Prenatal sex steroids play a vital role in the development of the whole organism, and therefore also the brain. Exposure of the fetus to testosterone seems to be of special importance both for typical development and pathology. The key factor impacting offspring development  appears to be diet, both in terms of shortage and excessive intake of certain food products. Prenatal steroid levels are measured using the ratio of the lengths of the second and fourth fingers (2D:4D). So far, the digit ratio (2D:4D) has been shown to correlate negatively with prenatal testosterone and positively with prenatal estrogen. Numerous correlational studies found relationships between the 2D:4D phenotype and differences in magnitude of many psychological traits. Certain social and demographic variables also correlate with the digit ratio. The present paper offers a preliminary analysis of correlations between diet, prenatal hormones’ levels (established based on the digit ratio), and selected social variables. One of the findings is that countries with high meat consumption present the so-called masculine digit ratio, while countries with plant-based diets – a feminine digit ratio. Since the ground-breaking work on organizational vs. activational effects of hormones , a signiin utero , and social functioning characteristics. In order to evaluate the possible effect of the main environmental factor, such as diet profile, in shaping the sexual dimorphism in the general populations across the countries, the multivariate approach has been adopted. The profile of the food consumption involves various food components, and therefore, all basic aspects of consumption should be taken into account. The first stage of the analysis involves clustering of countries into the homogeneous groups on the basis of their profile of food consumption . The second stage is a description of clusters, which is supposed to provide a general picture of how people utilize the food sources in their countries. This stage of analysis includes also the comparison of the groups of countries in terms of the sexual dimorphism measured by 2D:4D. Although this kind of analysis does not allow to formulate any casual explanations, it may provide a valuable hint for more detailed research on the role of diet in prenatal androgens level regulation.K-means method) was administered in order to group countries presenting similar food consumption profiles.Dietary profiles of selected countries were analyzed. For the analysis were selected countries for which there are reliable data on digit ratio in their populations . The anaInformation on basic food products and alcohol intake was obtained from the Food and Agriculture Organization of the United Nations database.Extracted in the cluster analysis groups of countries were compared in terms of the digit ratio profile, which dominates in the country’s population. Digit ratio values were sourced from Manning et al. . They puThe next step was to compare the extracted in the cluster analysis groups of countries in terms of demographic variables associated with the quality of life. The demographic variables previously described by Manning et al.  were excDemographic data were sourced from The World Factbook,All data used for the analyses in this paper are compiled in additional materials.The data described above were analyzed using the multidimensional approach. The initial step was cluster analysis conducted on selected countries.K-means method of clustering. The procedure used information on the intake of basic food products in 29 countries  and the lowest consumption of meat  of all clusters. Eggs consumption is low, similar to that for cluster 3.In cluster 2, pork (pig meat) was the most consumed type. The consumption of eggs was also the highest of all three clusters. The values for foods such as poultry, fish, bovine, and wheat were in the middle, although in the case of bovine, fish, and poultry, they approximate the low values in cluster 1, while wheat consumption is similar to the lowest value in cluster 3.Cluster 3 features a particularly high consumption of meat , with the figures significantly higher than in the remaining clusters. The consumption of wheat and vegetables is the lowest of all analyzed clusters. The statistics for eggs are also close to the lowest value of cluster 1.F test was used for cluster comparisons.The next step of the analysis was to compare the three extracted clusters in terms of digit ratio and demographic values. Fisher The clusters were compared in terms of digit ratio  – Table The clusters differed in the following parameters: left hand digit ratio in men (L2D:4D M) and women (L2D:4D F), right hand digit ratio in women (R2D:4D F), and residuals from the left hand digit ratio analysis (resL2D:4D). Mean values reported by Manning et al.  for the In cluster 1, L2D:4D M values were in the middle for clusters, but still higher than the mean value for men. In women, both L2D:4D and R2D:4D were the highest of all analyzed clusters and higher than the population mean. The resL2D:4D value was positive and higher than in the other clusters.L2D:4D of men in cluster 2 was the highest and higher than the general population mean compared to the other clusters. L2D:4D F and R2D:4D F values were average relative to the remaining clusters and approximated the population means. The value of resL2D:4D was negative but close to zero, which is the intermediate value out of the three clusters.In cluster 3, L2D:4D for men was the lowest of all clusters of interest and close to the population mean. In women, both L2D:4D and R2D:4D values were the lowest among the three clusters, lower even than the mean for men. The resL2D:4D value was negative, the lowest of all clusters.Since previous studies have indicated that the digit ratio is related to social variables , 52, 55,In terms of demographics, cluster 1 stood out due to very high  infant mortality rate , low GPI in secondary level enrollment, and the lowest education expenditures among clusters. In addition, alcoholic beverages supply was the lowest. The other variables are the middle values among the three clusters, with TFR close to the lowest value recorded in cluster 2.Of note in cluster 2 are the highest median age in male and female of all clusters, the lowest birthrate and highest death rate, the lowest TFR and highest alcoholic beverages supply. Infant mortality rate  is the average for clusters, although similar to the lowest value in cluster 3.Cluster 3 has a low  infant mortality rate , the highest birthrate and lowest death rate, as well as the highest TFR (significantly higher than in the other two clusters). Median age in males and females is the lowest of all analyzed clusters. GPI in secondary level enrollment is the highest among clusters and greater than one, meaning that there are more women at this stage of education. Education expenditure is also the highest among the three clusters.By means of cluster analysis, it was possible to identify three groups of countries that differ in the consumption of basic food products, i.e., meat, wheat, etc. Cluster 1, which includes countries in the Mediterranean and Black Sea region, is characterized by a plant-based diet (wheat and vegetables) and low consumption of animal source foods . Cluster 3 includes mainly Scandinavian, Northern European, and large non-European countries . Most of them are high-income Western countries, with wide access to sea. The diet in these countries is based on meat , with low consumption of plant products (wheat and vegetables) and eggs. Cluster 2 includes Central and Western European countries. They exhibit high consumption of pig meat and eggs, which constitute the main components of the diet. However, overall meat consumption is considerably lower than in cluster 3.in utero. Cluster 2 countries have average parameters for the clusters of interest, with sex-typical digit ratios.The next step of the analysis revealed that the clusters differed not only in the diet type but also in terms of morphometric data, in this case, the digit ratio. The differences were seen mostly in left hand 2D:4D, since the right hand only discriminated clusters for women. Detailed analysis of clusters showed that countries with predominantly plant-based diets (cluster 1) have high digit ratio values both in males and females, i.e., low level of prenatal estrogen and/or high level of prenatal testosterone. By contrast, in countries with mainly meat-based diets (cluster 3), the male pattern of digit ratio is dominant in both sexes, suggesting high prenatal estrogen and/or low prenatal testosterone exposure in utero through the effects of prenatal sex steroids  and sexual dimorphism measured by 2D:4D ratio. In countries with high consumption of meat, male-like pattern of digit ratio is observed in both sexes. It may suggest that, in these countries, a meat-based diet is followed by males and females. The same countries appear to be the most developed and wealthy. It must be noted, however, that it is not possible, on the basis of this type of data, to pinpoint the mechanisms underlying these relationships. Especially, that all analyzed variables are interlinked on multiple levels.The existing data support the presence of the processes running at various levels that have a significant impact on the evolution of contemporary human societies. In the light of the findings confirming the fast pace of evolutionary changes in Homo sapiens , the rolBoth authors contributed to study design, data analysis, and manuscript writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer KS and handling Editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.


Rats in each phase of the oestrous cycle were assigned to the following experiments: (1) an immunohistochemical study of the number of cells expressing m1AChR or m2AChR in the POA-AHA and (2) analysis of the effects of the unilateral blockade of the m1AChR  or m2AChR  on either side of the POA-AHA on the ovulation rate. The number of m2AChR-immunoreactive cells was significantly higher at 09:00 h on each day of the oestrous cycle in the POA-AHA region, while no changes in the expression profile of m1AChR protein were observed. The ovulation rate in rats treated with PZP on the oestrous day was lower than that in the vehicle group. Animals treated on dioestrous-1 with PZP or MTC had a higher ovulation rate than those in the vehicle group. In contrast, on dioestrous-2, the MTC treatment decreased the ovulation rate. These results suggest that m1AChR or m2AChR in the POA-AHA could participate in the regulation of spontaneous ovulation in rats.Muscarinic receptors types 1 (m In particular, m1AChRs and m2AChRs have been detected in several brain areas; the highest proportion of m1AChR was found in the hippocampus, while the cerebellum and hypothalamus were the regions with the highest m2AChR expression -N-methyl-scopolamine is 50% lower than that in those of the right portion, but the dissociation constant (Kd) is 118% lower, and low levels of ACh were observed [1AChR-ir and m2AChR-ir cells and the ability of the blockade of either receptor to modify spontaneous ovulation. The differences observed in the effects of m1AChR and m2AChR blockade on ovulation performed at dioestrous-2 or oestrous could result from differences in the affinity of each mAChR present in each side of the POA-AHA.The specific blockade of mimplants . Since, observed . In the 2AChR are higher on dioestrous-2 than on the day of oestrous, while m1AChR predominates on the day of oestrous, even when the number of cells positive for mAChR is unchanged at 13:00 h on dioestrous-2 and on the day of oestrous.From the effects of the unilateral microinjections of PZP and MTC on ovulation, we suggest that the number and affinity of m1AChR or m2AChR selective antagonists on the regulation of spontaneous ovulation showed different effects than that previously reported in a study of chronic treatment with an atropine implant in the POA-AHA region [μg on implanting atropine research); and (4) the microinjection of the antagonist in solution produces a short-lived effect because the cerebrospinal fluid flow washed away the drug that was not binding to the receptor, while the atropine crystal implants stayed longer, producing a longer-lasting effect than the microinjections.The acute microinjection of mA region . The difμL of PZP or MTC on dioestrous-2; these results suggest that ACh regulates the growth of ovarian follicles through m1AChR or m2AChR. Interestingly, we have previously shown that at this stage of the cycle, implants of atropine in the POA-AHA region produce low follicular growth and atresia in the left ovary, without any apparent effects on the right ovary [The number of ova shed reduced from each ovary in rats that were microinjected with 100 pg/ht ovary .LHRH- or EB-induced ovulation observed in rats treated with PZP or MTC in the POA-AHA region supports the idea that inhibition of the LH or GnRH surge results from the inhibition of GnRH or oestradiol secretion, respectively, and/or from the stimulating feedback effects of oestradiol on LH release.1AChR on either side of the POA-AHA region on the day of oestrous and the stimulation of m2AChR on dioestrous-2, while on dioestrous-1, the activation of both receptors inhibits the mechanisms that regulate the spontaneous ovulation process. Therefore, m1AChR and m2AChR activation is also required on dioestrous-2 for a complete restoration of ovulation.Based on the present results, we suggest that ovulation requires the stimulation of the m


Adapt and evaluate the psychometric properties of the Vulnerability to Abuse Screening Scale to identify risk of domestic violence against older adults in Brazil. The instrument was adapted and validated in a sample of 151 older adults from a geriatric reference center in the municipality of Belo Horizonte, State of Minas Gerais, in 2014. We collected sociodemographic, clinical, and abuse-related information, and verified reliability by reproducibility in a sample of 55 older people, who underwent re-testing of the instrument seven days after the first application. Descriptive and comparative analyses were performed for all variables, with a significance level of 5%. The construct validity was analyzed by the principal components method with a tetrachoric correlation matrix, the reliability of the scale by the weighted Kappa (Kp) statistic, and the internal consistency by the Kuder-Richardson estimator formula 20 (KR-20)., categorized by presence (score > 3) or absence (score < 3) of vulnerability to abuse, with clinical and health conditions, we found statistically significant differences for self-perception of health (p = 0.002), depressive symptoms (p = 0.000), and presence of rheumatism (p = 0.003). There were no statistically significant differences between sexes. The Vulnerability to Abuse Screening Scale acceptably evaluated validity in the transcultural adaptation process, demonstrating dimensionality coherent with the original proposal (four factors). In the internal consistency analysis, the instrument presented good results (KR-20 = 0.69) and the reliability via reproducibility was considered excellent for the global scale (Kp = 0.92). The average age of the participants was 72.1 years , with a maximum of 92 years, and they were predominantly female . When analyzing the relationship between the scores of the Vulnerability to Abuse Screening Scale The Vulnerability to Abuse Screening Scale proved to be a valid instrument with good psychometric capacity for screening domestic abuse against older adults in Brazil. For older adults, the literature presents consolidated evidence of mortality by violence associated with falls and transportation accidents,This situation demands the construction and validation of tools for screening violence against elderly peopleThere are currently two instruments for screening domestic violence against older adults, validated for Brazilian culture and including psychometric studies,In addition, the Vulnerability to Abuse Screening Scale (VASS),The decision to analyze VASS was also based on the premise that the scale has a variable validated for Brazilian culture in Conflict Tactics Scales (CTS)In this light, this study aimed to transculturally adapt the VASS – Vulnerability to Abuse Screening Scale for older adults to Portuguese and analyze its psychometric properties of validity and reliability.We performed a cross-sectional study grounded in the procedures for transcultural adaptation proposed by Reinchenhem and Moraes,A team of specialists evaluated the equivalence of concepts and items by assessing the pertinence of the terms and concepts used in the original instrument and the impact of their meanings in Brazilian culture. They also explored the adaptation of items from the original instrument in relation to their capacity of representing these dimensions in the population,,The semantic equivalence process involved a grammar and vocabulary analysis, where we verified if the translation expresses the same concept as the original instrument and is adapted to the local realityIn the first, bilingual translators performed two translations (T1 and T2), which moved on to the second step for reverse translation (RT1 and RT2), sent to two medical translators, blindly and independently, one of whom was a sworn translator and the other from the same country as the original instrument. In the third step, we forwarded the reverse translations to a fifth bilingual evaluator, who verified the general and referential meanings of the terms and expressions in the scale., for which equivalence is judged from zero to 100%. Thus, the greater the relationship of literal correspondence between the translated terms and the original version, the higher the equivalence to the referential meaning. In the fourth step, five specialized professionals, including the researchers and one medical geriatrician, formulated a final consolidated version in Portuguese, incorporating the necessary changes.The connotative  meaning was classified into four categories: unchanged, slightly changed, drastically changed, and completely changed. The referential meaning was analyzed according to the Visual Analogue ScaleLastly, the synthesized version was applied via a focal group to a sample of older adults to verify the acceptability and comprehension of the instrument. We read each item to the participants, who expressed understanding and suggested changes. The reformulation of items was based on a percentage equal to or higher than 15% of interviewees who did not understand the questions.varimax orthogonal rotationThe dimensional structure of the VASS was measured by exploratory factorial analysis with the extraction of principal components by the tetrachoric correlation matrixKR-1). We effected intra-observer reliability in a random simple sample of 55 older adults, to whom we reapplied the instrument approximately seven days after the first interview. Simple Kappa values were estimated for each item and the weighted Kappa for the general score, given the sum of the points in the 12 items of the scale, and for the factors extracted in the factorial analysis.The internal consistency was estimated by the Kuder-Richardson coefficient, formula 20 (KR-20), and by intra-observer reliability through concordance by simple Kappa (K) and Kappa with quadratic weighting (Kp), where the latter were interpreted as recommended by ShroutWe analyzed the degree to which the data was adjusted for the factorial analysis via Kaiser-Meyer-Olkin (KMO)We collected data from February to May 2014 at the Reference Center for Elderly People of Belo Horizonte (Minas Gerais). In addition to the VASS, we used a questionnaire consisting of variables of the following types: sociodemographic; clinical ; functionality ; mental health and mood . All participants signed the free and informed consent term.The sample of the pre-test corresponded to 17 older adults, 65% male, at an average age of 70.5, and with up to four years of study.The general and referential equivalence of meanings between the original version and the reverse translations (RT1 and RT2) varied from 50% to 100% .The general meaning of the reverse translations (RT1 and RT2) was satisfactory, with most questions having unchanged meanings (eight questions – 66.5%) in both versions. Item “1” in RT2 was problematic, as inappropriate use of the “someone” pronoun drastically changed its meaning. The second and ninth items were slightly changed in both versions. The independent reverse translations of item “2” presented the idea that the term “harm” in English could mean “offend”. Despite being synonyms, we noticed in this adaptation that the word “harm” would be nearer to the sense of aggravation or damage, as proposed in the original translation of the instrument. Thus, the word “harm” was used literally in the pre-test version.For item “9”, we preferred RT2 because of the idea of a feeling of discomfort caused “by anyone in your family” and not “by someone”. Thus, we maintained the same criteria used to judge item “2”, and the second reverse translation proved more coherent with the meaning of the question.Both reverse translations were balanced in terms of meaning percentages, with RT2 slightly more adequate than RT1 in the referential sense. Consequently, we chose more items from RT2 (eight items) when creating the synthesized version .During the pilot study, the questionnaire was tested and well accepted by the interviewees. It was easily applied, with an average of two minutes for completion of the questionnaire.Differences in terms of the clarity and objectivity of the instrument were discussed among the older people. We analyzed the suggested changes and observed the pertinence of the adjustments for the purpose of clarity and objectivity.Items 4, 7, 8, 9, and 11 were changed to use words that were easier to understand, and to include “Mr.” and “Mrs./Ms.” Instead of “you” in order to address the interviewee respectfully. We also included articles to avoid long phrases or repeated ideas. The final translation of the scale is shown in versus 73.7 years). The sample was predominantly female (76.2%) and presented MEEM scores above 12 points with an average of 25.3 (DP = 2.68). The subjects were active and had a 20.0 (DP = 0.13) score average in instrumental activities of daily life , minimum of 60 years, and maximum of 92 years. The average age of the women was slightly lower than that of the men  or absence (score ≤ 3) of vulnerability to abuse, there were statistically significant differences in the self-perception of health (p = 0.002), depressive symptoms (p = 0.001), and presence of rheumatism (p = 0.003). There were no statistically significant differences between the sexes. The Kaiser-Meyer-Olkin test indicated a median yet acceptable adaptation of the sample, with a value of 0.64 and satisfactory adaptation for the factorial analysis by the Bartlett sphericity test, which showed an identity matrix with statistical significance (p < 0.002).Three factors met the Kaiser criteria, for which dimensions were extracted with self-value higher than or equal to one. However, based on the assumption that factors with higher explanation potential for the subjacent dimension may contribute to the explanation of the model, we opted to keep the fourth dimension. This was because it strengthened the idea of the general construct of the instrument, agreeing with the model proposed by the authors of the VASSWe performed a factorial analysis via principal components with all 12 of the items in the scale. Initially, we found a low factor load of variable 12, in addition to the exclusive allocation of only one item in a factor (variable five in factor four). To adjust the model, we performed a new analysis without this variable.In the second model, we found changes in the allocation of the factors and the items, which did not compromise the main idea of the constructs, remaining the same designation for the four factors, namely: vulnerability, discouragement/depression, coercion, and dependence. The factor loads varied from 0.57 to 0.92, with communalities between 0.62 and 0.93.As in the original study, factor “1” preserved the “vulnerability” construct, and came in first place, carrying items 8 and 9, in addition to variables 1, 2, and 3. The communalities of this factor varied between 0.62 and 0.88 and corresponded to 28% of the explained variance.The “discouragement/depression” construct moved positions in this study. This was the third factor in the original study, represented by variables 7, 8, and 9, and came second in this analysis, aggregating questions 4 and 5. The third dimension corresponded to the “coercion” construct (completed by variables 10 and 11), and preserved the idea of stress due to potential violence experienced, as in the original study. The “dependence” dimension was the second factor in the original study and explained 32% of the variance in the validation of the VASS in 2003. In this analysis, it came in last place, with a higher explanatory potential (35% of the explained variance), despite one variable less (items 5 and 6).All factors in all items were determined .The internal consistency of the global VASS scale obtained a KR-20 of 0.69 (p < 0.001), showing good consistency, especially considering the brief structure of the instrument.For the four constructs, reliability was considered good to moderate, with KR-20 values of 0.715, 0.32, 0.51, and 0.35.In the “vulnerability” subscale, the simultaneous removal of each item reduces its internal consistency. We identified a similar finding when analyzing the impact of the removal of items from this dimension on the scale total – in other words, the removal of all items from this subscale reduces the internal consistency of the VASS global construct.As the remaining factors were composed of only two variables, the item-total correlations were emitted through the removal of each item with the evaluation of the impact on the KR-20 value of the VASS global score. In this regard, the internal consistency in each case either decreased or remained the same, showing that the items are correlated and help explain the VASS global construct idea.The intra-observer reliability considered three instances: the stability of the answer to each item; for the global scale given the sum of the points attributed on a scale from zero to 12; and for each subscale or subjacent dimension of the VASS. For each item analyzed individually, the K values varied between 0.37 and 0.93 (p < 0.001), reflecting good and adequate concordance. Only questions six and 10 obtained lower concordance estimates. The result of the Kp statistic for the total of the four constructs of the VASS was 0.97 (p < 0.001), proving that the global instrument has substantial reliability. For each factor or subscale, the results found varied from excellent  to moderately reliable (0.736 and 0.561) .,,This transcultural adaptation verified the semantic equivalence of the VASS for Brazilian culture. Observance of recommendations for the transcultural translation and adaptation of the scaleBy comparing the findings of transcultural adaptation of the VASS in this study with those found by Maia and Maia,As for the construct validity of the VASS, the dimensional analysis with four factors proves the findings of Schofield and MishraIn this regard, the performance of this study seems to have surpassed the findings of Reichenheim et al. in the adaptation process of the HS- EASTThe factors were also repositioned in the second phase of the VASS application in the cohort studied in Australia, after three years of follow-up from the first interview. On that occasion, despite the factors having been placed in distinct positions, the original items remained in themIn addition to this repositioning, the composition of the factors was also different. The “vulnerability” factor aggregated the highest number of variables (five of the 11 items), and its composition maintained the original values, but aggregated two others (items 8 and 9), which belonged to the discouragement/depression factor. Despite the changes, this allocation seems to have been pertinent, since variables 8 (“Do you feel that nobody wants you around?”) and 9 (“Do you feel uncomfortable with anyone in your family?”) presented, in the fifth phase of semantic equivalence, the impression that they are more closely associated with situations of interpersonal conflict than with depression. This reinforces the findings of the VASS validation in Australia, which showed high face validity for the “vulnerability” factor as a measure of susceptibility to mistreatment, as well as a strong association with stressful or abusive events, especially in relations with a partner/spouse, children, and other family membersWe highlight that these items are formulated exactly as the HS EAST instrument, and, thus, both the validation conducted by Neale et al.Another possible explanation for the repositioning of the items is the size and characterization of the sample of this study, represented by active older adults who participate in health promotion activities, and who have better health indicators than the samples of the original VASS studies.The greatest change in the structure of the factors occurred in the second dimension, “discouragement/depression”, which, in the original version, was composed of variables 7 (“Are you sad or lonely often?”), 8 (“Do you feel that nobody wants you around?”), and 9 (“Do you feel uncomfortable with anyone in your family?”). The current version kept item 7 and added question 4 (“Do you have enough privacy at home?”).,The internal consistency of the general VASS score (KR-20 = 0.69) is considered adequate and good. There are no reliability analyses, considering the sum of the items in the scale in the studies of the main authorWhen comparing reliability between the global VASS index and the similar instrument it was based on (HS EAST), both in its context of origin and in the Brazilian oneThe isolated removal of each of the 12 items decreased or maintained the internal consistency of the global scale, indicating that the variables contribute to the explanation of the general construct. It was not possible to compare these data with the previous VASS studies, as this analysis was presented only in this study.The vulnerability construct is the most substantial (KR-20 = 0.71), gaining explanation power and better reliability in relation to the values found in studies by Schofield et al.,Despite the lower number of items, in the remaining subscales , the internal consistency values remained very close to those found in previous studies, with the exception of the “dependence” factor, which had a lower score in this study (KR-20 = 0.32), contrasting with previous studies (α = 0.52 and 0.74) As for the reproducibility of the VASS, we can safely affirm that the general score is satisfactorily reliable (Kp = 0.92) and has better performance than the HS EASTUntil now in Brazil, no methods qualify as a gold standard for approaching domestic violence towards elderly people. The limitations of this study include our inability to test the sensibility and specificity of the triage instrument, and the fact that we did not perform a confirmatory factorial analysis using, for example, the structural equations method, since this analysis method requires a larger sample than that used in this study.However, the VASS behaved acceptably in terms of dimensionality, and was coherent with the original proposal. Thus, there do not seem to be any contraindications for its use, except in environments of primary health care or reference centers for the elderly.


The amount of NO3-N content was higher than that of NH4-N, however, under saturated moisture condition, the amount of NH4-N was increased with the incubation time regardless of the levels of Pb application. The amount of NO3-N content in the soil gradually decreased with the advance of incubation at saturated condition. Although application of urea increased the NO3-N content in the soil, such an increase was suppressed by the application of different levels of Pb at water saturated condition throughout the incubation period.Urea is applied to soil for plant growth and undergoes various changes while in the soil. The mobility of heavy metals changes with the transformation of urea applied to the soil that, in turn, affects the activity of microbial biomass. The objective of this study was to determine the interaction between lead (Pb) and urea under two moisture regimes (60% and saturated water contents). At 60% water content, the amount of NH


Reward devaluation resulted in a within-session decrement of the burst number, with the response rate dropping only after the contact with the devalued reward, as predicted. This response pattern was reliably observed only in subjects at their first devaluation experience. In contrast, exposure of separate groups of animals to the two different concentrations yielded lower levels of burst number in the low concentration group apparent since the beginning of the session, as previously observed with dopamine D1-like receptor blockade. These results show that the analysis of burst number, but not of burst size, reveals a specific activation pattern in response to reward devaluation, which differs from the pattern observed comparing the response to two different sucrose concentrations in separate groups of subjects, i.e. in a condition not involving reward devaluation. Finally, the characterisation of the experimental measures of the analysis of licking microstructure in behaviourally  meaningful functional terms, might be relevant for the investigation of the mechanisms underlying behavioural activation and the related evaluation processes.We previously observed that dopamine D2-like receptor blockade in rats licking for sucrose produced a within-session decrement of the emission of licking bursts similar to the effect of either reward devaluation, or neuroleptics, on operant responding for different rewards, which, accordingly, we interpreted as an extinction-like effect. This implies that exposing animals to reward devaluation would result in a drop of burst number taking place only after the contact with the devalued reward. To test this prediction, we compared the difference in the within-session time course of burst number in response to high (10%)  Moreover, a statistically significant 3 ways interaction between devaluation, trial, and group  and a statistically significant 4 ways interaction between devaluation, trial, time and group  were revealed. Further analyses (F-tests for contrasts) based on the 3 ways interaction showed that, in all the trials and both in G1 and G2, exposure to sucrose dilution resulted in a marked decrease of the total lick number (th min in response to the 2% sucrose solution compared to the 10% solution (nd and 3rd rows). As for the comparison between G1 and G2 in the first trial, in session I, with both groups being exposed to a 10% sucrose solution, the curves representing the lick number time course are almost superimposable, showing a small but statistically significant difference limited to one time bin (15–18 min). In session II, G2, i.e. the group exposed to the solution at reduced concentration, showed a reduced level of the number of licks since the beginning of the session up to the 15th min compared to G1, which was exposed to the 10% solution  = 14.17; P = 0.0005], due to the reduced burst number in session II in both groups regardless of trial   = 3.70; P = 0.029] and time  was revealed, with no significant effect of the factor group . A more clear picture emerged from the comparisons between the time course data based on the significant 4 ways interaction  (nd and 3rd row). In trial 1, exposure to the diluted solution in G2 (session II) resulted in a within-session decline which resulted in a significant reduction with respect to the response to the 10% solution (session I) from the 3rd to the 6th time bin . The within session-decline in response to sucrose dilution was apparent also in the comparison between G1 and G2 in session II of the trial I, due to a significant reduction of burst number from the 2nd to the 5th time bin, while a superimposable time course of the responses of the two groups was observed in session I, with both groups exposed to the 10% solution , while in G2, now at its second exposure to devaluation, the difference between the two solutions was anticipated, starting at the 2nd time bin (2nd and 3rd rows mid panels). In trial 3 the difference between the response to the two solutions (session I vs session II within each group) was apparent since the very beginning of the session in both groups (nd and 3rd rows right panels). The pattern emerging from these results shows a within-session decline of burst number in response to sucrose dilution, which is very clear at the first episode of reward devaluation in both groups, but the reduction of the response level appears to be anticipated following repeated experiences of reward devaluation.ANOVA of burst number data revealed a statistically significant effect of per burst data showed a statistically significant effect of devaluation , with no significant effects of group  and trial . Moreover, a statistically significant interaction between devaluation, trial and group was revealed . F-test for contrasts showed a statistically significant reduction of this parameter in response to the 2% sucrose solution, in both groups and in all trials  = 8.58, P = 0.007] and a significant interaction between devaluation and group , due to a reduced burst size after reward devaluation in the group G2, with no 3 ways interaction between these two factors and time . ANOVA of the data of the first two time bins  revealed a significant interaction between the factors group and devaluation . Post-hoc analysis (Newman-Keuls) showed a statistically significant reduction of the number of lick per burst in the group G2 in time bins T1 and T2. ANOVA of the data of the third time bin (T3) failed to show statistically significant effects . The analysis of the within-session time course of number of licks per burst of the second and third devaluation trials showed very similar results, with only the factor devaluation resulting in a statistically significant effect in all analyses, due to the reduced values observed in both groups in all time bins after reward devaluation   = 6.58; P = 0.014], due to a slight increase of this parameter in response to the 2% sucrose solution, with no statistically significant effects of trial  and group  and no significant interaction between the three factors   = 0.014, n.s.; trial: F = 2.7, n.s.; devaluation: F = 0.29, n.s.; interaction between the three factors: F = 0.24; n.s.], we report the whole experiment mean value for each group: 14.95 ± 0.43 (G1) and 14.61 ± 0.45 (G2).Since ANOVA of the latency to the first lick data failed to show any statistically significant main effect or relevant interactions  and session , with a statistically significant interaction between the two factors . Further analysis (F-tests for contrasts) revealed that the high concentration group showed a higher lick number level, compared with the low concentration group, from the second session (S2) up till the end of the experiment. Moreover, a reduction in the lick number was observed in this group when exposed to the lower sucrose concentration in the three devaluation sessions . Conversely, an increased lick number in the low concentration group was observed in the last session, when exposed to the 10% concentration (R1).ANOVA of lick number data (nd panel) showed a statistically significant effect of the factor session  but not group , with a statistically significant interaction between the two . The high concentration group showed higher levels of burst number in the first four sessions (S1-S4), with statistically significant decrements when exposed to the 2% concentration in the three devaluation sessions . Conversely, an increased burst number in the low concentration group was observed in the last session, when exposed to the 10% concentration (R1).ANOVA of burst number data  revealed a statistically significant effect of the factors group  and session , with a significant interaction between the two factors . F-tests for contrasts revealed a different response between the two groups starting at the 5th session (S5): in the sessions from S5 to S15, in 8 time points out of 11, the high concentration group showed a significantly higher level of number of licks per burst. Exposure of the high concentration group to the 2% concentration resulted in a decrement of this measure in the 2nd and 3rd devaluation sessions , but not in the first one (D1). Exposure of the low concentration group to the 10% concentration (R1) failed to induce statistically significant effects.ANOVA of the whole session number of licks per burst of the first two devaluation trials in the high concentration group  showed a significant interaction between the factors devaluation and time , due to a reduction in burst size in the course of the session at S8 and S12 (exposure at 10%) compared to a specular increase at D1 and D2 (exposure at 2%). Consistently, the ANOVA of the single time bins showed a significant main effect of devaluation at T1 in both trials , due to a reduction of burst size at D1 and D2, with no effect in the successive time bins in the trial S8-D1 , and a significant main effect in the third time bin (T3) in the trial S12-D2 , due to an increased burst size at D2. ANOVA of the within-session time course of number of licks per burst of the third devaluation trial in the high concentration group [S15 (10%) vs D3 (2%)] failed to show any statistically significant effects or interactions between the factors. Consistently with the first two trials, ANOVA of the single time bins showed a significant effect of devaluation in the first two time bins , due to a reduced burst size at D3, but not in the third   showed only a significant effect of time , due to the increased burst size in the last time bin (T3), regardless of group . The analyses of the within-session time course of the number of licks per burst relative to the 2nd (D2) and the 3rd (D3) successive negative contrasts failed to show any statistically significant effect, as well as the ANOVA of the single time bins of all the three contrast sessions.  = 2.83, P = 0.105, n.s.).per burst of the upshift trial  showed only a significant effect of upshift , due to the increased burst size after upshift from 2% to 10% sucrose concentration, regardless of group . Moreover, a significant effect of upshift (increased burst size at session R1) was revealed in the first time bin , again, with no interaction with the factor group . No statistically significant effects or interactions were revealed by the analysis of the data relative to the successive time bins.ANOVA of the within-session time course of number of licks group , while a statistically significant effect of session was revealed , due to slight differences between the sessions. The interaction between the two factors was not significant  = 8.56; P = 0.006] and session , with a significant interaction between the two . F-tests for contrasts revealed significantly lower latencies for the high concentration group from the 3rd session (S3) up till the first devaluation session (D1). For the remaining part of the experiment no differences between the two groups were observed, with the exception of the session D3 .ANOVA of the latency to the 1group was present , due to the higher level of burst number in the high concentration group. In the 2nd and 3rd sessions, an interaction between group and time was also present . F-test for contrasts revealed that in both cases the difference between the two groups exposed to the different concentration solutions was apparent since the beginning of the session. In the other sessions examined <2.5, n.s.]. However, in three of these sessions , a statistically significant interaction between the factors group and time  was revealed . In one case (S6), F-tests for contrasts revealed a statistically significant difference between the two groups in favour of the high concentration group, which was apparent since the beginning of the session and lasted for first six minutes (th time point for S8 and 2nd time point for S15) (In  minutes . In the for S15) .group and the within-group factors time and devaluation , have shown that, in the first devaluation episode, a statistically significant difference in favour of the 10% concentration is apparent only since the 2nd time point, and persists up till the fourth time point, thus resembling the response pattern of an extinction curve, as observed in Experiment 1. In the successive devaluation episodes , a significant difference is apparent since the beginning of the session, consistently with the observations from Experiment 1, showing an anticipated decrease of burst number after repeated devaluation episodes.time and upshift , with no three ways interaction between these factors and group , due to an increased burst number at the beginning of the R1 session,with respect to the preceding session (D3), in both groups. The lack of a significant interaction between group and upshift  indicates that the overall effect of the concentration upshift did not result in significant differences between the two groups. However, it should be recalled that in the whole experiment data analysis a statistically significant difference between the groups at session R1was revealed, based on the group × session interaction .st 9-min time bin but not in the whole session data). Most importantly in relation to the aim of the present study, a within-session decrement of burst number occurring after the contact with the reward was observed in the first devaluation session (D1).Consistently with earlier findings, the results of Experiment 1 showed that, in animals with daily access to a 10% sucrose solution, reward devaluation obtained by reducing the sucrose concentration solution to 2% resulted in a reduced ingestion, as indicated by the reduced lick number see , mainly versus low concentration group. We examined all the sessions preceding the 1st devaluation episode, and the single sessions preceding the successive two devaluation episodes . When the difference in total values was not present, the within-session burst number time courses of the high and of the low concentration group were virtually super-imposable.  Hence, these results show that the difference in the burst number within-session time course in two groups of subjects in response to two different sucrose concentrations, i.e. in a condition which does not involve changes in the reward value, is very different from the extinction pattern observed with reward devaluation.The most relevant comparison in relation to the aim of Experiment 2 was the comparison between the within-session burst number time course of the high des Figs  and 7. IThus, the results from this study, taken together with the results of our previous studies on the effect of dopamine D1- and D2-like receptor antagonists on licking microstructure , 9, provIn both experiments see Figs  and 9, rrd time bin. In Experiment 2, burst size reduction was consistently observed in the 3 devaluation trials  only in the first time bin . In the 3rd time bin of session D2, burst size was increased. Thus, the results from Experiment 2 might suggest a within-session attenuation of the effect of reward devaluation on burst size. Such an effect appears to depend (i) to the trend in decrease of burst size over time in the session preceding devaluation, when the animals are exposed to the familiar concentration (10%), and (ii) to a specular increase in burst size in the devaluation session . However, considering the lack of full consistency between the results from the two experiments, no conclusions can be drawn on this matter on the basis of the present set of data, which simply shows that, in our experimental conditions, the effect of reward devaluation of reducing burst size is more reliably observed early in the session.In Experiment 1 , the invversus low concentration group to two different sucrose concentrations, 10% vs 2%, respectively  level after a week. The time course of the latency to the 1st lick values across sessions showed both groups at the same level in the 1st two sessions. The high concentration group values progressively decreased across the first week sessions, reaching a low value which remained stable up to the end of the experiment. The decline of the values of the low concentration group was less steep, reaching the level of the high concentration group from the 9th session up to the end of the experiment . The difference between the two groups in the speed of the progressive increase in burst number across sessions, and of the almost specular progressive reduction of latency to the 1st lick, reflects the different reinforcing effect of the two sucrose concentrations. Thus, these results show that the response to reinforcement of burst number across sessions is similar to that of the response latency, which, in a number of experimental paradigms, is considered as a measure of behavioural activation [The comparison between the whole session data of the response of the high tivation , 31, 32.th session in the high concentration group is due to a progressive increase of this parameter across sessions, which might depend, at least in part, by an habituation process, possibly consisting in a gradual overcome of the inhibitory feed-back provided by novelty of taste [The increased burst size observed from the 5of taste . The higversus low concentration group to the low sucrose concentration solution , mainly due to increased whole session burst size in the high concentration group and to increased whole session burst number in the low concentration group. The analysis of the results of the single time bins suggests that the increase in burst size after the concentration upshift is accounted for by the response early in the session. The analysis of the within-session time course of burst number showed a similar response pattern for both groups, characterised by an increased response at the beginning of R1 session with respect to D3. The comparison between the two groups at session R1 showed a higher burst number in the low concentration group, paralleled by a lower  burst size value, with the result that lick number did not differ between the two groups. These results show that, while responding to an increase in the reward value, the level of emission of licking bursts is immediately updated to the level which is appropriate for the increased value reward. This is the opposite with respect to devaluation, with the response persisting for several minutes before dropping to a lower level. Thus, these observations provide support to the interpretation of burst number as a measure of behavioural activation. Moreover, this makes sense in adaptive terms. Indeed, it might be advantageous to persist in a response which is not particularly demanding in terms of effort while dealing with a devalued reward, since small reductions in the reward value might be compensated by an increased response rate , while tIn Experiment 1, we observed a slight increase in the intra-burst lick rate after reward devaluation, while in Experiment 2 we observed slight differences in this measure between sessions (regardless of group). This might seem somewhat surprising. Indeed, the rhythm of the tongue movements while licking is extremely stable, depending on a brainstem Central Pattern Generator (CPG) the activity of which can occur without proprioceptive feedback and without descending input from the cortex . HoweverThe results of this study might provide further elements for the interpretation of the effects of either drugs or other experimental manipulations on ingestive behaviour. Based on the lack of effect on burst size and initial lick rate, two previous studies reported the failure to observe anhedonic effects after administration either of the dopamine D2-like receptor antagonist eticlopride  or of thIn conclusion, these results show that the analysis of burst number, but not of burst size, reveals a specific activation pattern in response to reward devaluation, which differs from the pattern observed comparing the response to two different sucrose concentrations in separate groups of subjects. These response patterns are paralleled by similar ones induced by either D2-like (extinction mimicry) or D1-like (different activation level) receptor antagonism , 9. Thus


To compare the efficacy and safety of two filgrastim formulations for controlling chemotherapy-induced neutropenia and to evaluate the non-inferiority of the test drug relative to the originator.This phase III non-inferiority study had a randomized, multicenter, and open-label design. The patients were randomized at a ratio of 1:1 with a follow-up period of 6 weeks for each patient. In both study arms, filgrastim was administered subcutaneously at a daily dose of 5 mg/kg body weight. The primary endpoint was the rate of grade 4 neutropenia in the first treatment cycle. The secondary endpoints were the duration of grade 4 neutropenia, the generation of anti-filgrastim antibodies, and the rates of adverse events, laboratory abnormalities, febrile neutropenia, and neutropenia of any grade.The primary efficacy analysis demonstrated the non-inferiority of the test drug compared with the originator drug; the upper limit of the 90% confidence interval (CI) for the rate of neutropenia between the two groups (12.61%) was lower than the established margin of non-inferiority. The two treatments were similar with respect to the secondary endpoints and safety.The efficacy and safety profile of the test drug were similar to those of the originator product based on the rate of grade 4 neutropenia in the first treatment cycle. This study supports Anvisa's approval of the first biosimilar drug manufactured by the Brazilian industry (Fiprima®). Neutropenia is one of the most common chemotherapy-related adverse events, with a mortality rate of 10%, and it increases the costs associated with cancer treatment 3 are associated with an increased risk of infection or fever without the objective identification of microorganisms. It is estimated that 25%–40% of patients without previous treatment develop febrile neutropenia 3 and an oral temperature ≥38.2°C There is a direct correlation between absolute neutrophil count (ANC) and the risk of opportunistic infections in cancer patients undergoing chemotherapy The risk of febrile neutropenia varies according to the specific chemotherapy regimen The use of broad-spectrum antibiotics and hematopoietic growth factors is currently considered effective against chemotherapy-induced toxicity Escherichia coli using recombinant DNA technology to produce Filgrastim, which has been approved for clinical use since 1991 Granulocyte colony-stimulating factor (G-CSF) is the most commonly used hematopoietic growth factor for the prophylaxis of febrile neutropenia. The G-CSF class of therapeutic agents was developed by isolating, purifying, and cloning this hematopoietic regulatory factor Recently, a pegylated formulation of filgrastim was released that reduced the renal clearance of the drug and increased the terminal half-life compared with unpegylated filgrastim Currently, the American Society of Clinical Oncology, the European Organization for Research and Treatment of Cancer, and the National Comprehensive Cancer Network Primary prophylaxis for febrile neutropenia can also be given to patients at high risk of neutropenic complications, even when the expected rate of febrile neutropenia is <20%. The high-risk factors include age >65 years, low performance status, cytopenia due to tumor-induced impairment of the bone marrow, active infections, or open wounds. Secondary prophylaxis is indicated for patients who previously experienced febrile neutropenia and for those whom a lower chemotherapy dose or a prolonged interval between chemotherapy cycles may be associated with worse overall or disease-free survival in vitro biological activity and in vivo (rodent) toxicology and pharmacodynamics. A phase I, single-center, randomized trial was undertaken to demonstrate the equivalence of Eurofarma filgrastim and Roche filgrastim in terms of pharmacokinetic characteristics. Eurofarma filgrastim was well tolerated with no additional safety concerns compared with Roche filgrastim. Eurofarma filgrastim is bioequivalent to Roche filgrastim with regard to pharmacokinetics A previous study of experimental filgrastim produced by Eurofarma demonstrated comparability in terms of both The aim of this study was to compare the efficacy and safety of two filgrastim formulations for controlling chemotherapy-induced neutropenia and to evaluate the non-inferiority of the test drug in relation to the originator.This phase III, non-inferiority, randomized, open-label, multicenter study compared two products containing filgrastim: the test drug  and the originator drug . The primary endpoint was the rate of grade 4 neutropenia in the first treatment cycle . The secondary endpoints included the rates of febrile neutropenia and neutropenia of any grade, the duration of grade 4 neutropenia, the generation of anti-filgrastim antibodies, and the frequency of adverse events and laboratory abnormalities.This study was approved by the Research Ethics Committee of the participating institutions. All the participants provided written informed consent before undergoing any procedure in accordance with the Brazilian standards of Good Clinical Practice and the Declaration of Helsinki.2 docetaxel, 50 mg/m2 doxorubicin, and 500 mg/m2 cyclophosphamide; or AT regimen: 75 mg/m2 docetaxel and 60 mg/m2 doxorubicin); those with no more than one previous chemotherapy regimen for metastatic disease with a performance status of 0 or 1 on the Zubrod scale and adequate organ function; and those aged ≥18 years who signed the informed consent form.The selected patients included those with breast cancer stage II to IV; those with an indication for full-dose chemotherapy with one of the two eligible regimens  in other clinical studies involving medication of any type or in interventional studies; an intolerance or allergy to any component of the filgrastim formulations evaluated in this study; and pregnant or lactating women.The main demographic characteristics of the patients in the per-protocol (PP) and intention-to-treat (ITT) populations are shown in 3 or until the 15th day of the chemotherapy cycle (V14), whichever occurred first.After stratification according to clinical site and the previous use of chemotherapy, the patients were randomized at a ratio of 1:1 to receive filgrastim (Fiprima® or Granulokine®); the follow-up period for each patient was 6 weeks, except in cases of death, withdrawal of consent, or loss to follow-up. In both study arms, filgrastim was administered subcutaneously at a daily dose of 5 mg/kg body weight until the ANC recovered to ≥10,000/mmThe patients were evaluated at the screening visit (SV), at the randomization visit , and at daily visits during treatment. Filgrastim was administered on the odd visits , whereas on even visits (V2 to V14), drug administration was accompanied by health and safety assessments and laboratory tests. The final visit (FV) occurred 3 weeks (±5 days) after the first day of chemotherapy.To assess the non-inferiority of the test drug compared with the originator drug, the 90% CI for the difference in the rates of grade 4 neutropenia between the two groups was initially calculated. Non-inferiority was defined as an absolute value of the upper limit of the CI <15%. In an exploratory manner, the 95% CI for the difference in the rate of grade 4 neutropenia between the two groups was also calculated.th percentile [Q1] – 75th percentile [Q3]). Categorical variables are described as absolute and relative frequencies. The Kolmogorov-Smirnov test with the Lilliefors correction was used to evaluate the standard distribution of the outcome variables. The Lilliefors correction was also used to adjust the estimated population parameters (mean and variance or SD).Continuous variables are presented as the variation , mean, standard deviation (SD), median, and interquartile range  and failure to meet the eligibility criteria (n=12); these failures were due to inadequate organ function (n=8), the presence of serious comorbidities (n=1), radiation within 6 weeks prior to randomization (n=1), lack of an indication for chemotherapy with one of the eligible regimens (n=1), and performance status other than 0 or 1 on the Zubrod scale (n=1). Two randomized female patients did not receive at least one dose of the evaluated drugs; both patients discontinued the study prematurely, one for loss to follow-up and the other for withdrawal of consent. Of the 217 randomized patients who received at least one dose of the study treatment, two patients who had been randomized to receive the test drug instead received the originator drug. 3 after reaching the nadir; n=41). Therefore, the ITT, PP, and safety populations consisted of 217, 170 and 217 patients, respectively.Of the 217 eligible patients who received treatment, all belonged to the ITT and safety populations. Of these patients, 47 did not enter the PP population because they fulfilled at least one of the following criteria: received commercial filgrastim during the study period, were under medical surveillance, and did not perform all the tests specified in the study schedule (n=4); did not undergo an ANC evaluation at the SV (n=1); did not have at least 3 ANC results between V2 and V14 ; or irregular administration of filgrastim , withdrawal of informed consent (n=1), use of medications not allowed during the study period (n=1), and death (n=1).vs. originator) and the comparison of the rates of grade 4 neutropenia between the two treatment arms. There results was no significant difference (p=0.9971) in the rate of grade 4 neutropenia between the two groups in the first chemotherapy cycle.The primary efficacy endpoint was determined by evaluating the non-inferiority of the test drug compared with the originator drug with respect to the rate of grade 4 neutropenia in the PP population. This rate was defined as the ratio of the number of patients with grade 4 neutropenia to the total number of patients in each group during the first chemotherapy cycle. The 90% CI for the difference in the rate of grade 4 neutropenia between the test and originator drugs in the first chemotherapy cycle was between –12.67 and 12.61. The CI included zero, which indicates the absence of a significant difference in this rate between the two groups. To demonstrate the non-inferiority of the test drug compared with the originator drug, the upper limit of the 90% CI for the difference in the rate of neutropenia in the first cycle should be smaller than the margin of non-inferiority (M), which was set at an absolute value of 15%. In addition to the 90% CI, the 95% CI for the difference in the average rate of grade 4 neutropenia in the first cycle was also calculated in an exploratory manner. This interval varied between –15.06 and 15.00 and also included zero, demonstrating the lack of a significant difference between the two groups.p=0.6311), although the rate was slightly lower for the test drug than for the originator drug.With regard to the secondary endpoint, the number of patients with grade 4 neutropenia in the two groups in the ITT population was 56 (51.4%) for the test drug and 59 (54.6%) for the originator drug. The comparison of these rates indicated no significant difference between the two groups (p=0.669). In the ITT population, the rates of febrile neutropenia were 3.7% and 2.8% for the test and originator drugs, respectively; again, these rates were not significantly different (p=0.710).The rate of febrile neutropenia was defined as the ratio of the number of patients with febrile neutropenia to the total number of patients in each study group; only a single episode of febrile neutropenia during the study period was considered for each patient. The analysis of the PP population indicated rates of 3.49% and 2.38% in the test and originator drug groups, respectively; these rates were not significantly different (p=0.2768).The rate of neutropenia of any grade was defined as the ratio of the number of patients with neutropenia of any grade (1 to 5) to the total number of patients in each group; a single episode of neutropenia at its worst grade in the evaluated cycle was considered for each patient. The number of patients with neutropenia in the ITT population was 99 (90.8%) for the test drug and 93 (86.1%) for the originator drug; there was no significant difference between the treatment arms regarding this variable .The duration of grade 4 neutropenia in the two treatment groups was estimated as the difference in days between the date of the onset of grade 4 neutropenia and the response “Yes” to the question regarding changes in the grade of neutropenia during the even visits. Because the evaluation was not conducted daily, the duration categories considered for this analysis were “within 2 days” and “3 to 4 days”. In the PP population, the number of patients with grade 4 neutropenia that lasted <2 days and 2–4 days was 39 (88.6%) and 5 (11.4%), respectively, in the test drug group and 40 (95.2%) and 2 (4.8%), respectively, in the originator drug group. In the ITT population, these numbers were 41 (89.1%) and 5 (10.9%) in the test drug group and 39 (92.9%) and 3 [7.15] in the originator drug group. The chi-square test indicated no significant differences in the duration of neutropenia between the two treatments in the two populations  for IgA, IgG, and IgM at the SV and V8 in the safety population. In the characterization phase, the samples with a positive result in the screening stage were analyzed; in the confirmation phase, the samples positive for any Ig in the characterization phase were analyzed. Patients with samples from a single visit were not included. The results indicated no significant difference in antibody production between the treatment groups.In the safety population, 206 patients had mild-to-moderate adverse events during the study period: 101 in the test drug arm and 105 in the originator drug arm. The cases of neutropenia were not considered adverse events because neutropenia was a study endpoint and was therefore analyzed separately in the efficacy analyses.The most common adverse events in the two groups were nausea , diarrhea , leukopenia , vomiting , and asthenia . For events that occurred in both groups, the relative difference between groups in the number of patients with at least one occurrence of that event was evaluated. Hypothesis tests for equality of proportions were conducted only for events with a relative difference of ≥10% between the groups. There were no significant differences in the frequencies of these adverse events between the groups.Among the reported adverse events, 14 were considered strongly correlated with the study drug (11 in the test drug group and 14 in the originator drug group), 39 were considered possibly correlated with the study drug (26 in the test drug group and 13 in the originator drug group), and 15 were considered most likely correlated with the study medication (8 in the test drug group and 7 in the originator drug group).In the safety population (n=217), nine patients had serious adverse events: three in the test drug arm (three records) and six in the originator drug arm (nine records). All the reported serious adverse events were considered clearly uncorrelated with the study drug.There was one reported death during the study. In the database, this patient had documented abdominal pain and vomiting as serious adverse events; however, these events were considered uncorrelated with the study drugs.This study compared two filgrastim formulations to evaluate the non-inferiority of a test drug relative to an originator drug in the prevention and control of neutropenia associated with the TAC and AT chemotherapy regimens. The primary efficacy analysis of the primary endpoint in the PP population demonstrated the non-inferiority of the test drug compared with the originator drug; the upper limit of the 90% CI for the difference between groups in the rate of grade 4 neutropenia in the first treatment cycle (test drug–originator drug=12.61%) was lower than the absolute value of the pre-established M (15%).Notably, the incidence of grade 4 neutropenia in both groups was lower than that used in the sample size calculation, most likely because blood counts were analyzed every two days. Consequently, an exploratory analysis was conducted using the 95% CI, and the result of this analysis corroborated the primary analysis. The latter analysis was broader and therefore more conservative, and its upper limit coincided with the adopted margin. Although these results are considered borderline with respect to demonstrating non-inferiority, the fact that the 95% CI did not exceed the M is further evidence in favor of the test drug.p=0.9971). The other comparative analyses revealed no significant differences between the two formulations in the other efficacy variables or in the toxicity profile.It was not possible to demonstrate the superiority of the test drug because no significant differences were observed between the two formulations when the rates of grade 4 neutropenia in the first cycle were compared using the test for equality of proportions with the continuity correction  and to the secondary endpoints (rate of febrile neutropenia and duration of grade 4 neutropenia) evaluated in the PP and ITT populations.Randomized trials have demonstrated the benefit of prophylactic filgrastim for the treatment of cytotoxic chemotherapy-induced neutropenia based on reductions in the following criteria: rate of febrile neutropenia; duration of grade 4 neutropenia; depth of the neutrophil nadir; number of hospitalizations; and rate of antibiotic use. Chemotherapy cycles of doxorubicin followed by docetaxel are known to be associated with a high incidence of hematological toxicity (rate of febrile neutropenia: 33%) Despite a similar patient cohort in terms of median age and treatment regimens, the rate of febrile neutropenia among the patients in our population was almost half that reported in pivotal studies. The reasons for this observation are not evident, but there are numerous plausible explanations, including fundamental differences in the patient population, supportive care, and other unmeasured factors. In our study, the women were chemotherapy naïve, with less vulnerable bone marrow, and more responsive than chemotherapy-pretreated patients. In contrast, 20% of the patients in previously published studies had received prior chemotherapy, a known risk factor for severe neutropenia. Nevertheless, there was no significant difference between the groups in our study With regard to safety, there were certain numerical differences between the groups in some of the analyses. However, there was no apparent pattern that indicated which treatment was less toxic. During the study period, there was one recorded death in the originator drug group. There was no significant difference in the frequency of adverse events between the two treatment groups. In addition, although no hypothesis tests were performed, our descriptive analyses suggested that the groups were similar with respect to changes in the measured laboratory parameters.The efficacy of the test drug was similar to that of the originator drug with respect to non-inferiority in the rate of grade 4 neutropenia during the first chemotherapy cycle. There were no significant differences in the other efficacy and safety endpoints between the groups.This study supports Anvisa's approval of the first biosimilar drug manufactured by the Brazilian industry (Fiprima®).This study was sponsored by Eurofarma Laboratórios S.A.This study is not an off-label investigational study.The authors report that this study received financial support from Eurofarma Laboratórios S.A. to analyze an investigational drug.Hegg R and Mattar A wrote and revised the manuscript. Hegg R, Mattar A, van Eyll Rocha S, de Matos Neto JN, Pedrini JL, Aleixo SB, Rocha RO and Cramer-Junior RP were responsible for the clinical trial and revising the manuscript.


The results show that R2 and Q2LOO of 4 kinds of nonlinear models are more than 0.6 and SDEPext is 0.213, 0.222, 0.189, and 0.218, respectively. Compared with the multiple linear regression (MLR) model , the correlation coefficient and the standard deviation are both better than MLR. The reliability, stability, robustness, and external predictive ability of models are good, particularly of the model of linear kernel function and eps-regression type. This model can predict the antimicrobial activity of the compounds with similar structure in the applicability domain. Hydroxyl benzoic esters are preservative, being widely used in food, medicine, and cosmetics. To explore the relationship between the molecular structure and antibacterial activity of these compounds and predict the compounds with similar structures, Quantitative Structure-Activity Relationship (QSAR) models of 25 kinds of hydroxyl benzoic esters with the quantum chemical parameters and molecular connectivity indexes are built based on support vector machine (SVM) by using R language. The External Standard Deviation Error of Prediction (SDEP QSAR , 2 is usHydroxyl benzoic esters are important kinds of preservatives, which are widely used in medicine, food, cosmetics, pesticides, and other fields . At pres t0.5 in the range of atomic number (the number of C among 1–4 on the ester chain of MIC and 1–3 of t0.5). Qiu et al. [ELUMO and the increase of dipole moment μ were the main independent factors contributing to the antifungal activity of the compounds. SVM has shown obvious advantages in the QSAR research, but QSAR study of the compound of hydroxyl benzoic esters is confined to the linear model at present; there is no literature on the nonlinear QSAR analysis of the system.SVM is a machine learning algorithm based on statistical learning theory proposed by Cortes et al. –17. SVM u et al.  optimize Escherichia coli under the most stable configuration, which provides a basis of predicting the antibacterial activity of similar compounds.In this paper, we use the quantum chemical parameters and molecular connectivity indexes to analyze the antibacterial activity of the hydroxyl benzoic esters. The QSAR model is established by the SVM algorithm in the R software. We obtain the structure-activity relationship between the molecular structural parameters and the antibacterial activity ofThis paper took the 25 hydroxyl benzoate group compounds as the research object, including 10 o-hydroxyl benzoic esters, 2 m-hydroxyl benzoic esters, and 13 p-hydroxyl benzoic esters. Their details are shown in t1/2) (h) at the condition of minimum inhibition concentration of 25 hydroxyl benzoic esters was collected from the literature [1/2) to express its antibacterial activity. The results are shown in The antimicrobial half-life   is used Through the R software program, the training set with 22 compounds is used to build the nonlinear models by SVM algorithm based on the selected descriptors. Firstly, we standardize the data and then establish 4 models of kernel for radial, linear, eps-regression, and nu-regression type, respectively.Model validation is very important for QSAR research, which consists of two aspects: internal validation to test the fitting ability and robustness of models and external validation to test the model's predictive ability. Both internal and external validations are equally important .R2) between the experimental and predicted values of the training dataset and leave-one-out cross-validation (Q2LOO) are used to test the reliability, robustness, stability, and whether the models are overfitting or not.There are many methods to estimate a model's stability, robustness, and internal predictive ability, such as the fitting correlation coefficient, cross-validation, random model test, Y random, and various residual errors (like Root Mean Squared Errors (RMSEs), standard residual error, etc.) . In thisQ2cv is a necessary but nonsufficient condition for the high external predictive ability [R2ext, external Q2ext, and SDEPext. In this paper, the test set is used to predict the corresponding lgt1/2 and external predictive ability of the models is evaluated by SDEPext.A very important purpose of the QSAR models is to predict the related activity data of new or even nonsynthetic compounds, in order to guide the design and synthesis of compounds with desirable activity, or to screen the compounds. This requires that the model has good predictive ability and generalization ability; however, cross-validation can only explain the internal predictive ability of models and good internal prediction ability does not mean the excellent external prediction ability –36; that ability . The onlWe use principal component analysis to extract the most critical molecular descriptors of the hydroxyl benzoic esters for antibacterial half-life.1/2 are shown in Four nonlinear SVM models based on the selected descriptors are established by using training set. Experimental values and internal prediction results of lgt See 1/2 of the test set is predicted, respectively, by 4 SVM models and the results are shown in ext of the models and the residual between experimental values and the predicted results of lgt1/2 are displayed in 1/2 are shown in lgt See Tables R2 was only 0.421, but the equation had good linear relationship when the number of C atoms was less than 4. When the number of C atoms in the ester group is more than 4, the influencing factors become more complex and cannot be described by simple linear relationship and may be in nonlinear or diversified relationship. So we use the R language to write the program and establish 4 kinds of nonlinear models through the SVM machine algorithm for 25 hydroxyl benzoic esters and predict lgt1/2. Predicted results of training set are shown in 1/2 is drawn by using R software. R2 is greater than 0.6 [Q2 is greater than 0.5, the model is good, and model is excellent when the values are more than 0.9 [R2 and Q2 to be greater than 0.6. R2 and Q2LOO are greater than 0.6 and R2 and Q2LOO of two models with linear kernel function are close to 0.75, so we may think that the stability, robustness, and internal predicted ability of the 4 models are better and the models are not overfitting because R2 is larger than Q2LOO by no more than 25%. By RS extracting, the para-, ortho-, and metacompound from 25 hydroxyl benzoic esters make up external test set to test the models, and the prediction results are shown in 1/2 of the test set are in the range of −0.037244~0.322733 and SDEPext is 0.213, 0.222, 0.189, and 0.218, respectively. The results indicate that the 4 models have high external predictive ability among themselves; in particular the model of the linear kernel function and eps-regression type is better than the other 3 models. Scatter plots of experimental values and prediction results by 4 SVM models of 25 compounds of lgt1/2 are shown in The degree of freedom and the speed of the preservative molecule determine the effective collision between the central atom of reactivity and the group or atom of microbial molecular activity. As a result, the antimicrobial property of the preservative is essentially determined by the electronic behavior of the preservative and the microorganism, that is, the quantum biochemical characterization of preservative. Therefore, from the perspective of quantum chemistry to study the relationship between the structure and properties of compound, the effective antimicrobial groups of preservative can be explained in essence . Jiang ethan 0.6 , 38 and than 0.9 . TropshaE , ZPE , and p (polarizability). We consider that E, ZPE, and p are the key factors for antibacterial half-life of hydroxyl benzoic esters. p is a kind of structural parameter characterized by molecular deformation tensor under the action of external electric field. It is the most important property that p is related to the volume of the molecule and p contains information about the molecular interaction that is able to characterize the properties of the molecule as an electron acceptor. Since the coefficients of p and ZPE are negative, this indicates that the value of p and ZPE is greater and the antibacterial half-life of hydroxyl benzoic esters is shorter but E is just the opposite because the coefficient is positive.In In summary, QSAR nonlinear model obtained by quantum chemical parameters and molecular connectivity indexes can better predict the antibacterial activity of hydroxyl benzoic esters. The introduction of SVM algorithm solves the problem of poor correlation of QSAR and complex nonlinear relationship between the molecular descriptors when formula weight is large, which provides a basis for the prediction of the antibacterial activity of compounds with similar structure.The establishment of the 4 kinds of nonlinear models using 25 hydroxyl benzoic acid esters by SVM method, through internal and external validation, the stability, and robustness, and internal and external predictive ability of 4 kinds of models are good; that is, the models are available and may predict new compounds in the applicability domain.R2 and Q2LOO, the minimum SDEPext, and the optimal linear relationship between predictive and experimental value of lgt1/2 in 4 kinds of SVM models, which is the optimal model.The model of linear kernel function and eps-regression type has the largest SVM algorithm is a good method to solve the problem of multicollinearity and complex nonlinear relationship between molecular descriptors in QSAR modeling.E, ZPE, and p are the key factors for antibacterial half-life of hydroxyl benzoic esters.Therefore, the main conclusions of this paper are as follows: