of oncogenic p21 Ras proteins which are found in nearly 30% of all human cancers and necessary for cellular development and growth. The recent discovery and progression to phase II clinical trials of trihalobenzocycloheptapyridine Sch-66336 as a potent inhibitor of FPT with oral, in vivo efficacy in mice have spawned extensive structure-activity relationship studies (SAR) of this class of compounds. Of the many trihalobenzocycloheptapyridine analogues prepared, we have identified several which inhibit FPT and cellular proliferation at single-digit nanomolar concentrations and which have good pharmacokinetic properties in mice.

Tricyclic farnesyl protein transferase inhibitors: crystallographic and calorimetric studies of structure-activity relationships.

Crystallographic and thermodynamic studies of farnesyl protein transferase (FPT) complexed with novel tricyclic inhibitors provide insights into the observed SAR for this unique class of nonpeptidic FPT inhibitors. The crystallographic structures reveal a binding pattern conserved across the mono-, di-, and trihalogen series. In the complexes, the tricycle spans the FPT active site cavity and interacts with both protein atoms and the isoprenoid portion of bound farnesyl diphosphate. An amide carbonyl, common to the tricyclic compounds described here, participates in a water-mediated hydrogen bond to the protein backbone. Ten high-resolution crystal structures of inhibitors complexed with FPT are reported. Included are crystallographic data for FPT complexed with SCH 66336, a compound currently undergoing clinical trials as an anticancer agent (SCH 66336, 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5, 6]cyclohepta[1, 2-b]pyridin-11-yl)-1-piperidinyl]-2-oxoethyl]-1-piperidinecarbo xamide ). Thermodynamic binding parameters show favorable enthalpies of complex formation and small net entropic contributions as observed for 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-11H-benzo[5, 6]cyclohepta[1, 2-b]pyridin-11-ylidene)-1-piperidinyl]-2-oxoethyl]pyridine N-oxide where DeltaH degrees bind = -12.5 kcal/mol and TDeltaS degrees bind = -1.5 kcal/mol.

Effects of SCH 59228, an orally bioavailable farnesyl protein transferase inhibitor, on the growth of oncogene-transformed fibroblasts and a human colon carcinoma xenograft in nude mice.

The products of the Ha-, Ki-, and N-ras proto-oncogenes comprise a family of 21 kDa guanine nucleotide-binding proteins which play a crucial role in growth factor signal transduction and in the control of cellular proliferation and differentiation. Activating mutations in the ras oncogenes occur in a wide variety of human tumors. Ras proteins undergo a series of posttranslational processing events. The first modification is addition of the 15-carbon isoprene, farnesyl, to a Cys residue near the carboxy-terminus of Ras. Prenylation allows the Ras oncoprotein to localize to the plasma membrane where it can initiate downstream signalling events leading to cellular transformation. Inhibitors of the enzyme which catalyzes this step, farnesyl protein transferase (FPT), are a potential class of novel anticancer drugs which interfere with Ras function. SCH 59228 is a tricyclic FPT inhibitor which inhibits the farnesylation of purified Ha-Ras with an IC50 of 95 nM and blocks the processing of Ha-Ras in Cos cells with an IC50 of 0.6 microM. SCH 59228 has favorable pharmacokinetic properties upon oral dosing in nude mice. The in vivo efficacy of SCH 59228 was evaluated using a panel of tumor models grown in nude mice. These included several rodent fibroblast lines expressing mutationally-activated (val12) forms of the Ha-Ras oncogene. In some cases, these proteins contain their native C-terminal sequence (CVLS) which directs farnesylation. In one model, the C-terminal sequence was altered to CVLL, making the expressed protein a substrate for a distinct prenyl transferase, geranylgeranyl protein transferase-1. When dosed orally at 10 and 50 mg/kg (four times a day, 7 days a week) SCH 59228 significantly inhibited tumor growth of cells expressing farnesylated Ha-Ras in a dose-dependent manner; over 90% growth inhibition was observed at the 50 mg/kg dose. Tumor growth of cells expressing the geranylgeranylated form of Ha-Ras was less potently inhibited. Growth of tumors derived from a rodent fibroblast line expressing activated Ki-Ras containing its native C-terminal sequence (CVIM), which preferentially directs farnesylation, was also inhibited by SCH 59228. Inhibition in the Ki-Ras model was less than that observed in the Ha-Ras model. In contrast, tumors derived from cells transformed with the mos oncogene were not significantly inhibited even at the highest dose level. SCH 59228 also significantly and dose-dependently inhibited the growth of human colon adenocarcinoma DLD-1 xenografts (which express activated Ki-ras). These results indicate that SCH 59228 possesses in vivo antitumor activity upon oral dosing in tumor models expressing activated ras oncogenes. This is the first report of oral antitumor activity with an FPT inhibitor. These results are discussed in light of recent observations on alternative prenylation of some Ras isoforms.

(+)-4-[2-[4-(8-Chloro-3,10-dibromo-6,11-dihydro-5H-benzo[5, 6]cyclohepta[1,2-b]- pyridin-11(R)-yl)-1-piperidinyl]-2-oxo-ethyl]-1-piperidinecarboxamid e (SCH-66336): a very potent farnesyl protein transferase inhibitor as a novel antitumor agent.

We have previously shown that appropriate modification of the benzocycloheptapyridine tricyclic ring system can provide potent farnesyl protein transferase (FPT) inhibitors with good cellular activity. Our laboratories have also established that incorporation of either pyridinylacetyl N-oxide or 4-N-carboxamidopiperidinylacetyl moieties results in pharmacokinetically stable inhibitors that are orally efficacious in nude mice. We now demonstrate that further elaboration of the tricyclic ring system by introducing a bromine atom at the 7- or the 10-position of the 3-bromo-8-chlorotricyclic ring system provides compounds that have superior potency and selectivity in FPT inhibition. These compounds have good serum levels and half-lives when given orally to rodents and primates. In vitro and in vivo evaluation of a panel of these inhibitors has led to identification of 15 (SCH 66336) as a highly potent (IC50 = 1.9 nM) antitumor agent that is currently undergoing human clinical trials.

Inhibitors of farnesyl protein transferase. 4-Amido, 4-carbamoyl, and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]- cyclohepta[1,2-b]pyridin-11-yl)piperazine and 1-(3-bromo-8-chloro-6,11- dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)piperazine.

The synthesis of a variety of novel 4-amido, 4-carbamoyl and 4-carboxamido derivatives of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl) piperazine to explore the SAR of this series of FPT inhibitors is described. This resulted in the synthesis of the 4- and 3-pyridylacetyl analogues 45a and 50a, respectively, both of which were orally active but were found to be rapidly metabolized in vivo. Identification of the principal metabolites led to the synthesis of a variety of new compounds that would be less readily metabolized, the most interesting of which were the 3- and 4-pyridylacetyl N-oxides 80a and 83a. Novel replacements for the pyridylacetyl moiety were also sought, and this resulted in the discovery of the 4-N-methyl and 4-N-carboxamidopiperidinylacetyl derivatives 135a and 160a, respectively. All of these derivatives exhibited greatly improved pharmacokinetics. The synthesis of the corresponding 3-bromo analogues resulted in the discovery of the 4-pyridylacetyl N-oxides 83b (+/-) and 85b [11S(-)] and the 4-carboxamidopiperidinylacetamido derivative 160b (+/-), all of which exhibited potent FPT inhibition in vitro. All three showed excellent oral bioavailability in vivo in nude mice and cynomolgus monkeys and exhibited excellent antitumor efficacy against a series of tumor cell lines when dosed orally in nude mice.

A randomized, placebo-controlled, double-blind trial of mesalamine in the maintenance of remission of Crohn's disease. The Canadian Mesalamine for Remission of Crohn's Disease Study Group.

BACKGROUND & AIMS: The efficacy of mesalamine for the maintenance of remission in patients with Crohn's disease is controversial. The aim of this study was to conduct a double-blind, placebo-controlled study of mesalamine (750 mg four times a day for 48 weeks) in maintaining remission in 293 patients with Crohn's disease. Patients were stratified according to the method of induction of remission (medical or surgical). METHODS: Patients were assessed at weeks 4, 12, 24, 36, and 48. Relapse was defined as a Crohn's Disease Activity Index of >150 (+60 points over baseline). RESULTS: Of the 293 patients, 246 (84%) returned for at least 4 weeks of follow-up and were included in the final analysis. Thirty of the 118 (25%) who received mesalamine had a relapse compared with 47 of 128 (36%) receiving placebo (P = 0.056). Among those with relapse, the time to relapse was 119 days for the mesalamine-treated patients compared with 109 days for placebo-treated patients (P = NS). However, 25% of mesalamine-treated patients had relapsed by 249 days of follow-up compared with 154 days for placebo-treated patients. Subgroup analysis showed that patients with ileocecal-colonic disease or patients who were women had fewer relapses on mesalamine therapy than placebo-treated patients (21% vs. 41%, P = 0.018; and 19% vs. 41%, P = 0.003, respectively). CONCLUSIONS: Mesalamine treatment reduced relapse compared with placebo treatment, although conventional statistical significance was not achieved.

Antitumor 8-chlorobenzocycloheptapyridines: a new class of selective, nonpeptidic, nonsulfhydryl inhibitors of ras farnesylation.

Ras farnesylation by farnesyl protein transferase (FPT) is an intracellular event that facilitates the membrane association of the ras protein and is involved in the signal transduction process. FPT inhibition could be a novel, noncytotoxic method of treating ras dependent tumor growth. We report here three structural classes of 8-chlorobenzocycloheptapyridines as novel, nonpeptidic, nonsulfhydryl FPT inhibitors having antitumor activity in mice when dosed orally. We discuss structural and conformational aspects of these compounds in relation to biological activities as well as a comparison to the conformation of a bound tetrapeptide FPT inhibitor.

Reliability and validity of infant speech-sound discrimination-in-noise thresholds.

Infants were tested on a speech-sound discrimination-in-noise task using the visual reinforcement infant speech discrimination (VRISD) procedure with an adaptive (up-down) threshold protocol. An adult control group was tested using the same stimuli and apparatus. The speech sounds were synthetic magnitude of ba and magnitude of ga. The masker was band-passed presented continuously at 48 dB SPL. Test-retest reliability was good for both groups, although test-retest differences were smaller for adults. For infants the mean of the absolute values of the differences between tests was only 5.2 dB, and there was less than a 10-dB difference between the two tests of 14 (87.5%) of the 16 infants completing the study. The infant-adult difference in discrimination threshold in noise was 6.9 dB, which agrees well with detection-in-noise thresholds from earlier studies and with discrimination-in-noise thresholds obtained on a subset of subjects in our earlier work. Advantages of the adaptive threshold procedure and its possible applications both in research studies and in the clinic are discussed.

Infant-adult differences in unmasked thresholds for the discrimination of consonant-vowel syllable pairs.

Speech sound discrimination thresholds were obtained for two speech sound contrasts (/ba/ vs. /da/ and /ba/ vs. /ga/) for infant and adult subjects. The stimuli were computer-generated synthetic tokens. An adaptive (one-up, one-down) threshold procedure was used with the visual reinforcement infant speech discrimination procedure for the infant subjects. Adults were tested using the same apparatus and threshold-tracking protocol as the infants. There was a 28-dB difference in threshold for discrimination of /ba/ versus /da/ and a 25-dB difference in threshold for discrimination of /ba/versus/ga/ between the infants and the adults. The differences reveal that to reach a criterion level of performance on a simple speech perception task, infants require much greater stimulus intensity than do adults. This has implications for our understanding of normal auditory development, for our notions of hearing impairment in infants and for the role of intensity in research studies of infant speech perception.

Infant speech-sound discrimination in noise.

The effects of noise on 7- to 11-month-old infants' speech-sound discrimination (/ba/vs/ga/) were determined using a conditioned head-turn procedure. Variation in performance as a function of signal-to-noise ratio (S/N) was estimated by testing each infant at four S/N's (-8, 0, 8, and 16 dB). Adults were tested for comparison at four S/N's (-12, -8, -4, and 0 dB). The S/N's were chosen based on pilot data. Performance varied monotonically with S/N for both age groups, but infants required greater S/N than adults to achieve comparable levels of performance. Both groups were also tested using an adaptive (1-up, 1-down) threshold procedure with a 3-dB step size. There was a group mean difference in threshold of 5.8-dB S/N favoring the adults. Weighted group psychometric functions, derived from the responses obtained in the adaptive runs, showed good correspondence with the data points at the four S/N's. The slopes of these functions were the same (7.5%/dB) for infants and adults. The results suggest that infants are at a greater disadvantage than adults when processing speech in noise and that concern over the effects of a noisy environment on the acquisition of language is justified. In addition, the adaptive threshold procedure can be used as an efficient way to estimate the limits of discrimination ability as a function of S/N or intensity, both for individual subjects and for groups of subjects, in developmental research.

Mutagenic metabolites of benzene detected in the Microscreen assay.

The reactive metobolite responsible for benzene hematotoxicity and carcinogenicity is unknown. It can be hypothesized that the ultimate carcinogen derived from benzene metabolism might also act as a mutagen. This laboratory has recently developed a new assay that can detect mutagens of all types, using a single strain of bacteria, E. coli WP2s (lambda), as a target. Different genetic end points can be monitored in the same exposed population of bacteria. When a number of known metabolites of benzene were assayed, only trans,trans-muconic acid gave a strong positive response. Mutations were induced at two genetic loci (Trp+ revertants and T5 resistance). The mutagenic activity was greatly increased when a rat liver metabolizing system was added. We speculate that trans,trans-muconic acid is metabolized to a diepoxide, which may be the ultimate mutagen and possibly the ultimate carcinogen.

Articulation index predictions of contextually dependent words.

Three investigations were conducted to determine the application of the articulation index (AI) to the prediction of speech performance of hearing-impaired subjects as well as of normal-hearing listeners. Speech performance was measured in quiet and in the presence of two interfering signals for items from the Speech Perception in Noise test in which target words are either highly predictable from contextual cues in the sentence or essentially contextually neutral. As expected, transfer functions relating the AI to speech performance were different depending on the type of contextual speech material. The AI transfer function for probability-high items rises steeply, much as for sentence materials, while the function for probability-low items rises more slowly, as for monosyllabic words. Different transfer functions were also found for tests conducted in quiet or white noise rather than in a babble background. A majority of the AI predictions for ten individuals with moderate sensorineural loss fell within +/- 2 standard deviations of normal listener performance for both quiet and babble conditions.

Inter-interpreter agreement for ABR tracings. Results from 100 patients under otoneurologic investigation.

The auditory brainstem response (ABR) tracings of 100 patients under investigation for a neural lesion were examined independently by two audiologists. Each tracing for the ear under question was classified as normal or abnormal. There was 94% agreement. Suggestions are made as to how to establish quality control in audiology clinics.

Effect of recent hearing aid improvements on management of the hearing impaired.

Recent developments in hearing aid technology and earmold acoustics have improved the outlook for the hearing impaired. Advances involving the electric microphone, integrated circuit, and earmold/hearing aid coupling system have affected such features as compression amplification, filtering, and frequency characteristics of the amplified signal at the eardrum. The resulting changes in hearing aid fitting approaches are described, including factors which are considered in such fittings as in-the-ear, binaural, CROS and BICROS, with examples of difficult cases. Hearing aids are then discussed in the context of a broader rehabilitation strategy.

Cerebellopontine angle lesions: an audiological test protocol.

Audiological test protocols were devised for use in patients suspected of having a cerebellopontine angle tumor. Data supporting the logic for inclusion of each test are presented. Protocol design was based on test results obtained in 45 tumor and 554 non-tumor patients. In summary, we found: 1) speech-in-noise results were of some value in those patients with normal hearing and of no value in those patients with hearing loss, 2) the acoustic reflex test gave misleading information in 25% of patients, and 3) the ABR test proved to be the most effective test in separating tumor and non-tumor patients. All individuals having cerebellopontine angle tumors and 11% of those without such lesions had abnormal ABR results in this series.

Diagnostic features of the auditory brainstem response in identifying cerebellopontine angle tumours.

This report concerns the diagnostic features of the ABR which are most effective in identifying patients with cerebellopontine angle tumours. The conclusions are based on the results of 40 normal subjects, 35 patients without a cerebellopontine angle tumour and 68 patients without a tumour. One hundred per cent of the tumour patients and ten per cent of the non-tumour patients had abnormal ABR results.

A comparison of three modes of hearing aid-telephone coupling.

The stray magnetic field emitted by telephone receivers has been used by hearing aid wearers to improve telephone communication. Recent technical advances by Bell Canada have eliminated the magnetic field. The company sponsored the design of a hearing aid modification to compensate for the loss of this magnetic field. This report concerns the evaluation of the modification. The results of two experiments indicated that it did not provide a satisfactory substitute for the magnetic field. A by-product of this project was the development of a device (audiometer-telephone interface) which permits simulation of telephone listening, thus allowing clinical assessment of a patient's telephone communication ability.