Using second harmonic generation to predict patient outcome in solid tumors.

BACKGROUND: Over-treatment of estrogen receptor positive (ER+), lymph node-negative (LNN) breast cancer patients with chemotherapy is a pressing clinical problem that can be addressed by improving techniques to predict tumor metastatic potential. Here we demonstrate that analysis of second harmonic generation (SHG) emission direction in primary tumor biopsies can provide prognostic information about the metastatic outcome of ER+, LNN breast cancer, as well as stage 1 colorectal adenocarcinoma. METHODS: SHG is an optical signal produced by fibrillar collagen. The ratio of the forward-to-backward emitted SHG signals (F/B) is sensitive to changes in structure of individual collagen fibers. F/B from excised primary tumor tissue was measured in a retrospective study of LNN breast cancer patients who had received no adjuvant systemic therapy and related to metastasis-free survival (MFS) and overall survival (OS) rates. In addition, F/B was studied for its association with the length of progression-free survival (PFS) in a subgroup of ER+ patients who received tamoxifen as first-line treatment for recurrent disease, and for its relation with OS in stage I colorectal and stage 1 lung adenocarcinoma patients. RESULTS: In 125 ER+, but not in 96 ER-negative (ER-), LNN breast cancer patients an increased F/B was significantly associated with a favorable MFS and OS (log rank trend for MFS: p = 0.004 and for OS: p = 0.03). On the other hand, an increased F/B was associated with shorter PFS in 60 ER+ recurrent breast cancer patients treated with tamoxifen (log rank trend p = 0.02). In stage I colorectal adenocarcinoma, an increased F/B was significantly related to poor OS (log rank trend p = 0.03), however this relationship was not statistically significant in stage I lung adenocarcinoma. CONCLUSION: Within ER+, LNN breast cancer specimens the F/B can stratify patients based upon their potential for tumor aggressiveness. This offers a "matrix-focused" method to predict metastatic outcome that is complementary to genomic "cell-focused" methods. In combination, this and other methods may contribute to improved metastatic prediction, and hence may help to reduce patient over-treatment.

Gene signature critical to cancer phenotype as a paradigm for anticancer drug discovery.

Malignant cell transformation commonly results in the deregulation of thousands of cellular genes, an observation that suggests a complex biological process and an inherently challenging scenario for the development of effective cancer interventions. To better define the genes/pathways essential to regulating the malignant phenotype, we recently described a novel strategy based on the cooperative nature of carcinogenesis that focuses on genes synergistically deregulated in response to cooperating oncogenic mutations. These so-called 'cooperation response genes' (CRGs) are highly enriched for genes critical for the cancer phenotype, thereby suggesting their causal role in the malignant state. Here, we show that CRGs have an essential role in drug-mediated anticancer activity and that anticancer agents can be identified through their ability to antagonize the CRG expression profile. These findings provide proof-of-concept for the use of the CRG signature as a novel means of drug discovery with relevance to underlying anticancer drug mechanisms.

Rasagiline-associated motor improvement in PD occurs without worsening of cognitive and behavioral symptoms.

BACKGROUND: Cognitive and behavioral adverse events (AEs) such as hallucinations, confusion, depression, somnolence and other sleep disorders commonly limit effective management of motor symptoms in PD. Rasagiline (N-propargyl-1(R)-aminoindan) mesylate is a novel, second-generation, selective, irreversible monoamine oxidase type B inhibitor, demonstrated in monotherapy and adjunctive trials to be effective for PD with excellent tolerability. METHODS: The occurrence of cognitive and behavioral AEs and the change from baseline in the Unified Parkinson's Disease Rating Scale (UPDRS) part I mental subscores were reviewed in two multicenter, randomized, placebo-controlled, 26-week trials of rasagiline for early and moderate-to-advanced patients with PD. The UPDRS is a multi-item rating scale specific to PD; part I rates the patient's intellectual impairment, thought disorders, depression and motivation/initiative. RESULTS: The TEMPO study evaluated rasagiline monotherapy in early PD patients (n=404). The PRESTO study evaluated rasagiline as adjunctive therapy in moderate-to-advanced PD patients with motor complications who were receiving optimized levodopa/carbidopa (n=472). In the analysis of adverse event reporting for both studies, no cognitive and behavioral AE in either the rasagiline 1 mg or placebo groups exceeded 10% of the study population and the frequency differences between rasagiline 1 mg and placebo never exceeded 3%. There was no adverse effect on the UPDRS mental subscore relative to placebo in either of the two studies. CONCLUSION: Rasagiline 1 mg once daily improves PD symptoms and motor fluctuations in early and moderate-to-advanced PD patients without causing significant cognitive and behavioral AE or adverse changes in mentation, behavior and mood.

The efficacy and safety of oral desmopressin in children with primary nocturnal enuresis.

PURPOSE: We confirmed findings that oral desmopressin safely decreases the number of wet nights in children with enuresis and identified doses at which acceptable responses can be obtained. MATERIALS AND METHODS: We evaluated the safety and efficacy of oral desmopressin in a double-blind, placebo controlled, parallel group, randomized, multicenter trial of 193 children 6 to 16 years old with documented primary nocturnal enuresis. The study was conducted in 2 phases: 1) a 2-week dose ranging phase in which children received desmopressin (0.2, 0.4 or 0.6 mg.) or placebo at bedtime and 2) an 8-week dose titration phase that followed a 2-week placebo washout. Patients received 0.2 mg. desmopressin or placebo for the first 2 weeks and then the dose was increased in 0.2 mg. increments at 2-week intervals until the patient was completely dry or was receiving 0.6 mg. Patients were instructed to limit fluid intake. Mean decrease from baseline in the number of wet nights, percentage of responding patients and safety were assessed at 2-week intervals. RESULTS: There was a statistically significant linear response to oral desmopressin at doses from 0.2 to 0.6 mg. during the dose ranging phase (p < or =0.05). The decrease in wet nights after 2 weeks of treatment with desmopressin was 27%, 30% and 40% at 0.2, 0.4 and 0.6 mg. doses, respectively, compared to 10% with placebo. All doses were statistically significantly different from placebo (p < or =0.05). During the dose titration phase all placebo treated and 87% of desmopressin treated patients were receiving the maximum dose of 3 tablets nightly because they had not been completely dry in the previous 2 weeks. Nevertheless, 44% of desmopressin treated patients had achieved at least a 50% reduction from baseline in the number of wet nights per 2 weeks at the lower doses of 0.2 and 0.4 mg. Most adverse events (rhinitis, pharyngitis, headache and increased cough) were mild to moderate in severity, unrelated to treatment and resolved before the study was completed. CONCLUSIONS: Oral desmopressin administered at bedtime to children with primary nocturnal enuresis was significantly better than placebo for decreasing episodes of bed-wetting (p <0.05). A linear dose-response relationship was observed (p <0.05). An acceptable response to treatment (50% or greater reduction from baseline in wet nights per 2 weeks) was seen at all doses of desmopressin. Oral desmopressin, up to 0.6 mg. for 8 weeks, was well tolerated.

The Rilutek (riluzole) Global Early Access Programme: an open-label safety evaluation in the treatment of amyotrophic lateral sclerosis.

OBJECTIVES: This study had two main objectives: 1. To enable patients with amyotrophic lateral sclerosis (ALS) who had not participated in previous riluzole trials to receive riluzole therapy, and 2. To expand safety experience with the drug in a broad patient population. METHODS: This was a Phase IIIb multicentre, multinational, open-label, uncontrolled single treatment study of riluzole. Patients with diagnosed possible or probable ALS were administered 100 mg of riluzole/day (50 mg b.i.d.). Clinical and laboratory adverse events were recorded every month for the first 3 months and thereafter at 3-monthly intervals. RESULTS: 8383 patients from 44 countries were entered into the study; 7916 of these patients with recorded data were administered the study drug. The mean duration of riluzole treatment was 202.1 days, with a range of 1-630 days. The most frequently reported serious and non-serious adverse events were common symptoms of ALS (respiratory symptoms and dysphagia), and only 1.9% of serious adverse events were considered to be related to the study drug. CONCLUSIONS: The safety results with this broad population (over 10% of the estimated ALS population worldwide) were consistent with those previously reported from placebo-controlled trials. No increase in adverse events and no unexpected adverse events were observed.

Clinical trials of riluzole in patients with ALS. ALS/Riluzole Study Group-II.

Two double-blinded, placebo-controlled clinical trials of riluzole have now been carried out in more than 1,100 patients with ALS. The results of both studies show a modest benefit in prolonging survival that is statistically significant. These results led to the availability of this drug by the Food and Drug Administration for use in the United States beginning in early 1996. This is the first drug that has been available for ALS. It begins a new era in both basic and clinical research in an attempt to find a cure for this disease.

In vivo recovery and survival of monoclonal-antibody-purified factor VIII concentrates.

In response to reports of discrepant in vitro assays of high-purity concentrates, a double-blind crossover study of in vivo recovery and half-life of two brands of monoclonal-antibody-purified factor VIII concentrates (Monoclate and Hemofil-M) was performed in 23 patients with hemophilia A. In vivo recoveries were close to values predicted from the labelled unitage when plasma samples were assayed by a one-stage method. When a two-stage assay was used, lower recoveries were calculated and the recovery with Hemofil-M was slightly but significantly lower than that with Monoclate. The concentrates were re-assayed in vitro by the two-stage method. Monoclate (which is assayed by the manufacturer using a two-stage method) contained 97% of the labelled potency and Hemofil-M (which is assayed by the manufacturer using a one-stage method) contained 81% of the labelled potency. Differences in in vitro and in vivo assay methods contribute to disparities between expected and observed factor VIII recovery. Clearance of Hemofil-M was significantly faster than that of Monoclate, but volume of distribution at the steady state, mean residence time, and plasma half-disappearance times of the two concentrates were not significantly different.

Lack of immune response to mouse IgG in hemophilia A patients treated chronically with Monoclate, a monoclonal antibody affinity purified factor VIII preparation.

Hemophilia A is caused by factor VIII deficiency that historically has been treated with either a cryoprecipitate fraction of serum or factor VIII concentrate. Recently, the availability of affinity isolated factor VIII (Monoclate) has allowed for a highly purified preparation for the chronic therapy of hemophilia A. This factor VIII preparation contains a trace quantity (less than 50 ng/100 I. U.) of mouse IgG. Immunoassays for the measurement of human IgG, IgM and IgE anti-mouse IgG antibody (HAMA) were developed and used to measure HAMA levels in hemophilia A patients undergoing chronic therapy with Monoclate in three different clinical studies. Natural antibodies to mouse IgG were observed in patient sera prior to Monoclate infusion. Data is presented demonstrating that induction of HAMA upon Monoclate treatment does not occur. The low level of mouse IgG contained in Monoclate appears to be below the threshold of immunogenicity. Most importantly, clinical symptoms related to hypersensitivity or anaphylaxis were never observed in any patient undergoing chronic therapy with Monoclate in these clinical studies.

Initial clinical experience with a new pasteurized monoclonal antibody purified factor VIIIC.

Heat treatment of lyophilized factor VIII and factor IX concentrates has been found to eliminate HIV virus infectivity in plasma-derived products. Pasteurization of factor VIII in solution has recently been used to reduce the risk of hepatitis transmission in concentrates prepared by standard fractionation methods. This report presents early experience with factor VIIIC prepared by monoclonal antibody immunoaffinity chromatography following pasteurization of the factor VIIIC/von Willebrand factor complex (Monoclate-P). Twelve patients were treated in three centers with Monoclate-P. Recovery and survival of factor VIII clotting activity were determined and patients were closely monitored for infusion safety. The mean half-life was 14.2 +/- 5.0 while recovery in predicted plasma volume was 72 +/- 12% corresponding to a response of 1.99 +/- 0.66 U/dL for every U/kg administered. These values are very similar to those found for Monoclate in previous studies indicating that pasteurized factor VIIIC purified by immunoaffinity chromatography retains satisfactory pharmacokinetic properties with an added margin of viral safety.

Viral safety and inhibitor development associated with factor VIIIC ultra-purified from plasma in hemophiliacs previously unexposed to factor VIIIC concentrates. The Monoclate Study Group.

A multicenter study evaluated the potential for transmitting non-A/non-B hepatitis, as well as other viruses, with the use of the factor VIIIC product Monoclate. This product is purified from plasma via the monoclonal process that includes heat treatment for ultra-purification as a final step. Twenty different lots of Monoclate were used, and each patient received the assigned lot for the first 6 months of the trial. Nineteen of 38 patients adhered strictly to International Committee on Thrombosis and Hemostasis criteria in that they had normal liver enzymes, no evidence of hepatitis prestudy, and had no previous blood product use. Fourteen hemophilia centers from the United States, the United Kingdom, the Netherlands, and Israel participated in this study. Development of factor VIII inhibitor occurred in six of 38 patients, which was within the statistically expected range. Adverse events were mild, and Monoclate was well tolerated in this group. All 38 patients remained HIV seronegative.

Prostacyclin and thromboxane A2 synthesis by rabbit pulmonary artery.

Superfused spiral strips of rabbit intrapulmonary artery (i.p.a.) were contracted by arachidonic acid (AA) and by the following substances in order of potency: prostaglandin (PG) endoperoxide analog (U46619) > PGH2 > PGF2 alpha. Intrapulmonary artery strips were consistently relaxed by PGE2 and by the enzyme inhibitors, indomethacin, aspirin, meclofenamic acid and 1-pentylimidazole. These latter inhibitors of cyclooxygenase and thromboxane (TX) synthetase also blocked the AA-induced contraction of rabbit i.p.a. Prostacyclin had no effect on the i.p.a. or produced either a small contraction or relaxation. TXA2, formed by incubating horse platelet microsomes with PHG2, always contracted the tissue and was more potent than the parent endoperoxide. Incubations of [14C]AA with i.p.a. produced mainly [14C]-6-keto-PGF1 alpha (he breakdonw product of prostacyclin ) and [14C]TXB2 (the breakdown product of TXA2); the identities of these products were confirmed by radioimmunoassay and by gas chromatography-mass spectrometry. The synthesis of TXB2 by i.p.a. cannot be attributed to adhering lung tissue or platelets and appears to be produced by the vascular tissue itself. It is concluded that, although both prostacyclin and thromboxane may contribute to the resting tone of the rabbit i.p.a., the response to AA is mainly due to production of TXA2.

Poststimulation catecholamine synthesis and tyrosine hydroxylase activation in central noradrenergic neurons. I. In vivo stimulation of the locus coeruleus.

The ability of increased neuronal activity to accelerate catecholamine biosynthesis and tyrosine hydroxylase activity in the rat brain was tested. Noradrenergic neurons of the locus coeruleus (LC) were stimulated unilaterally at 20 Hz and the cortex and/or hippocampus from stimulated and contralateral (control) sides of the brain were analyzed and compared. Rats were injected with a dopa decarboxylase inhibitor and the accumulation of endogenously synthesized dopa used as an in vivo index of tyrosine hydroxylase activity. Thirty minutes after termination of 15 min of unilateral LC stimulation, dopa accumulation was 35% greater in the ipsilateral cortex + hippocampus. In untreated rats, at the end of 15 min of LC stimulation, there was an ipsilateral depletion of cortical norepinephrine (NE) which recovered within 30 min. When rats were injected with [3H]tyrosine (i.v.) during this half-hour recovery period, a poststimulation increase in [3H]catecholamine synthesis was observed in both the cortex (63%) and hippocampus (55%). In the cortex, there was more newly synthesized [3H]dopamine than [3H]NE, but LC stimulation preferentially increased the synthesis of [3H]NE. The hippocampus contained negligible amounts of [3H]dopamine and was used in subsequent studies. Tyrosine hydroxylase activity was assayed in vitro in supernatants derived from stimulated and control hippocampi. Ten minutes of LC stimulation (20 Hz) maximally activated hippocampal tyrosine hydroxylase and this activation was maintained for up to 20 min after stimulation was terminated. The results illustrate a stimulation-induced activation of NE biosynthesis and tyrosine hydroxylase activity in central NE neurons in vivo. This activation is maintained in the immediate poststimulation period and is not necessarily due to removal of end product inhibition by NE.

Poststimulation catecholamine synthesis and tyrosine hydroxylase activation in central noradrenergic neurons. II. Depolarized hippocampal slices.

The ability of neuronal depolarization to increase catecholamine biosynthesis in the poststimulation period was investigated in a preparation of central noradrenergic tissue, maintained in vitro. Rat hippocampal slices were superfused with oxygenated Krebs-Ringer phosphate saline (KRP) or depolarized with KRP containing 55 mM KCl. Slices were then transferred to fresh, nondepolarizing KRP containing [3H]tyrosine for further incubation. Ten minutes of K+ depolarization resulted in a 78% increase in [3H]catecholamine synthesis, measured in the poststimulation period, relative to nondepolarized, control slices. This activation of catecholamine synthesis was maintained for up to 10 min following termination of K+ depolarization. Depolarization in the presence of tetrodotoxin did not block the poststimulation increase in catecholamine synthesis. The increased catecholamine synthesis in the poststimulation period can be accounted for by increased tyrosine hydroxylation since: 1) the synthesis of [14C]catecholamines from [14C]dopa was not increased by K+ depolarization and 2) K+ depolarization led to a 71% increase in the accumulation of [3H]dopa newly synthesized from [3H]tyrosine in the presence of the decarboxylase inhibitor, brocresine. Under these conditions, no significant depletion of tissue norepinephrine could be detected. The depolarization-induced increase in catecholamine synthesis was independent of the presence of Ca++ in the superfusion and/or incubation media, suggesting its dissociation from Ca++-dependent transmitter release. The absence of enhanced [3H]catecholamine synthesis following depolarization of slices in a Ca++-free K+-KRP containing 1.0 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) suggested that there is an absolute requirement for tissue Ca++ during the stimulation-induced synthesis activation process. There appears to be a depolarization-related phenomenon whose triggering is Ca++-independent, but which, in the presence of Ca++, is manifested as an increase in catecholamine biosynthesis (tyrosine hydroxylase activity).

Tyrosine hydroxylase: allosteric activation induced by stimulation of central noradrenergic neurons.

Electrical stimulation of the rat locus coeruleus cases about a 300% increase in the activity of the tyrosine hydroxylase prepared from the hippocampus on the stimulated side and assayed in the presence of subsaturating concentrations of tyrosine and pteridine cofactor. Addition of calcium or cAMP to soluble preparations of tyrosine hydroxylase isolated from the hippocampus produces a similar activation of tyrosine hydroxylase. The activation of tyrosine hydroxylase produced by calcium is reversed by addition of the calcium chelator, EGTA, while the activation produced by cAMP addition or by electrical stimulation of the locus coeruleus is unaffected by addition of EGTA to the assay medium. The activation of tyrosine hydroxylase produced by electrical stimulation or by addition of calcium or cAMP to the assay medium appears to be mediated in part by alterations in the kinetic properties of the enzyme. All treatment causes the enzyme to have an increased affinity for substrate and pteridine cofactor and a decreased affinity for the endproduct inhibitor, norepinephrine. These results are suggestive that the activation of tyrosine hydroxylase which occurs during periods of increased impulse flow in noradrenergic neurons may be initiated by alterations in calcium fluxes or by changes in the steady state levels of cAMP which accompany neuronal depolarization.