Dose-dependent Treatment Effect Research Articles

THU156 Significantly Improved Annual Height Velocity With Once-Weekly TransCon CNP In Children With Achondroplasia: The ACcomplisH Phase 2, Randomized, Double-Blind, Placebo-Controlled, Dose-Escalation Trial

THU156 Significantly Improved Annual Height Velocity With Once-Weekly TransCon CNP In Children With Achondroplasia: The ACcomplisH Phase 2, Randomized, Double-Blind, Placebo-Controlled, Dose-Escalation Trial

Evaluation of dose-dependent treatment effects after mid-trial dose escalation in biomarker, clinical, and cognitive outcomes for gantenerumab or solanezumab in dominantly inherited Alzheimer's disease.

While the Dominantly Inherited Alzheimer Network Trials Unit (DIAN-TU) was ongoing, external data suggested higher doses were needed to achieve targeted effects; therefore, doses of gantenerumab were increased 5-fold, and solanezumab was increased 4-fold. We evaluated to what extent mid-trial dose increases produced a dose-dependent treatment effect. Using generalized linear mixed effects (LME) models, we estimated the annual low- and high-dose treatment effects in clinical, cognitive, and biomarker outcomes. Both gantenerumab and solanezumab demonstrated dose-dependent treatment effects (significant for gantenerumab, non-significant for solanezumab) in their respective target amyloid biomarkers (Pittsburgh compound B positron emission tomography standardized uptake value ratio and cerebrospinal fluid amyloid beta 42), with gantenerumab demonstrating additional treatment effects in some downstream biomarkers. No dose-dependent treatment effects were observed in clinical or cognitive outcomes. Mid-trial dose escalation can be implemented as a remedy for an insufficient initial dose and can be more cost effective and less burdensome to participants than starting a new trial with higher doses, especially in rare diseases. We evaluated the dose-dependent treatment effect of two different amyloid-specific immunotherapies.Dose-dependent treatment effects were observed in some biomarkers.No dose-dependent treatment effects were observed in clinical/cognitive outcomes, potentially due to the fact that the modified study may not have been powered to detect such treatment effects in symptomatic subjects at a mild stage of disease exposed to high (or maximal) doses of medication for prolonged durations.

Identification of Novel Targets Based on Splicing Alterations for Undruggable RAS/CDK Signaling Cascade in Multiple Myeloma

Background: RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis. Targeting these pathways represents a novel therapeutic strategy in MM. Our ongoing studies (>420 patients) demonstrate that aberrantly spliced transcript expressions can predict MM patient survival outcomes better than gene expression alone, indicating a significant role of splicing mechanism in MM pathophysiology. These studies also identified intron retentions as the predominant recurrent alterations (~32% of spliced genes were retained introns) in MM. We evaluated splicing alterations associated with pathway-level responses after RAS/CDK inhibition in order to identify and validate novel molecular targets.Methods/results: MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to inhibit RAS and CDK pathways. Our studies demonstrated strong synergistic (IC<0.5) MM cytotoxicity triggered by this combination treatment, which triggered dose-dependent manner G0/G1 phase growth arrest. We assessed early death cascade in MM cells after Ei+Ci treatment, and demonstrated significant priming to selective peptides BIM, BAD, and MS1 or HRK, suggesting dependency on BCL2 and MCL1 or on BCL-XL proteins. Our studies showed that Ei+Ci treatment induced inhibition of key target molecules in Erk1/2 and CDK4/6 signaling including c-myc, p-RSK, p-S6, p-RB, and E2F1, suggesting on-target activity of Ei and Ci. Patient MM cells co-cultured with or without autologous BM stromal cells remain equally sensitive to Ei+Ci, suggesting that this combination can overcome the protective effects of the MM BM milieu. Moreover, our in vivo study demonstrated a significant (P=0.0004) MM burden decrease in Ei+Ci-treated mice. We evaluated the effect of Ei+Ci treatment on target gene expression in BM cells isolated from flushed femurs of treated animals with Ei, Ci or Ei+Ci, and observed downregulation of Erk1/2-CDK4/6-dependent gene signature. Therefore, we suggest that these inhibitors selectively target Erk1/2, CDK4/6 and their downstream substrates both in vitro/vivo.We next evaluated aberrantly spliced transcript expression in MM cells, with/without Erk1/2 knockdown (KD) or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. This observation was further supported by principal component analyses: upregulation in response to Erk1/2 KD and downregulation due to treatment with Ei+Ci were considered spliced gene-signatures linked to RAS/CDK modulation. Gene/pathway enrichment analyses of these genes showed their involvement in cell proliferation and regulation of epigenetic networks in MM. Importantly, these analyses suggest that overexpression of RAVER1/SNRPB core splicing regulator genes are associated with RAS/CDK pathway regulation. These genes encode subunits of U1/2/4/5 spliceosome complex and are involved in intron retention processes, a marker of malignant transformation. We compared signature-gene expressions from 558 MM patient samples to the signature-genes in plasma cells from normal donors and observed significant (p<2e-11) upregulation of genes with progression from MGUS to sMM, and, also to overt MM . SNRPB overexpression is associated with shorter overall patient survival (p<0.01), while RAVER1 is linked with poor outcomes. SNRPB proteins are also overexpressed in MM cells. Our studies evaluating SNRPB effects on RNA splicing showed both upregulation of transcripts with full intron retention and transcripts with cryptic stop codons utilizing intronic sequences causing their partial retention. We evaluated RAVER1 and SNRPB expression in BM cells from animals treated with Ei and Ci alone or in combination. We observed significant downregulation of RAVER1/SNRPB (p=0.001) in BM samples obtained from animals treated with Ei+Ci. We observed decreased intron retention events in genes in treated samples, consistent with our in vitro analyses in MM cell lines and patient samples. Thus, RAVER1/SNRPB overexpression contributes to the aberrant transcriptome splicing associated with RAS/CDK cascade in MM.Conclusions: Our studies 1) show an association between RNA processing and RAS-CDK pathways in MM, 2) identify a core splicing protein, SNRPB/RAVER1, as a novel target for modulating this cascade, and 3) suggest that targeting spliceosome complexes represents a promising therapy in MM. DisclosuresLetai: Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board. Anderson: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

P-089: Identification of novel targets in multiple myeloma for “undruggable” RAS/CDK signaling cascade

<h3>Background</h3> RAS/CDK-dependent pathways play essential roles in multiple myeloma (MM) pathogenesis; both pathways are undruggable. We evaluated molecular changes associated with pathway-level responses after RAS/CDK inhibition to identify novel molecular targets. <h3>Method</h3> in our previous studies MM cells were treated with selected Erk1/2 and CDK4/6 inhibitors (Ei, Ci) to target RAS/CDK pathways. Our studies indicate strong synergistic (IC<0.5) MM cytotoxicity triggered by Ei+Ci treatment, which in a dose-dependent manner arrested MM cells in G0/G1 phase and activated mitochondrial apoptotic signaling. Ei+Ci treatment decreased Erk1/2, CDK4/6, and p-Erk1/2 levels in MM cells, and also induced inhibition of key targets (c-myc, p-RSK,-RB, E2F1) of RAS/CDK cascade. Our studies in patient samples indicate that MM cells co-cultured with or without autologous BM stromal cells remain equally sensitive to Ei+Ci, suggesting that Ei+Ci combination can overcome the protective effects of the MM BM milieu. An in vivo study demonstrated a significant (P=0.0004) MM burden decrease in Ei+Ci-treated mice. Our studies therefore suggesting on-target activity of these inhibitors in vitro/vivo. <h3>Results</h3> We evaluated mRNA splicing changes in MM cells, with and without Erk1/2 knockdown or with Ei+Ci treatment. Unsupervised clustering of deregulated genes showed dose-dependent treatment effects. Upregulation in response to Erk1/2 knockdown and downregulation due to treatment with Ei+Ci were considered spliced gene signatures linked to RAS/CDK modulation. Gene/pathway enrichment analyses of these genes showed their involvement in cell proliferation and regulation of epigenetic networks in MM. Importantly, these analyses suggest that overexpression of RAVER1/SNRPB core splicing regulator genes are associated with RAS/CDK pathway regulation. These genes encode subunits of U1/2/4/5 spliceosome complexes and are involved in intron retention processes, a marker of malignant transformation. We evaluated expressions of RAVER1 and SNRPB in 558 MM patent samples and 10 normal donor BM PCs and observed significant (p<2e-11) upregulation of both genes in clonal PCs with progression from MGUS to sMM, and to overt MM. SNRPB overexpression is associated with shorter overall patient survival (p<0.01), while RAVER1 has a trend toward poor outcomes. SNRPB proteins are also overexpressed in MM cells. Evaluating SNRPB effects on RNA splicing showed upregulation of transcripts with full intron retention or transcripts with cryptic stop codons utilizing intronic sequences causing their partial retention. Thus, SNRPB overexpression contributes to aberrant transcriptome splicing associated with RAS/CDK cascade in MM. <h3>Conclusions</h3> Our studies show an association between RNA processing and RAS-CDK pathways in MM, identify a core splicing protein, SNRPB, as a novel target for modulating this undruggable cascade, and suggest that targeting spliceosome complexes is a promising therapy.

Quantifying the effects of antibiotic treatment on the extracellular polymer network of antimicrobial resistant and sensitive biofilms using multiple particle tracking

Novel therapeutics designed to target the polymeric matrix of biofilms requires innovative techniques to accurately assess their efficacy. Here, multiple particle tracking (MPT) was developed to characterize the physical and mechanical properties of antimicrobial resistant (AMR) bacterial biofilms and to quantify the effects of antibiotic treatment. Studies employed nanoparticles (NPs) of varying charge and size (40–500 nm) in Pseudomonas aeruginosa PAO1 and methicillin-resistant Staphylococcus aureus (MRSA) biofilms and also in polymyxin B (PMB) treated Escherichia coli biofilms of PMB-sensitive (PMBSens) IR57 and PMB-resistant (PMBR) PN47 strains. NP size-dependent and strain-related differences in the diffusion coefficient values of biofilms were evident between PAO1 and MRSA. Dose-dependent treatment effects induced by PMB in PMBSensE. coli biofilms included increases in diffusion and creep compliance (P < 0.05), not evident in PMB treatment of PMBRE. coli biofilms. Our results highlight the ability of MPT to quantify the diffusion and mechanical effects of antibiotic therapies within the AMR biofilm matrix, offering a valuable tool for the pre-clinical screening of anti-biofilm therapies.

Apple polyphenol-rich drinks dose-dependently decrease early-phase postprandial glucose concentrations following a high-carbohydrate meal: a randomized controlled trial in healthy adults and in vitro studies

BackgroundPrevious research demonstrated that a high dose of phlorizin-rich apple extract (AE) can markedly inhibit early-phase postprandial glycemia, but efficacy of lower doses of the AE is unclear. ObjectiveTo determine whether lower AE doses reduce early-phase postprandial glycemia in healthy adults and investigate mechanisms. DesignIn a randomized, controlled, double-blinded, cross-over acute trial, drinks containing 1.8 g (HIGH), 1.35 g (MED), 0.9 g (LOW), or 0 g (CON) of a phlorizin-rich AE were consumed before 75 g starch/sucrose meal. Postprandial blood glucose, insulin, C-peptide, glucose-dependent insulinotropic polypeptide (GIP) and polyphenol metabolites concentrations were measured 0–240 min, acetaminophen concentrations to assess gastric emptying rate, and 24 h urinary glucose excretion. Effects of AE on intestinal glucose transport were investigated in Caco-2/TC7 cells. ResultsAE significantly reduced plasma glucose iAUC 0–30 min at all doses: mean differences (95% CI) relative to CON were −15.6 (−23.3, −7.9), −11.3 (−19.6, −3.0) and −8.99 (−17.3, −0.7) mmol/L per minute for HIGH, MEDIUM and LOW respectively, delayed Tmax (HIGH, MEDIUM and LOW 45 min vs. CON 30 min), but did not lower Cmax. Similar dose-dependent treatment effects were observed for insulin, C-peptide, and GIP. Gastric emptying rates and urinary glucose excretion did not differ. Serum phloretin, quercetin and epicatechin metabolites were detected postprandially. A HIGH physiological AE dose equivalent decreased total glucose uptake by 48% in Caco-2/TC7 cells. ConclusionsPhlorizin-rich AE, even at a low dose, can slightly delay early-phase glycemia without affecting peak and total glycemic response.

Evaluating the structural effects of intra-articular sprifermin on cartilage and non-cartilaginous tissue alterations, based on sqMRI assessment over 2 years

Sprifermin (recombinant human fibroblast growth factor-18), a potential disease-modifying osteoarthritis (OA) drug, demonstrated dose-dependent effects on femorotibial cartilage thickness (by quantitative magnetic resonance imaging [MRI]) in the phase II FORWARD study. This post-hoc analysis evaluated the potential effects of sprifermin on several articular structures in the whole joint over 24 months using semi-quantitative MRI assessment. Patients aged 40-85 years with symptomatic radiographic knee OA, Kellgren-Lawrence grade 2 or 3, and medial minimum joint space width ≥2.5mm in the target knee were randomized (1:1:1:1:1) to receive three double-blinded, once-weekly, intra-articular injections of sprifermin 30μg or 100μg or placebo every 6 (q6mo) or 12 months. 1.5- or 3T MRIs were read using the Whole-Organ Magnetic Resonance Imaging Score (WORMS) system at baseline and 24 months. Change from baseline at 24 months on compartment and/or whole knee level was assessed for cartilage morphology, bone marrow lesions (BMLs), and osteophytes by delta-subregional and delta-sum (DSM) approaches. Menisci, Hoffa-synovitis, and effusion-synovitis were also evaluated for worsening. 549 patients were included. Dose-dependent treatment effects from baseline to 24 months were observed on cartilage morphology (sprifermin 100μg q6mo vs placebo; mean DSM (95% confidence interval [CI])-0.6 (-1.5, 0.2); less cartilage worsening) in the entire knee and BMLs sprifermin 100μg q6mo vs placebo; mean DSM (95% CI)-0.2 (-0.5, 0.1) in the patellofemoral compartment. No effects over 24 months were observed on osteophytes, menisci, Hoffa-synovitis or effusion-synovitis. Positive effects associated with sprifermin were observed for cartilage morphology changes, and BML improvement. There were no meaningful negative or positive effects associated with sprifermin in the other joint tissues examined.

254. Community Level Interventions for Pre-eclampsia (CLIP) in Pakistan: A cluster randomised controlled trial

In Pakistan, an important contributor to maternal, fetal, and neonatal mortality is pregnancy hypertension. Its early detection and initial management may be amenable to task-sharing by Lady Health Workers (LHWs). To reduce by 20%, one or more of: maternal death/morbidity, stillbirth, or neonatal death/morbidity in the intervention clusters. The Pakistan Community-Level Interventions for Pre-eclampsia (CLIP) cluster randomised control trial (NCT01911494) recruited pregnant women in 20 union councils (clusters) in Sindh Province. The CLIP intervention (10 clusters) consisted of community engagement and LHW-provided mobile health-guided clinical assessment and initial treatment (MgSO4 or methyldopa, as appropriate), and referral by LHWs to facility for further management. Data were collected by quarterly household survey. Treatment effect was estimated by multi-level logistic regression modeling, adjusting for baseline cluster- and individual-level variables of prognostic significance. A priori defined secondary analyses included evaluation of temporal and dose-dependent treatment effects. Of 39,444 women recruited (20,264 women intervention, 19,180 control), losses to follow-up were 3·7% and 4·5%, respectively. The primary outcome did not differ between arms (N = 5381 women, 26·6% vs. N = 4195, 21·9%); adjusted odds ratio (aOR) 1·20, 95% confidence interval (CI) [0·84–1 72]. In both arms, an estimated reduction in the odds of primary outcome of 7.0% per quarter was observed (OR = 0.93, 95% CI 0.93,0.94, p < 0.001). In the intervention arm, the temporal trend-adjusted primary outcome rate decreased by 8.0% (aOR = 0.92 [0.90–0.95], p < 0.001) for each CLIP visit beyond three. While there was no difference in the primary outcome in intervention vs. control clusters, outcomes improved over time in all clusters, particularly maternal morbidity; suggesting that quarterly household surveillance served as an intervention. Receipt of more than three CLIP visits was associated with fewer adverse outcomes, suggesting a possible threshold of intervention that must be delivered to achieve positive health outcomes.

The role of monocyte subpopulations in vascular injury following partial and transient depletion.

The innate immunity system plays a critical role in vascular repair and restenosis development. Liposomes encapsulating bisphosphonates (LipBPs), but not free BPs, suppress neointima formation following vascular injury mediated in part by monocytes. The objective of this study was to elucidate the role of monocyte subpopulations on vascular healing following LipBP treatment. The potency- and dose-dependent treatment effect of clodronate (CLOD) and alendronate (ALN) liposomes on restenosis inhibition, total monocyte depletion, and monocytes subpopulation was studied. Rats subjected to carotid injury were treated by a single IV injection of LipBPs at the time of injury. Low- and high-dose LipALN treatment (3 and 10mg/kg, respectively) resulted in a dose-dependent effect on restenosis development after 30days. Both doses of LipALN resulted in a dose-dependent inhibition of restenosis, but only high dose of LipALN depleted monocytes (-60.1±4.4%, 48h post injury). Although LipCLOD treatment (at an equivalent potency to 3mg/kg alendronate) significantly reduced monocyte levels (72.1±6%), no restenosis inhibition was observed. The major finding of this study is the correlation found between monocyte subclasses and restenosis inhibition. Non-classical monocyte (NCM) levels were found higher in LipALN-treated rats, but lower in LipCLOD-treated rats, 24h after injury and treatment. We suggest that the inhibition of circulating monocyte subpopulations is the predominant mechanism by which LipBPs prevent restenosis. The effect of LipBP treatment on the monocyte subpopulation correlates with the dose and potency of LipBPs.

Effects of 1,25-dihydroxyvitamin D3 and vitamin D3 on the expression of the vitamin d receptor in human skeletal muscle cells.

Vitamin D receptor (VDR) expression and action in non-human skeletal muscle have recently been reported in several studies, yet data on the activity and expression of VDR in human muscle cells are scarce. We conducted a series of studies to examine the (1) effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on VDR gene expression in human primary myoblasts, (2) effect of 16-week supplementation with vitamin D3 on intramuscular VDR gene expression in older women, and (3) association between serum 25-hydroxyvitamin D (25OHD) and intramuscular VDR protein concentration in older adults. Human primary myoblasts were treated with increasing concentrations of 1,25(OH)2D3 for 18 h. A dose-dependent treatment effect was noted with 1 nmol/L of 1,25OH2D3 increasing intramuscular VDR mRNA expression (mean fold change±SD 1.36±0.33; P=0.05). Muscle biopsies were obtained at baseline and 16 weeks after vitamin D3 supplementation (4,000 IU/day) in older adults. Intramuscular VDR mRNA was significantly different from placebo after 16 weeks of vitamin D3 (1.2±0.99; -3.2±1.7, respectively; P=0.04). Serum 25OHD and intramuscular VDR protein expression were examined by immunoblot. 25OHD was associated with intramuscular VDR protein concentration (R=0.67; P=0.0028). In summary, our study found VDR gene expression increases following treatment with 1,25OH2D3 in human myoblasts. 25OHD is associated with VDR protein and 16 weeks of supplementation with vitamin D3 resulted in a persistent increase in VDR gene expression of vitamin D3 in muscle tissue biopsies. These findings suggest treatment with vitamin D compounds results in sustained increases in VDR in human skeletal muscle.

The Stage-specific Testicular Germ Cell Apoptotic Response to Low-dose Radiation and 2,5-hexanedione Combined Exposure. II

Testicular effects of chemical mixtures may differ from those of the individual chemical constituents. This study assessed the co-exposure effects of the model germ cell- and Sertoli cell-specific toxicants, X-irradiation (x-ray), and 2,5-hexanedione (HD), respectively. In high-dose studies, HD has been shown to attenuate x-ray-induced germ cell apoptosis. Adult rats were exposed to different levels of x-ray (0.5 Gy, 1 Gy, and 2 Gy) or HD (0.33%), either alone or in combination. To assess cell type-specific attenuation of x-ray effects with HD co-exposure, we used laser capture microdissection (LCM) to enrich the targeted cell population and examine a panel of apoptosis-related transcripts using PCR arrays. The apoptosis PCR arrays identified significant dose-dependent treatment effects on several genes, with downregulation of death receptor 5 (DR5), Naip2, Sphk2, Casp7, Aven, Birc3, and upregulation of Fas. The greatest difference in transcript response to exposure was seen with 0.5 Gy x-ray exposure, and the attenuation effect seen with the combined high-dose x-ray and HD did not persist into the low-dose range. Examination of protein levels in staged tubules revealed a significant upregulation in DR5, following high-dose co-exposure. These results provide insight into the testis cell-specific apoptotic response to low-dose co-exposures of model testicular toxicants.

Transcriptional Profiling of the Dose Response: A More Powerful Approach for Characterizing Drug Activities

The dose response curve is the gold standard for measuring the effect of a drug treatment, but is rarely used in genomic scale transcriptional profiling due to perceived obstacles of cost and analysis. One barrier to examining transcriptional dose responses is that existing methods for microarray data analysis can identify patterns, but provide no quantitative pharmacological information. We developed analytical methods that identify transcripts responsive to dose, calculate classical pharmacological parameters such as the EC50, and enable an in-depth analysis of coordinated dose-dependent treatment effects. The approach was applied to a transcriptional profiling study that evaluated four kinase inhibitors (imatinib, nilotinib, dasatinib and PD0325901) across a six-logarithm dose range, using 12 arrays per compound. The transcript responses proved a powerful means to characterize and compare the compounds: the distribution of EC50 values for the transcriptome was linked to specific targets, dose-dependent effects on cellular processes were identified using automated pathway analysis, and a connection was seen between EC50s in standard cellular assays and transcriptional EC50s. Our approach greatly enriches the information that can be obtained from standard transcriptional profiling technology. Moreover, these methods are automated, robust to non-optimized assays, and could be applied to other sources of quantitative data.

Double-Blind, Randomized, Placebo-Controlled, Dose-Response Study of the Safety and Efficacy of Botulinum Toxin Type A in Subjects with Crow's Feet

Published evidence suggests that botulinum toxin type A (BTX-A) is an effective treatment for crow's feet. However, few dose-ranging studies have been performed. To assess the safety and efficacy of a single treatment with one of four doses of BTX-A (Botox/Vistabel, Allergan Inc) compared with placebo for the improvement of crow's feet. Subjects received a single bilateral treatment of 18, 12, 6, or 3 U of BTX-A or placebo injected into the lateral aspect of the orbicularis oculi muscle (parallel-group, double-blind design). Investigators and subjects rated crow's feet severity at maximum smile on day 7 and at 30-day intervals from days 30 to 180. As observed by both investigators and subjects, all doses of BTX-A resulted in improvements in crow's feet severity when compared with placebo. A dose-dependent treatment effect for efficacy was observed, with higher doses having an increased magnitude and duration of effect. However, a clear differentiation between the 18 U and 12 U doses was not apparent. Few adverse events were reported, with no statistically significant differences between BTX-A and placebo in the incidence of subjects experiencing adverse events. BTX-A is safe and effective in decreasing the severity of crow's feet, with 12 U per side suggested as the most appropriate dose.

Double-Blind, Randomized, Placebo-Controlled, Dose-Response Study of the Safety and Efficacy of Botulinum Toxin Type A in Subjects with Crowʼs Feet

Background Published evidence suggests that botulinum toxin type A (BTX-A) is an effective treatment for crow's feet. However, few dose-ranging studies have been performed. Objectives To assess the safety and efficacy of a single treatment with one of four doses of BTX-A (Botox/Vistabel, Allergan Inc) compared with placebo for the improvement of crow's feet. Methods Subjects received a single bilateral treatment of 18, 12, 6, or 3 U of BTX-A or placebo injected into the lateral aspect of the orbicularis oculi muscle (parallel-group, double–blind design). Investigators and subjects rated crow's feet severity at maximum smile on day 7 and at 30-day intervals from days 30 to 180. Results As observed by both investigators and subjects, all doses of BTX-A resulted in improvements in crow's feet severity when compared with placebo. A dose-dependent treatment effect for efficacy was observed, with higher doses having an increased magnitude and duration of effect. However, a clear differentiation between the 18 U and 12 U doses was not apparent. Few adverse events were reported, with no statistically significant differences between BTX-A and placebo in the incidence of subjects experiencing adverse events. Conclusion BTX-A is safe and effective in decreasing the severity of crow's feet, with 12 U per side suggested as the most appropriate dose.

Gene expression profile induced by 17 alpha-ethynyl estradiol in the prepubertal female reproductive system of the rat.

The profound effects of 17beta-estradiol on cell growth, differentiation, and general homeostasis of the reproductive and other systems, are mediated mostly by regulation of temporal and cell type-specific expression of different genes. In order to understand better the molecular events associated with the activation of the estrogen receptor (ER), we have used microarray technology to determine the transcriptional program and dose-response characteristics of exposure to a potent synthetic estrogen, 17 alpha-ethynyl estradiol (EE), during prepubertal development. Changes in patterns of gene expression were determined in the immature uterus and ovaries of Sprague-Dawley rats on postnatal day (PND) 24, 24 h after exposure to EE, at 0.001, 0.01, 0.1, 1 and 10 micro g EE/kg/day (sc), for four days (dosing from PND 20 to 23). The transcript profiles were compared between treatment groups and controls using oligonucleotide arrays to determine the expression level of approximately 7000 annotated rat genes and over 1740 expressed sequence tags (ESTs). Quantification of the number of genes whose expression was modified by the treatment, for each of the various doses of EE tested, showed clear evidence of a dose-dependent treatment effect that follows a monotonic response, concordant with the dose-response pattern of uterine wet-weight gain and luminal epithelial cell height. The number of genes whose expression is affected by EE exposure increases according to dose. At the highest dose tested of EE, we determined that the expression level of over 300 genes was modified significantly (p < or = 0.0001). A dose-dependent analysis of the transcript profile revealed a set of 88 genes whose expression is significantly and reproducibly modified (increased or decreased) by EE exposure (p < or = 0.0001). The results of this study demonstrate that, exposure to a potent estrogenic chemical during prepubertal maturation changes the gene expression profile of estrogen-sensitive tissues. Furthermore, the products of the EE-regulated genes identified in these tissues have a physiological role in different intracellular pathways, information that will be valuable to determine the mechanism of action of estrogens. Moreover, those genes could be used as biomarkers to identify chemicals with estrogenic activity.

Initial monoamine oxidase-A inhibition by moclobemide does not predict the therapeutic response in patients with major depression

It is generally accepted that the clinical efficacy of monoamine oxidase inhibitors (MAOI) is related to inhibition of this enzyme. In order to evaluate the predictive ability of monoamine oxidase-A inhibition for therapeutic efficacy, the start of treatment effects of moclobemide, a selective, reversible monoamine oxidase-A inhibitor, on plasma concentrations of monoamines and monoamine metabolites were determined. The plasma levels of 3,4-dihydroxy-phenylglycol (DHPG, deaminated metabolite of noradrenaline), 5-hydroxyindoleacetic acid (5-HIAA, deaminated metabolite of serotonin), 3,4-dihydroxyphenylacetic acid and homovanillic acid (DOPAC and HVA, deaminated metabolites of dopamine),l-dihydroxyphenylalanine (l-dopa) and noradrenaline were investigated and related to treatment outcome. This was a randomized double blind parallel group study in 47 patients with criteria of major depression according to DSM III R. Moclobemide 300 mg/day, 450 mg/day or 600 mg/day was administered continuously for 6 wecks. Plasma concentrations of monoamine metabolites and monoamines were determined just before treatment by moclobemide, 4 h after the first dose, 24 h after the first dose, before the first dose on day 7, and 4 h after the first dose on day 7. Each moclobemide dose improved depression as measured by MADRS (Montgomery-Asberg Depression Rating scale) but there was no difference between the three doses. Moclobemide dose-dependently reduced plasma concentration of DHPG,l-dopa and HVA. No dose-dependent treatment effect was observed for plasma 5-HIAA, noradrenaline and DOPAC. The clinical outcome as defined by the final MADRS score was not related to any start of treatment changes in plasma monoamine metabolites reflecting inhibition of MAO-A. It is concluded that monoamine oxidase-A inhibition at the beginning of the treatment does not predict clinical outcome.