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Continuous Glucose Monitoring in Hospitalized Patients: Is It Time to Embrace a Paradigm Shift?

DOI: 10.31038/EDMJ.2025934

 

The use of continuous glucose monitoring (CGM) has transformed outpatient diabetes care, yet its implementation in the inpatient setting remains limited [1,2]. In our recent systematic review and meta-analysis of six randomized controlled trials (RCTs) (n=979), we evaluated whether adding unblinded CGM (real-time or intermittently scanned) to point-of-care (POC) glucose testing, to assist with insulin adjustment, improves outcomes for non-critically ill, hospitalized adults with diabetes mellitus. We found that the addition of CGM significantly increased time in range (TIR, 70–180 mg/dL) by a mean difference (MD) of +7.24% (P <0.00001), supported by high-certainty evidence. It also reduced time below range (TBR) <70 mg/dL (MD: −1.23%, P=0.02; moderate certainty) and <54 mg/dL (MD: −0.95%, P<0.00001; high certainty). Time above range (TAR) >250 mg/dL decreased by −3.70% (P=0.003), and mean glucose levels declined by −10.93 mg/dL (P=0.0003), both with high-certainty evidence. In terms of safety outcomes, CGM use reduced hypoglycemic events <70 mg/dL (MD: −1.21 events per patient, P=0.001; low certainty) and <54 mg/dL (MD: −1.24 events per patient, P=0.03). Notably, nocturnal hypoglycemic events were also reduced for both <70 mg/dL (MD: −0.16 events per patient, P=0.002) and <54 mg/dL (MD: −0.11 events per patient, P=0.006), although the certainty of evidence was not formally graded for these specific outcomes [1-3].

We believe these findings mark an inflection point in hospital glucose management. While point-of-care (POC) glucose testing has long been the mainstay, CGM offers high frequency of data points, rate-of-change of glycemia over time, direction of blood glucose levels and potential for proactive hypo-and hyperglycemia prevention [4]. Although the six included RCTs in our review varied in population, diabetes types, insulin protocols, and device models (Dexcom G6 and Guardian Sensor 3), our sensitivity analyses demonstrated robust, consistent benefit across subgroups. Nevertheless, we recognize that further research is warranted to address remaining knowledge gaps and to further validate these findings across diverse clinical settings. The implementation of CGM in the general ward still faces several challenges. It requires integration of CGM with electronic medical records and training staff and providers on initiation of download and interpretation. Ideally software integration should include the use of artificial intelligence to pick up patterns of hypo and hyperglycemia and a new “glucose monitoring team,” like a telemetry unit should review those flags in real time and inform the treatment team to make clinical decisions. But even more basic integration can provide retrospective data that will assist providers in making insulin adjustments based on better quality of that than what is provided by POC glucose alone [5].

The 2025 Standards of Care in Diabetes from the American Diabetes Association (ADA) now endorse universal use of CGM for all patients with diabetes, regardless of insulin therapy, as well as the continued use of CGM during hospitalization in those already utilizing the technology [6]. Our findings reinforce this recommendation to continue CGM during hospitalization in patients admitted already using the technology but also as a primary tool that could be started in hospital to assist glycemic management in patients with hyperglycemia. With evidence mounting improved outcomes, fewer hypoglycemic events, and no apparent safety concerns, we believe CGM will eventually be implemented as standard practice for most non-ICU inpatients with hyperglycemia.

In conclusion, continuous glucose monitoring (CGM) represents a significant advancement in diabetes management in non-critically ill hospitalized patients. By enabling continuous insight into glycemic patterns, it facilitates optimized glycemic management, resulting in improved safety and clinical outcomes compared to the use of POC glucose testing alone. With supportive policies, appropriate infrastructure, and comprehensive training, CGM integration into routine inpatient care is warranted – particularly for patients at the highest risk of hypoglycemia. Advancing beyond intermittent glucose measurements to leverage continuous data represents a critical evolution in optimizing inpatient glycemic management.

References

  1. American Diabetes Association Professional Practice Committee (2025) Diabetes Technology: Standards of Care in Diabetes-2025. Diabetes Care. [crossref]
  2. Zelada H, Perez-Guzman MC, Chernavvsky DR, Galindo RJ (2023) Continuous glucose monitoring for inpatient diabetes management: an update on current evidence and practice. Endocr Connect. [crossref]
  3. Cavalcante Lima Chagas G, Teixeira L, Clemente MRC, et al. (2025) Use of continuous glucose monitoring and point-of-care glucose testing in hospitalized patients with diabetes mellitus in non-intensive care unit settings: A systematic review and meta- analysis of randomized controlled trials. Diabetes Res Clin Pract. [crossref]
  4. Agarwal S, Galindo RJ, Shah AV, Abreu M (2024) Diabetes Technology in People with Type 2 Diabetes: Novel Curr Diab Rep. [crossref]
  5. Tian T, Aaron RE, Yeung AM, et al. (2023) Use of Continuous Glucose Monitors in the Hospital: The Diabetes Technology Society Hospital Meeting Report J Diabetes Sci Technol. [crossref]
  6. American Diabetes Association Professional Practice Committee (2025) Diabetes Care in the Hospital: Standards of Care in Diabetes-2025. Diabetes Care. [crossref]

Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of the Novel Long-Acting FGF21 Analog Zalfermin: A Multiple Ascending Dose Study in Healthy Participants with Overweight/Obesity

DOI: 10.31038/EDMJ.2025933

Abstract

Introduction: This phase I multiple ascending dose study investigated the safety, pharmacokinetic, and pharmacodynamic properties of zalfermin in healthy participants.

Methods: Male participants aged 22–55 years and female participants aged 22–45 years with a body mass index between 27.0–39.9 kg/m2 were randomized 3:1 to receive either once-weekly subcutaneous zalfermin in multiple ascending doses of 3 mg, 9 mg, 27 mg, 60 mg, and 120 mg, or placebo for 12 weeks. Blood samples were obtained for endpoint assessments. The primary endpoint was the total number of treatment-emergent adverse events (TEAEs) from treatment administration to end of follow-up.

Results: Overall, 57 participants were enrolled. A total of 237 TEAEs were reported in 87.7% of participants; these were all mild to moderate in severity. TEAEs were mainly gastrointestinal-related and most prevalent in the 120 mg group; thus, treatment at this dose level was terminated prematurely. Dose proportionality was established for the maximum concentration of zalfermin in serum at steady state, and the geometric mean for the time to maximum concentration of zalfermin in serum at steady state ranged from 29–38 hours. The plasma half-life of zalfermin was approximately 120 hours and significant improvements in plasma lipid profiles were observed. The maximum tolerated (multiple ascending) dose of zalfermin was 60 mg and was compatible with once-weekly dosing.

Conclusions: The pharmacodynamic profile of zalfermin, particularly the observed improvement in lipids, is promising for the treatment of a range of cardiometabolic diseases including metabolic dysfunction-associated steatohepatitis. Further clinical development and investigations into zalfermin are warranted.

Keywords

Pharmacokinetics, Pharmacodynamics, Phase I, Safety, Tolerance

Introduction

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive form of metabolic dysfunction-associated steatotic liver disease (MASLD) [1], characterized by abnormal accumulation of fat in the liver (steatosis), inflammation, hepatocellular ballooning, and fibrosis [2,3]. The prevalence of both MASLD and MASH is increasing worldwide [4-6]. Approved pharmacotherapies that effectively treat MASLD and MASH alongside other cardiometabolic-related comorbidities are limited [7]; thus, there is a high unmet need for new therapies.

Fibroblast growth factor 21 (FGF21) was discovered as a metabolic regulator in 2005 [8]. Based on preclinical trials investigating obesity- related metabolic conditions, activation of the FGF21 receptor complex has been associated with several beneficial effects. These include sustained weight loss, reduced blood glucose and triglyceride (TG) levels, improvements in insulin sensitivity and hepatic steatosis [9], reduced low-density lipoprotein cholesterol (LDL-C) levels, and increased high-density lipoprotein cholesterol (HDL-C) levels [10-12]. This has led to the pursuit of FGF21 as a potential pharmacotherapy for the treatment of MASH and other metabolic-related conditions [13,14]. Zalfermin is a long-acting, proteolytically stabilized FGF21 analog, which has been preclinically shown to induce pharmacologically mediated weight loss accompanied by an improved serum lipid profile [15].

In a first-in-human, single ascending dose (SAD) study investigating the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of zalfermin in healthy males, zalfermin, administered in the dose range of 2 to 180 mg, was found to have an acceptable safety profile regardless of participants’ race and ethnicity, a plasma half-life of approximately 120 hours, and demonstrated improvements in plasma lipid profiles [16]. However, to date, zalfermin has not been tested in a human female population nor via a repeated dosing regimen.

Given the long plasma half-life of zalfermin and improvement in the plasma lipid profile observed in the SAD study [16], this phase I trial investigated the safety, tolerability, PK, and PD of zalfermin when administered multiple times weekly in a healthy male and female population.

Furthermore, literature has shown that increases in FGF21 can cause a decrease in the luteinizing hormone surge and female fertility in mice [17-19]; therefore, zalfermin may mediate a reversible pause in the menstrual cycle, potentially contributing to hypothalamic amenorrhea. Thus, we collected reproductive hormone and menstrual cycle data from female participants to evaluate this.

Materials and Methods

Study Design and Participants

This was a phase I, randomized, double-blind, placebo-controlled trial (NCT03479892) conducted at a single clinical research site that enrolled female participants (aged 22–45 years) and male participants (aged 22–55 years) with overweight or obesity (body mass index 27.0–39.9 kg/m2) who were otherwise healthy. All participants were required to be generally healthy as judged by the investigator based on medical history; physical examination; and the results of vital signs, electrocardiogram, and clinical laboratory tests performed during the screening visit.

Based on nonclinical findings and previous literature [15,17-20], the menstrual cycle and reproductive hormones were monitored, and a pelvic ultrasound scan was conducted for female participants. Female participants were required to have regular menstrual cycles (defined as 24–35 days between the first day of menses and the end of the cycle, for the two most recent menstrual periods; this was self- reported), and have bilateral tubal ligation or bilateral salpingectomy or use a nonhormonal intrauterine device.

Key exclusion criteria for all participants included any clinically significant disease history and use of any prescription or non- prescription medication. The full list of exclusion criteria is in the supplementary appendix.

Participants were randomized in a 3:1 ratio to receive either once-weekly subcutaneous zalfermin in multiple ascending doses (MADs) of 3, 9, 27, 60, and 120 mg or placebo for 12 weeks. Sequential initiation was included to allow for safety clearance evaluation within each cohort before proceeding to the next cohort. The trial design can be found in Figure S1.

Safety Outcomes

The primary endpoint was the total number of treatment-emergent adverse events from the time of first zalfermin administration at baseline (day 1) to the end of the follow-up period (day 112). A treatment-emergent adverse event was defined as any event that either had onset after administration of the trial product but no later than the follow-up visit or was present before the trial product was administered and increased in severity during the treatment period but no later than the follow-up visit. Stopping criteria are listed in the supplementary appendix. Hereafter, treatment-emergent adverse events will be referred to as adverse events (AEs).

Secondary safety endpoints were changes from baseline to follow- up in vital signs, clinical laboratory safety parameters (biochemistry, hematology, and coagulation), electrocardiogram parameters, number of injection site reactions, and the presence of anti-zalfermin antibodies. Additional exploratory safety endpoints are listed in the supplementary appendix.

Supportive Secondary PK Endpoints

Blood samples for the PK analysis of zalfermin were collected from each participant on days 1 and 2 at baseline and at the start of the treatment period. Further blood samples were taken on days 8, 15, 22, 36, 50, 64, 78, 79, and 80 during the treatment phase; on day 85 (end of treatment [EOT]); days 92, 99, and 106 post-treatment; and on day 112 (follow-up). Parameters assessed were the terminal serum half-life of zalfermin at steady state (t½,SS), the maximum zalfermin serum concentration at steady state (Cmax, SS), the time to maximum zalfermin serum concentration at steady state (tmax, SS), and the apparent total serum clearance of zalfermin at steady state (CL/FSS). Serum zalfermin concentrations were measured from last dose (day 78, pre-dose) until follow-up (day 112). Exploratory PK parameters are listed in the supplementary appendix.

PK Sampling

The bioanalysis of zalfermin was performed by the Department of Development Bioanalysis, Novo Nordisk A/S. Zalfermin was assessed in serum by a validated enzyme-linked immunosorbent assay according to departmental procedures, the US Food and Drug Administration 2001 guidance on validation of bioanalytical methods [21], and the European Medicines Agency guideline on bioanalytical methods validation and current practice [22]. The lower limit of quantification for the assay was defined as 1.00 nmol/L. However, to avoid positive pre-dose samples, the lower limit of quantification was raised from 1.00 nmol/L (version 2.0) to 2.00 nmol/L.

Study samples were analyzed using an analyte-specific capture antibody and a second (detection) analyte-specific biotin- labeled antibody. The antibody–antigen complex was visualized by 3,3’,5,5’-Tetramethylbenzidine substrate. Quantification was performed using the optical density values at 450 nm (reference wavelength at 620 nm was subtracted). All calibration standards and unknown samples were analyzed in duplicate determinations (2 wells), and quality control samples were analyzed in two duplicates (2 × 2 wells). Sunrise (Tecan), controlled by Magellan version 7.1, was the instrument software used and Thermo Scientific Corporation Watson™ Bioanalytical LIMS version 7.4.2 (also called DReS), was the computer application software used for the testing systems.

Exploratory PD Endpoints and Sampling

Body weight, waist circumference, whole body fat mass, and whole body lean mass were assessed from baseline (day 1) to EOT (day 85). Blood samples were obtained for the analysis of TG, total cholesterol (TC), HDL-C, LDL-C, very low-density lipoprotein cholesterol (VLDL-C), and beta-hydroxybutyrate levels from baseline to EOT. Estimated treatment ratios (ETRs) for zalfermin treatment groups versus placebo were calculated for all lipids. Blood samples were also taken for the analyses of leptin and soluble leptin receptor, fasting FGF21, and glucose metabolism parameters (fasting serum glucose [FSG], fasting serum insulin [FSI], fasting plasma glucagon [FPG], and glycated hemoglobin [HbA1c]) from baseline to EOT. ETRs for zalfermin treatment groups versus placebo were calculated for hormones. Changes in insulin and glucose area under the curves (AUCs) related to the oral glucose tolerance test (OGTT) from baseline to EOT were also assessed as exploratory endpoints, and ETRs for zalfermin treatment groups versus placebo were also calculated for the OGTT parameters.

Statistical Analyses

Safety endpoints were analyzed using the safety analysis set (SAS; all participants who had been exposed to ≥1 dose of the trial product). Participants in the SAS contributed to the evaluation ‘as treated’. AEs were summarized using descriptive statistics. All AEs were coded using Medical Dictionary for Regulatory Activities (MedDRA) version 20.1. The statistical analyses for the secondary and exploratory safety endpoints are listed in the supplementary appendix.

PK and PD endpoints were analyzed using the full analysis set (including all randomized participants who received ≥1 dose of the trial product). Participants in the full analysis set contributed to the evaluation ‘as treated’. Further details on the statistical analyses for the supportive secondary PK endpoints are described in the supplementary appendix. All PD endpoints were summarized by zalfermin dose and placebo using descriptive statistics. Further details on the statistical analyses for the PD endpoints are described in the supplementary appendix.

Results

Baseline Characteristics

Overall, 57 participants were enrolled with a mean age (standard deviation [SD]) of 37.9 (6.6) years. The majority were male (54.4%) and White (80.7%). Mean (SD) body weight and body mass index were 93.8 (14.1) kg and 32.5 (3.2) kg/m2, respectively (Table 1). Mean (SD) fasting FGF21 in plasma was 240.2 (248.9) pg/mL. The full list of baseline characteristics is presented in Table 1.

Table 1: Baseline characteristics

Characteristic

Zalfermin
3 mg(n = 10) 9 mg(n = 9) 27 mg(n = 9) 60 mg(n = 9) 120 mg(n = 6) Placebo(n = 14)

Total

(N = 57)

Age, years

37.2 (6.1)

36.2 (4.4) 39.2 (5.4) 34.1 (8.6) 41.5 (5.3) 39.4 (7.4)

37.9 (6.6)

Sex, n (%)

Male

3 (30.0)

3 (33.3) 5 (55.6) 6 (66.7) 6 (100.0) 8 (57.1) 31 (54.4)

Female

7 (70.0) 6 (66.7) 4 (44.4) 3 (33.3) 0 (0.0) 6 (42.9)

26 (45.6)

Female of childbearing potential

Yes

4 (57.1)

4 (66.7) 3 (75.0) 2 (66.7) 0 (0.0) 2 (33.3) 15 (57.7)

No

3 (42.9) 2 (33.3) 1 (25.0) 1 (33.3) 0 (0.0) 4 (66.7)

11 (42.3)

Race, n (%)

White

8 (80.0)

7 (77.8) 9 (100.0) 7 (77.8) 4 (66.7) 11 (78.6) 46 (80.7)

Asian

0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

0 (0.0)

Black or African American

2 (20.0)

2 (22.2) 0 (0.0) 2 (22.2) 1 (16.7) 3 (21.4) 10 (17.5)

American Indian or Alaska Native

0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (16.7) 0 (0.0)

1 (1.8)

Body weight, kg

89.1 (10.8)

89.4 (17.8) 98.8 (13.6) 97.2 (19.9) 99.1 (9.5) 92.1 (10.9) 93.8 (14.1)

BMI, kg/m2

32.5 (2.5) 32.1 (4.0) 34.1 (2.7) 33.7 (4.1) 32.1 (3.7) 31.2 (2.5)

32.5 (3.2)

Waist circumference, cm

103.2 (6.2)

103.9 (13.1) 108.1 (8.9) 106.9 (12.8) 108.4 (11.4) 101.3 (11.0) 104.7 (10.6)

Triglycerides, mg/dL

120 (41) 224 (110) 162 (56) 180 (147) 199 (39) 124 (59)

162 (89)

LDL cholesterol, mg/dL

116 (34)

126 (23) 123 (23) 153 (39) 162 (31) 129 (37) 133 (34)

HDL cholesterol, mg/dL

57 (17) 45 (9) 51 (12) 45 (7) 40 (6) 50 (12)

49 (12)

FGF21 in plasma, pg/mL

310.1

(285.5)

306.6(277.2) 310.4(329.3) 257.7(310.3) 136.8(47.0) 135.6(95.6)

240.2

(248.9)

HbA1c, %

5.2 (0.3)

5.3 (0.2) 5.4 (0.3) 5.3 (0.3) 5.6 (0.3) 5.4 (0.4)

5.3 (0.3)

Data are mean (standard deviation) unless otherwise stated.
BMI, body mass index; FGF21, fibroblast growth factor 21; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein.

Safety and Tolerability

Primary Endpoint AEs

In total, 237 AEs were reported for 50 participants (87.7%), and were mild to moderate in severity and transient (Table 2). Of the 237 events, 204 AEs in 40 participants were reported across the five zalfermin treatment groups, and 33 events in 10 participants were reported in the placebo group. The highest number of AEs was reported in the 9 mg treatment group, with 61 events in nine participants; no clear dose dependency in the number of AEs was observed (Table 2). AEs were mainly gastrointestinal (GI)-related, with 49 events recorded in 27 participants across all treatment groups including placebo. GI- related AEs were most prevalent in the zalfermin 120 mg treatment group, with four events of vomiting recorded in four participants and six events of nausea recorded in five participants (Table 2). As a result of this, treatment with the 120 mg dose was terminated prematurely. Overall, the most frequently reported AEs by preferred term were increased appetite (Table 2) and nausea, and injection site reactions.

Five participants withdrew from the trial and five participants discontinued treatment due to AEs (Table 2). A total of 134 AEs in 42 participants were assessed to have probable or possible relation to the trial product (Table 2). No deaths, serious AEs, or AEs related to technical complaints were reported.

Table 2: AEs from time of first zalfermin administration (day 1) to the end of the follow-up period (day 112).

Zalfermin
3 mg(n = 10) 9 mg(n = 9) 27 mg(n = 9) 60 mg(n = 9) 120 mg(n = 6) Placebo (n = 14)

Total (N = 57)

n (%) | E

n (%) | E n (%) | E n (%) | E n (%) | E n (%) | E

n (%) | E

AEs

8 (80.0) | 23

9 (100.0) | 61 8 (88.9) | 42 9 (100.0) | 50 6 (100.0) | 28 10 (71.4) | 33 50 (87.7) | 237

Serious AEs

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

0 (0.0) | 0

AEs leading to withdrawal

0 (0.0) | 0

2 (22.2) | 2 0 (0.0) | 0 1 (11.1) | 1 2 (33.3) | 2 0 (0.0) | 0 5 (8.8) | 5

AEs leading to treatment discontinuation

0 (0.0) | 0 3 (33.3) | 3 0 (0.0) | 0 2 (22.2) | 2 0 (0.0) | 0 0 (0.0) | 0

5 (8.8) | 5

Related to trial product

Probable

5 (50.0) | 9

7 (77.8) | 21 7 (77.8) | 22 8 (88.9) | 18 5 (83.3) | 12 4 (28.6) | 5 36 (63.2) | 87

Possible

4 (40.0) | 7 5 (55.6) | 10 3 (33.3) | 3 4 (44.4) | 10 4 (66.7) | 7 6 (42.9) | 10

26 (45.6) | 47

Unlikely

7 (70.0) | 7

9 (100.0) | 30 6 (66.7) | 17 8 (88.9) | 22 4 (66.7) | 9 9 (64.3) | 18

43 (75.4) | 103

Severity

Severe

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

Moderate

5 (50.0) | 5 7 (77.8) | 20 5 (55.6) | 8 7 (77.8) | 17 5 (83.3) | 15 8 (57.1) | 9

37 (64.9) | 74

Mild

8 (80.0) | 18

9 (100) | 41 8 (88.9) | 34 9 (100) | 33 6 (100) | 13 10 (71.4) | 24 50 (87.7) | 163

Injection site reactions

1 (10.0) | 2 4 (44.4) | 10 6 (66.7) | 18 5 (55.6) | 11 0 (0.0) | 0 2 (14.3) | 3

18 (31.6) | 44

Symptoms

Itching

1 (100.0) | 1

2 (50.0) | 4 2 (33.3) | 5 2 (40.0) | 2 0 (0.0) | 0 0 (0.0) | 0 7 (38.9) | 12

Redness

1 (100.0) | 1 4 (100.0) | 4 5 (83.3) | 11 3 (60.0) | 3 0 (0.0) | 0 0 (0.0) | 0

13 (72.2) | 19

Ecchymosis

0 (0.0) | 0

1 (25.0) | 1 1 (16.7) | 1 2 (40.0) | 3 0 (0.0) | 0 2 (100.0) | 3 6 (33.3) | 8

Related to technical complaint

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

0 (0.0) | 0

AEs by system organ class and MedDRA Preferred Term
Metabolism and nutrition disorders

5 (50.0) | 5

6 (66.7) | 8 5 (55.6) | 6 9 (100.0) | 10 1 (16.7) | 1 4 (28.6) | 4 30 (52.6) | 34

Increased appetite

5 (50.0) | 5 5 (55.6) | 6 5 (55.6) | 5 8 (88.9) | 8 1 (16.7) | 1 4 (28.6) | 4

28 (49.1) | 29

Glucose tolerance impaired

0 (0.00) | 0

1 (11.1) | 1 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 2 (3.5) | 2

Hyperphagia

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Lack of satiety

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Type 2 diabetes mellitus

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Gastrointestinal disorders

3 (30.0) | 3

6 (66.7) | 9 3 (33.3) | 6 3 (33.3) | 7 6 (100.0) | 15 6 (42.9) | 9 27 (47.4) | 49

Vomiting

0 (0.0) | 0 2 (22.2) | 2 2 (22.2) | 2 3 (33.3) | 3 4 (66.7) | 4 0 (0.0) | 0

11 (19.3) | 11

Nausea

0 (0.0) | 0

3 (33.3) | 3 1 (11.1) | 1 1 (11.1) | 2 5 (83.3) | 6 0 (0.0) | 0 10 (17.5) | 12

Diarrhea

1 (10.0) | 1 1 (11.1) | 1 0 (0.0) | 0 2 (22.2) | 2 2 (33.3) | 2 1 (7.1) | 1

7 (12.3) | 7

Abdominal distension

1 (10.0) | 1

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 2 (14.3) | 2 4 (7.0) | 4

Lower abdominal pain

1 (10.0) | 1 2 (22.2) | 2 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

3 (5.3) | 3

Gastro-esophageal reflux disease

0 (0.0) | 0

1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 2 (33.3) | 2 0 (0.0) | 0 3 (5.3) | 3

Abdominal pain

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 2 (14.3) | 2

2 (3.5) | 2

Flatulence

0 (0.0) | 0

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1 2 (3.5) | 2

Upper abdominal pain

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1

1 (1.8) | 1

Constipation

0 (0.0) | 0

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Dry mouth

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1

1 (1.8) | 1

Food poisoning

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1 1 (1.8) | 1

Retching

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (16.7) | 1 0 (0.0) | 0

1 (1.8) | 1

Investigations

4 (40.0) | 4

6 (66.7) | 6 3 (33.3) | 4 3 (33.3) | 7 2 (33.3) | 2 4 (28.6) | 7 22 (38.6) | 30

Weight increased

3 (30.0) | 3 4 (44.4) | 4 2 (22.2) | 2 2 (22.2) | 2 0 (0.0) | 0 1 (7.1) | 1

12 (21.1) | 12

DBP increased

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 2 (22.2) | 3 1 (16.7) | 1 0 (0.0) | 0 3 (5.3) | 4

ALT increased

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1

2 (3.5) | 2

Blood creatine phosphokinase increased

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1

2 (3.5) | 2

CRP increased

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 1 (16.7) | 1 0 (0.0) | 0

2 (3.5) | 2

AST increased

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1 1 (1.8) | 1

Blood glucose decreased

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1 1 (1.8) | 1

Blood pressure increased

0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

SBP increased

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Blood triglycerides increased

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1

1 (1.8) | 1

GFR decreased

1 (10.0) | 1

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

LDL cholesterol increased

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1

1 (1.8) | 1

General disorders and administration site conditions

2 (20.0) | 3

5 (55.6) | 12 6 (66.7) | 19 5 (55.6) | 13 0 (0.0) | 0 3 (21.4) | 4 21 (36.8) | 51

Injection site erythema

1 (10.0) | 1 4 (44.4) | 5 5 (55.6) | 11 3 (33.3) | 3 0 (0.0) | 0 0 (0.0) | 0

13 (22.8) | 20

Injection site hemorrhage

0 (0.0) | 0

1 (11.1) | 1 1 (11.1) | 1 2 (22.2) | 3 0 (0.0) | 0 2 (14.3) | 3 6 (10.5) | 8

Injection site pruritus

1 (10.0) | 1 2 (22.2) | 4 2 (22.2) | 5 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0

6 (10.5) 11

Early satiety

0 (0.0) | 0

0 (0.0) | 0 1 (11.1) | 1 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 2 (3.5) | 2

Fatigue

1 (10.0) | 1 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

2 (3.5) | 2

Asthenia

0 (0.0) | 0

1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Injection site bruising

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Injection site hematoma

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Injection site induration

0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Injection site edema

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Injection site reaction

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Non-cardiac chest pain

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1 1 (1.8) | 1

Suprapubic pain

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Infections and infestations

1 (10.0) | 1

2 (22.2) | 5 2 (22.2) | 2 5 (55.6) | 7 3 (50.0) | 4 2 (14.3) | 2 15 (26.3) | 21

Upper respiratory tract infection

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 3 (33.3) | 3 0 (0.0) | 0 1 (7.1) | 1

5 (8.8) | 5

Gastroenteritis

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 2 (22.2) | 2 1 (16.7) | 1 1 (7.1) | 1 4 (7.0) | 4

Viral upper respiratory tract infection

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 3 (50.0) | 3 0 (0.0) | 0

3 (5.3) | 3

Urinary tract infection

0 (0.0) | 0

1 (11.1) | 1 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 2 (3.5) | 2

Anal abscess

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Chlamydial infection

1 (10.0) | 1

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Cystitis

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Ear infection

0 (0.0) | 0

1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Lower respiratory tract infection

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Rhinitis

0 (0.0) | 0

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Vulvovaginal mycotic infection

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Nervous system disorders

1 (10.0) | 1

2 (22.2) | 2 2 (22.2) | 2 1 (11.1) | 1 2 (33.3) | 2 2 (14.3) | 2 10 (17.5) | 10

Headache

0 (0.0) | 0 2 (22.2) | 2 2 (22.2) | 2 1 (11.1) | 1 1 (16.7) | 1 2 (14.3) | 2

8 (14.0) | 8

Dizziness

1 (10.0) | 1

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Lethargy

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (16.7) | 1 0 (0.0) | 0

1 (1.8) | 1

Reproductive system and breast disorders

3 (30.0) | 4

2 (22.2) | 5 0 (0.0) | 0 1 (11.1) | 2 0 (0.0) | 0 1 (7.1) | 2 7 (12.3) | 13

Polymenorrhea

3 (30.0) | 3 1 (11.1) | 1 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 1 (7.1) | 1

6 (10.5) | 6

Hypomenorrhea

1 (10.0) | 1

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1 2 (3.5) | 2

Menorrhagia

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Irregular menstruation

0 (0.0) | 0

1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Metrorrhagia

0 (0.0) | 0 1 (11.1) | 2 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 2

Vaginal hemorrhage

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Respiratory, thoracic, and mediastinal disorders

0 (0.0) | 0

2 (22.2) | 2 2 (22.2) | 2 2 (22.2) | 2 1 (16.7) | 1 0 (0.0) | 0

7 (12.3) | 7

Cough

0 (0.0) | 0

1 (11.1) | 1 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 2 (3.5) | 2

Allergic rhinitis

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0

2 (3.5) | 2

Dyspnea

0 (0.0) | 0

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Rhinorrhea

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (16.7) | 1 0 (0.0) | 0

1 (1.8) | 1

Upper respiratory tract congestion

0 (0.0) | 0

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Skin and subcutaneous tissue disorders

0 (0.0) | 0 4 (44.4) | 4 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1

5 (8.8) | 5

Hyperhidrosis

0 (0.0) | 0

3 (33.3) | 3 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 3 (5.3) | 3

Pruritus

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1

1 (1.8) | 1

Urticaria

0 (0.0) | 0

1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Injury, poisoning, and procedural complications

0 (0.0) | 0 3 (33.3) | 3 0 (0.0) | 0 0 (0.0) | 0 1 (16.7) | 1 0 (0.0) | 0

4 (7.0) | 4

Contusion

0 (0.0) | 0

2 (22.2) | 2 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 2 (3.5) | 2

Injection-related reaction

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (16.7) | 1 0 (0.0) | 0

1 (1.8) | 1

Injury

0 (0.0) | 0

1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Musculoskeletal and connective tissue disorders

0 (0.0) | 0 2 (22.2) | 2 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

3 (5.3) | 3

Back pain

0 (0.0) | 0

1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Musculoskeletal discomfort

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Tendonitis

0 (0.0) | 0

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Blood and lymphatic system disorders

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0

2 (3.5) | 2

Anemia

0 (0.0) | 0

1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Lymphadenopathy

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Psychiatric disorders

0 (0.0) | 0

1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1 2 (3.5) | 2

Insomnia

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1

1 (1.8) | 1

Decreased libido

0 (0.0) | 0

1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Cardiac disorders

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (16.7) | 1 0 (0.0) | 0

1 (1.8) | 1

Postural orthostatic tachycardia syndrome

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (16.7) | 1 0 (0.0) | 0 1 (1.8) | 1

Ear and labyrinth disorders

0 (0.0) | 0 1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Ear discomfort

0 (0.0) | 0

1 (11.1) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Eye disorders

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (16.7) | 1 0 (0.0) | 0

1 (1.8) | 1

Cataract

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (16.7) | 1 0 (0.0) | 0 1 (1.8) | 1

Immune system disorders

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1

1 (1.8) | 1

Allergy to chemicals

0 (0.0) | 0

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (7.1) | 1 1 (1.8) | 1

Neoplasms benign, malignant, and unspecified (including cysts and polyps)

1 (10.0) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Uterine leiomyoma

1 (10.0) | 1

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 1 (1.8) | 1

Renal and urinary disorders

1 (10.0) | 1 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Hematuria

1 (10.0) | 1

0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0 0 (0.0) | 0

1 (1.8) | 1

Treatment with the 120 mg dose was discontinued early due to AEs.
AE, adverse event; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CRP, C-reactive protein; DBP, diastolic blood pressure; E, number of events; GFR, glomerular filtration rate; LDL, low-density lipoprotein; MedDRA, Medical Dictionary for Regulatory Activities; SBP, systolic blood pressure.

Supportive Secondary Safety Endpoints

Vital Signs and ECG

Administration of zalfermin was not associated with any clinically relevant changes in vital signs such as body temperature, pulse, or respiratory rate (Table S1). Slight increases in systolic blood pressure (SBP) and diastolic blood pressure (DBP) were observed in participants who received the two highest doses of zalfermin; one AE of increased SBP in the zalfermin 60 mg treatment group and four AEs of increased DBP (three in the 60 mg treatment group, one in the 120 mg treatment group; Table 2). No AEs of increased heart rate were reported at follow-up (Table S1).

Clinical Laboratory Parameters

In total, six AEs related to clinical laboratory parameters across the zalfermin treatment groups were reported (ie, glomerular filtration rate decrease [n = 1], blood creatine phosphokinase increase [n = 1], alanine aminotransferase increase [n = 1], blood cholesterol increase [n = 1], and C-reactive protein increase [n = 2]); these events were deemed to be unlikely related to the trial product, and all were mild to moderate in severity.

Injection Site Reactions

In total, 44 injection site reactions were reported in 18 participants (31.6%), but did not appear to be dose dependent, with the highest number of events (6) reported in the zalfermin 27 mg dose (Table 2). All injection site reactions were mild; included mainly redness, ecchymosis, and itching; and duration ranged between 16 minutes and 13 days. The dose volume injected per dose is presented in Table S2.

Anti-zalfermin Antibodies

Across all zalfermin treatment groups, anti-zalfermin antibodies were detected in 14 participants (24.6%) post-baseline, all of whom had cross reactivity to FGF21 (Table S3). Of these 14 participants, 10 were positive for neutralizing antibodies toward FGF21 (Table S3).  Presence of anti-zalfermin antibodies was not related to any AEs and they were not associated with any effects on PK.

Exploratory Safety Endpoints

Results for exploratory safety outcomes including menses assessments, pelvic ultrasound, and reproductive hormone assessments in females; bone mineral density; and patient-reported outcomes are presented in the supplementary appendix (Tables S4– S7).

Pharmacokinetics

The concentration–time profiles across zalfermin doses are presented in Figure 1. Across the zalfermin 3 mg to 60 mg treatment groups, geometric mean Cmax, SS ranged between 20.4 nmol/L and 399 nmol/L (Table 3) and geometric mean area under the serum concentration–time curve from time 0 to 168 hours at steady state (AUC0-168,SS) ranged between 2612 nmol*h/L and 53,369 nmol*h/L (Table S8). Across all zalfermin treatment groups, dose proportionality was established for Cmax,SS (2β estimate: 1.94; 95% confidence interval: 1.80–2.10; P = 0.4439) and AUC0-168,SS (estimate 1.97; 95% confidence interval: 1.83–2.12; P = 0.6712). The geometric mean t1/2,SS of zalfermin ranged between 120 and 127 hours and apparent CL/FSS ranged from 0.0347 to 0.0599 L/h for the 3 mg to 60 mg doses (Table 3). Geometric mean tmax,SS ranged between 29 and 38 hours (Table 3). Further results for the exploratory PK parameters are presented in Table S8.

Figure 1: Pharmacokinetics of zalfermin after MADs in healthy male and female participants.
(A) shows the geometric mean for the full concentration–time profiles. (B) shows the geometric mean for concentration–time profiles at steady state and elimination on the logarithmic scale. The dotted lines in both graphs are reference lines for the LLOQ. Values below the LLOQ were imputed. LLOQ, lower limit of quantification; MAD, multiple ascending dose.

Table 3: Pharmacokinetics of zalfermin at steady state after MADs in healthy male and female participants.

Zalfermin
3 mg(n = 10) 9 mg(n = 9) 27 mg(n = 9)

60 mg

(n = 9)

t1/2,SS, h

122 (23.0)

127 (21.3) 120 (12.6)

122 (5.2)

Cmax,SS, nmol/L

20.4 (24.8)

98.1 (31.4) 162 (23.1)

399 (24.1)

tmax,SS, h

29 (71.9)

38 (41.9) 37 (30.2)

37 (29.3)

CL/FSS, L/h

0.0565 (22.1)

0.0347 (31.9) 0.0599 (25.1)

0.0553 (24.8)

Data are geometric mean (CV). Data are not shown for the 120 mg treatment group as treatment at this dose was discontinued early due to adverse events. CL/FSS, total apparent serum clearance at steady state; Cmax,SS, maximum plasma concentration at steady state; CV, coefficient of variation; MADs, multiple ascending doses; t1/2,SS, terminal serum half-life at steady state; tmax,SS, time to Cmax at steady state.

Pharmacodynamics

Body Weight, Waist Circumference, Whole Body Fat Mass, and Whole Body Lean Mass

Changes in body weight over time are shown in Figure 2. A statistically significant weight gain of 4.5 %-points (placebo-adjusted) was observed in the zalfermin 3 mg treatment group (P = 0.0008). For the zalfermin 60 mg treatment group, a weight loss of 1.9 %-points was observed; however, this was not statistically significant compared with placebo (Figure S2). No clinically relevant change or dose dependency was observed across any zalfermin dose for waist circumference, whole body fat mass, and whole body lean mass (Figure S2).

Figure 2: Change in body weight according to zalfermin dose over time: (A) 3 mg, (B) 9 mg, (C) 27 mg, and (D) 60 mg.
Data are not shown for the 120 mg treatment group as treatment at this dose was discontinued early due to adverse events.

Lipids

Sustained improvements in TG, HDL-C, and LDL-C levels were observed at EOT (Figure 3). The change from baseline to EOT in TG and VLDL-C was significant across the zalfermin 3 mg to 60 mg treatment groups versus placebo (P < 0.05) (Figure 4). Improvements in HDL-C and LDL-C were dose dependent, with the greatest improvements achieved with the zalfermin 60 mg dose versus placebo (P = 0.0113 and P = 0.0135, respectively; Figures 3 and 4).  Improvements in TC were significant for the zalfermin 27 and 60 mg doses versus placebo (P = 0.0023 and P = 0.0116, respectively). For beta-hydroxybutyrate, non-significant increases favored zalfermin versus placebo with the exception of the 27 mg dose (Figure 4).

Figure 3: Change from baseline in lipids: (A) triglycerides, (B) LDL cholesterol, and (C) HDL cholesterol after MADs of zalfermin in healthy participants.
Data are not shown for the 120 mg treatment group as treatment at this dose was discontinued early due to adverse events.
Vertical dotted reference lines represent first and last doses of zalfermin.
HDL, high-density lipoprotein; LDL, low-density lipoprotein; MAD, multiple ascending dose.

Placebo-adjusted changes in the 3, 9, 27, and 60 mg treatment groups were −33%, −52%, −61%, and −48%, respectively, for TGs; −1%, −4%, −17%, and −14%, respectively, for TC; 7%, −1%, −13%, and −20%, respectively, for LDL-C; −32%, −52%, −61%, and −48%, respectively, for VLDL-C; 51%, 10%, −26%, and 12%, respectively, for beta-hydroxybutyrate; and −1%, 11%, −1%, and 20%, respectively, for HDL-C (Figure 4).

Figure 4: Estimated treatment ratios for change from baseline to end of treatment in lipids for zalfermin versus placebo.
Data are not shown for the 120 mg treatment group as treatment at this dose was discontinued early due to adverse events.
CI, confidence interval; ETR, estimated treatment ratio; HDL, high-density lipoprotein; LDL, low-density lipoprotein; VLDL, very low-density lipoprotein.

Hormones

Decreases in plasma leptin levels were observed at EOT in all zalfermin treatment groups with no clear dose dependency (Table S9). Plasma soluble leptin receptor levels increased across all the zalfermin treatment groups at EOT; changes were not dose dependent (Table S9). The ETRs for plasma leptin and plasma soluble leptin receptor levels across all zalfermin treatment groups are presented in Figure S3. Changes in plasma leptin levels favored zalfermin except for the 3 mg dose (Figure S3). Changes in plasma soluble leptin receptor levels favored zalfermin across all treatment groups (Figure S3).

Glucose Metabolism

Variations in mean glucose metabolism parameters were observed across the zalfermin treatment groups, with no clear dose dependency. No clinically meaningful changes in FSG, FSI, or FPG were observed across the zalfermin treatment groups (Table S10). A reduction in HbA1c was observed in all zalfermin treatment groups, with greatest reduction observed in the 27 and 60 mg treatment groups (Table S10).

The ETRs for the glucose AUCs were consistently but not statistically significantly slightly above 1 for the 3 mg to 60 mg treatment groups, indicating a minimally higher glucose excursion with the OGTT. This was accompanied by a slight non-significant lowering below 1 in the ETRs for insulin AUCs in the two highest treatment groups (27 and 60 mg) at EOT, compared with baseline. This tendency was not confirmed for the incremental AUCs for insulin and glucose for the highest dose of zalfermin (Table S11).

Discussion

In the current study, AEs were mainly GI related across all MADs of zalfermin and were mild to moderate in severity. Zalfermin was generally well tolerated, except for the 120 mg treatment group that was terminated early due to GI-related AEs (vomiting and nausea). No deaths, serious AEs, or AEs related to technical complaints were reported across any of the zalfermin treatment groups. Overall, zalfermin had a safety profile that was consistent with the FGF21 analog class [13,23-26]. No clinically relevant effect on the menstrual cycle, female reproductive organs, or sex hormones were observed, and no clinically relevant changes at follow-up were observed in other safety parameters. Although slight increases in SBP and DBP were observed in the higher-dose zalfermin treatment groups, these were not regarded as events directly caused by treatment but likely incidental findings. Of note, one other FGF21 analog (PF-05231023) has reported slight increases in blood pressure [27]. Low-titer anti- zalfermin antibodies were detected in 14 participants with no clinical impact. Given that the 120 mg dose in this current study was terminated prematurely, the 60 mg dose appears to be the maximum tolerated dose for weekly administration.

Dose proportionality was observed for AUC0-168,SS and Cmax,SS. The geometric mean t½,SS of zalfermin ranged from 120 to 127 hours, and tmax,SS ranged from 29 to 38 hours across the zalfermin 3 mg to 60 mg treatment groups. The approximate plasma half-life of zalfermin of 120 hours (5 days) was determined in the SAD study [16] and corroborated in this study. Therefore, zalfermin may be capable of sustained PD activity with a once-weekly dosing regimen. By comparison, other FGF21 analogs such as efruxifermin and pegozafermin have reported half-lives in humans of 3–3.5 days, which were suitable for once-weekly dosing [14,28]. Other PK parameters, for example Cmax and tmax, were also similar between this study and the SAD study [16].

Participants in the lower-dose zalfermin treatment groups experienced some weight gain, likely mediated by an increase in appetite and patient-reported changes in food preferences, which has also been described in preclinical species [29,30]. In addition, some other FGF21 analogs have shown an increase in appetite with no clinically meaningful change in body weight [13,31]. In this current study, no clinically meaningful change in body weight was observed in the 60 mg treatment group, where increase in appetite was observed, indicating a potential treatment effect on energy expenditure. By comparison, no clinically meaningful weight loss was observed in the SAD study [16]. In a previous study, efruxifermin did not demonstrate meaningful weight loss at lower doses of 7 and 21 mg compared with the higher doses of 70 and 140 mg [13]. Furthermore, in another study, efruxifermin 50 mg did show a trend toward body weight reduction [32]. Thus, it is likely that studies involving higher doses of zalfermin and of longer duration may be needed for clinically significant weight loss to be observed; however, the effect on body weight reduction would need to be carefully balanced with the observed GI-related AEs. At the time of writing, the phase II study (NCT05016882) of zalfermin 30 mg and semaglutide combination therapy is in progress to determine their effect on MASH resolution and fibrosis improvement in patients with MASH and fibrosis stages 2–4 [33]. Semaglutide has previously demonstrated glucagon-like peptide-1 receptor agonist- mediated weight reduction [34], and it is hypothesized that this would confer additional benefit alongside zalfermin treatment.

Changes in leptin favored zalfermin versus placebo across all doses (except the 3 mg dose) despite no clinically significant weight loss across zalfermin treatment groups. This may be related to the mechanism of action of zalfermin with modulation of adipokines in adipose tissue, resulting in alterations in secretion and systemic leptin sensitivity [35]. A decrease in leptin may also be related to the decrease in female fertility that was observed preclinically [15,17-20], suggesting that this may not translate to patients with overweight/ obesity.

Positive effects on the lipid profile in humans have previously been demonstrated with other FGF21 analogs [13,24-26]. The percentage changes from baseline in TG, HDL-C, LDL-C, and VLDL-C levels for the 60 mg treatment group in this analysis were –48%, 20%, –20%, and –48%, respectively. Specifically for TG and VLDL-C, significant treatment effects were observed across all zalfermin treatment groups versus placebo (3 mg to 60 mg). These results are consistent with the lipid profile improvement reported in the SAD study [16]. Although this study was conducted in healthy volunteers with overweight/ obesity, these results indicate that zalfermin may be promising not only as pharmacotherapy for the treatment of MASH but for other cardiometabolic-related disorders such as dyslipidemia and severe hypertriglyceridemia [36].

No clinically meaningful changes in FSG, FSI, and FPG were observed across the zalfermin treatment groups. This is consistent with the results from the SAD study, where no clinically relevant changes were observed in glucose metabolism parameters [16]. This could be explained by the fact that in both the current study and the SAD study, analyses were conducted in populations without diabetes, and therefore, clinically meaningful changes in glucose metabolism parameters were not expected. However, in this current analysis, reduction of HbA1c was observed and was greatest in the zalfermin 27 and 60 mg treatment groups. This is likely attributable to the MAD design of this study, with participants having a higher exposure to zalfermin compared with the SAD study [16], and where an effect on HbA1c was harder to observe with once-weekly single dosing. In addition, participants in this current study had obesity and therefore may also have been insulin resistant. Furthermore, the results of the OGTT, with a slight increase in glucose excursion rate across all doses of zalfermin and a lowering of insulin, could suggest a reduction of glucose tolerance. This finding is not supported by the HbA1c results where a reduction was observed at the higher zalfermin dose levels. One explanation could be that the relationship observed between zalfermin and glucose tolerance may be influenced by body weight; however, further investigation is needed to assess this.

Some FGF21 analogs have shown sustained effects on insulin sensitivity and trends in the lowering of FPG [13,24]. However, in two previous studies, the results observed were in populations with type 2 diabetes, and therefore, the effects on glycemic parameters may be more pronounced than in the current study [13-24]. Nevertheless, further studies are warranted to determine potential effects of zalfermin on glucose metabolism parameters in participants with types 2 diabetes.

This study had several limitations. Although the purpose of the trial was to primarily assess safety and tolerability to establish a well- tolerated dose range for zalfermin to be investigated in larger trials, the relatively small sample size of 57 participants, and short duration and follow-up time, may affect the generalizability of the safety and efficacy assessments. Furthermore, most of the population was White and no Asian participants were included, thus there was limited diversity in this trial. In addition, fewer females were included in the 27 and 60 mg treatment groups, thus, there are limited data on the effects of zalfermin on the menstrual cycle and reproductive organs at higher doses. Longer, larger clinical studies may provide further insights into the efficacy and safety of zalfermin in more diverse populations.

Based on the safety and PK profile established during this MAD study of subcutaneous zalfermin, the maximum tolerated dose was 60 mg, and this was compatible with a once-weekly dosing regimen. The PD profile of zalfermin, particularly the improvement in the plasma lipid profile, is promising for the treatment of a range of cardiometabolic diseases such as MASH and dyslipidemia. The results obtained from this trial support further clinical development of zalfermin.

Acknowledgments

The authors thank the trial participants, the investigators, and trial site staff who conducted the trial. Medical writing support was provided by Casey McKeown, RVN, FdSc, and Liam Gillies, PhD, of Titan, OPEN Health Communications, and funded by Novo Nordisk, in accordance with Good Publication Practice (GPP) guidelines (www.ismpp.org/gpp-2022).

Data availability Access request proposals can be found at novonordisk-trials.com

Funding

This study was funded by Novo Nordisk A/S.

Conflict of interest disclosure

K.D., J.S.H., M.S.P., S.L.L., O.B., S.T., and B.A. are all employees of and shareholders in Novo Nordisk. C.K. is an employee of and shareholder in ICON.

Ethics approval

The trial was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. The protocol for this trial was approved by an independent ethics committee/institutional review board.

Participant consent

All participants provided written informed consent.

Clinical trial registration

This study is registered with ClinicalTrials.gov (NCT03479892).

Author contributions

K.D., J.S.H., M.S.P., S.L.L., O.B., S.T., and B.A. wrote the manuscript; K.D., J.S.H., M.S.P., S.T., and B.A. designed the research for the SAD and MAD studies; K.D., J.S.H., S.T., and B.A. performed the research for the SAD and MAD studies; K.D., J.S.H., M.S.P., S.T., and B.A. analyzed the data; S.L.L. and O.B. contributed new reagents and analytical tools; C.K. was the principal investigator and responsible for assessment of adverse events as well as the medical care of the participants during the study.

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Staying Under the Radar: Poison and the Female Serial Killer

DOI: 10.31038/AWHC.2025833

Brief Commentary

This brief commentary will explore the archetype of the female serial killer and her use of poison as a means to achieve her ends in the landscape of patriarchal, Western culture. A serial killer is a person who has killed two or more people (some definitions indicate that the person has killed three or more [1]) with cooling off periods between each murder and the murders generally taking place in different locations [2-4]. With origins in the Victorian era, poisoning has been predominantly perceived as a feminine means of murder, as women were believed to be instinctively passive creatures and belonged in the domestic sphere [5]. Thus, poison (e.g., arsenic) was a useful tool to rid the home of unwanted vermin, children, or husbands either for the sake of the woman’s reputation or financial gain [1,4,6]. In the past, poisoning would have been a convenient means of murder, as the untimely deaths of loved ones could be attributed to heart failure or consumption. However, as [7] identified, as cultural expectations of gender change, so too does the gendered nature of murder.

Typically, serial killers are cast as masculine actors who are active agents in the world motivated by fear, anger, jealously, or desire [8]. These cultural conceptions are reinforced through large- scale data on serial murders, which tend to be overwhelmingly committed by men, with only 5-8.6% of instances perpetrated by women [3]. In these cases, the women are often concealed within the archetypes of traditional womanhood, tethered to their relationship to men or children (e.g., mother, nurse, wife, daughter, prostitute). However, as these archetypes break down and women are liberated from patriarchal forms of control, so too will their reasons for and methods of murder change. Thus, there is a need to explore the role of poison as it pertains to feminine archetypes in the 21st century.

References

  1. Tracy SK (2025) Serial killing: A psychological Cognella (in press).
  2. Cullen E (2020) American evil: The psychology of serial killers. Waterside Press.
  3. S. Department of Justice, Morton, R. J., Tillman, J. M., & Gaines, S. J (2019) Serial murder: Pathways for investigation. AbeBooks.
  4. Ramsland K (2006) Inside the minds of serial killers: Why they Praeger.
  5. Sterling E (2019) Desperate motives for murder: Mercenary female baby killers in Victorian England. The Lincoln Humanities Journal, 7(1), 136-153. Available from: https://www.lincoln.edu/_files/academics/Lincoln-Humanities-Journal-Vol-7-2019. pdf#page=137
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Attachment and Trauma in Therapy: A Neuroaffective Developmental Perspective

DOI: 10.31038/PSYJ.2025731

Introduction

In psychotherapy, attachment and trauma are not abstract concepts—they are felt realities that enter the room through the body, the relational field, and the therapist-client interaction. From the perspective of neuroaffective developmental psychology [1], both attachment and trauma are seen as embodied processes that unfold across three interconnected levels of functioning: the autonomic – arousalregulating and sensing level, the limbic – emotional level, and the prefrontal – selfcontrol and emotional intelligence systems. This article offers an adjunct to the article Dances of connection: Neuroaffective development in clinical work with attachment (2015). It is a brief sketch of how psychological trauma and attachment patterns interact, and how these dynamics show up in therapy. The neuroaffective approach helps clinicians work not only with what clients say, but with how they regulate, feel and relate moment to moment.

Three Levels of Neuroaffective Functioning

This felt reality of attachment and trauma is particularly noticable when the client suffers from severe trauma, neglect or abuse in childhood. On a CT scan, the brain of the client with severe history is smaller than the brain of a normally attached person. This is not primarily because there is a lack of neurons; we are born with most of our neurons already developed. It is instead because the wiring between those neurons has not developed [2]. The neuroaffective model describes human development as unfolding, a growth of neuronal connections, through interaction between child and caregiver at three brain–body levels:

  1. The Autonomic Level – This is the body’s basic regulation system: arousal, movement, safety/danger detection. Trauma states generally show up as chronic hyperarousal, exaggerated startle responses or collapse, or disconnection from the body. When this level is intensely activated, either through trauma or through severe neglect or abuse, the limbic and prefrontal levels are shut off. In severe cases of childhood dysfunction, the basic neuronal growth is inhibited [3].
  2. Limbic Level – This level governs affect regulation, social bonding, and emotional resonance. Relational trauma, common in early insecure attachment, will intensely activate this In these circumstances, there is a deep disruption in emotional resonance. This may cause relational insecurity, intense agitation or a strong desire to control the beloved other.
  3. Prefrontal Level – This level is responsible for self-control, executive function, language, reflection, values, and The autonomic trauma response can make it difficult to manage daily chores. It can also fragment identity, so the person feels like floating pieces instead of a person, activating intense fear of going insane. Trauma and severe attachment dysfunction also impairs the ability to mentalize, i.e have insight into, one’s own or others’ inner states.

When trauma or insecure caregiving occurs—especially in early attachment relationships—it interrupts the integration between these levels. Throughout life, the person then develops strategies that keep them functioning but block relational depth.

Attachment Patterns and Trauma Responses

Attachment systems are our embodied adaptations to our early relational environments. Traumatic experiences shape how these systems become wired into our neural network (Table 1).

Disorganized attachment is often a marker of severe complex or developmental trauma, where the caregiver is also the source of fear [4]. This creates internal conflict with no solution—a condition that easily repeats itself in therapy.

Table 1: Attachment patterns and trauma responses.

Attachment Pattern

Somatic Tone Emotional Signature

Relational System

Secure Regulated Trusting, flexible Open, responsive
Avoidant Controlled Flat, disconnected Self-sufficient, distant
Anxious ambivalent Hyperaroused Overactivated, upset Preoccupied, ruminating
Anxious dependent Hyperaroused Overactivated, fearful Clingy, fearful
Disorganized Freeze/collapse Fear-without-solution Fragmented, chaotic

How These Patterns Show Up in Therapy

Clients do not “talk about” trauma and attachment—they live them. In the session, therapists may encounter:

  • Sudden shifts in presence or affect (dissociation, collapse or severe startle-response)
  • Fear of closeness
  • Intense attachment bids
  • Sudden conflict and complete loss of trust
  • Idealization followed by devaluation
  • Somatic cues like tension, dissociation, fidgeting or holding breath

These responses are not ‘resistance’—they are self protection responses as the client literally is living in the map from the past instead of in real time. Therapists, too, may be drawn into these reenactments—emotionally pulled into being caretaker, overwhelmed, rescuer, rejector, or withdrawing. Being aware of these dynamics in the client and in onself is central to effective therapy.

Healing Approach

The key to healing attachment trauma is not primarily insight or technique. It is a relational experience that will allow the nervous system to integrate (Table 2).

The therapist must become a regulating presence, offering consistent, attuned responses – especially when the client mistrusts the relationship. Repair after rupture or conflict is often the most powerful healing moment [5].

Table 2: Healing approach.

Level

Clinical Focus

Tools and Interventions

Autonomic Safety, arousal regulation Breath, grounding, containment, music, somatic tracking, synchronisation
Limbic Co-regulating emotions and emotional activities Voice tone, eye contact, emotional mirroring, shared activities and games
Prefrontal Impulse control, reflection and mentalization Playing with self-control, mentalizing questions, value clarification

Final Reflections

Neuroaffective developmental psychology reminds us that trauma is not only remembered. It is where the client lives. The therapist’s job is to become a “regulating other”, offering what was missing: safety, resonance, repair and mentalization. It is not about what we do, it is about who we are while we are doing it. Through this embodied, attuned presence, clients can begin to reorganize their inner experience—and gradually, resolve trauma responses and earn secure attachment from the inside out [6-10].

References

  1. Hart S, Bentzen M (2012) Through Windows of Karnac.
  2. Teicher MH, Samson JA, Anderson CM,Ohashi K (2016) The effects of childhood maltreatment on brain structure, function and connectivity. Nature Reviews Neuroscience, 17: 652–666. [crossref]
  3. Perry BD (2009) Examining child maltreatment through a neurodevelopmental lens: Clinical applications of the Neurosequential Model of Journal of Loss and Trauma, 14: 240-255.
  4. Perry BD, Szalavitz M (2006) The Boy Who Was Raised as a Dog: And Other Stories from a Child Psychiatrist’s Notebook – What Traumatized Children Can Teach Us About Loss, Love, and Basic Books
  5. Tronick E,Gianino A (1986) Interactive mismatch and repair: Challenges to the coping Social Perception in Infants.
  6. Bentzen M (2021) The Neuroaffective Picture North Atlantic Books.
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β-Mannanase Enzyme Supplementation in Reformulated Grow-Finishing Diets Resulted in Retained Performance and an Economic Benefit

DOI: 10.31038/JCRM.2025825

Abstract

Many vegetable feed ingredients – such as wheat, corn, barley, palm kernel meal, sun flower meal and others – contain β-mannans, known as strongly antinutritive polysaccharide fibres. The content of soluble β-mannans in swine diets commonly ranges between 0.15% to 0.40%. As little as 0.05% soluble β-mannans in feed can elicit a strong innate immune response. Hemicell HT (Elanco) is a β-mannanase enzyme used to supplement animal feed, breaking down β-mannans and thus preventing economic losses due to the wasteful immune response elicited by these β-mannans. The current field study compared fattening pig performance on a control diet to a reformulated diet, including a β-mannanase enzyme combined with a 45 kcal/kg reduction in net energy (NE). A 130-day feeding trial was conducted on a commercial grow-finishing facility with DanBred x Piétrain pigs starting at 70 days of age. Standard four-phase (0-30 d, 31-60 d, 61-90 d, and 91-130 d) control diets were compared to reformulated diets with a 45 kcal/kg NE reduction along with the inclusion of a β-mannanase enzyme (Hemicell HT; Elanco) at 300 g/tonne. Standard production data were collected and analysed using JMP 17.0 statistical software. Overall, pig performance and carcass quality data did not differ significantly between treatment groups during the entire grow-finishing period. Hemicell HT had an overall benefit of € 5.24 per grow-finishing pig and € 2.38 per tonne of feed due to the 45 kcal/kg NE reduction. The current trial demonstrated that the inclusion of Hemicell HT in reformulated lower-energy diets (- 45 kcal/kg NE) was able to retain production performance in grow-finishing pigs with an additional economic benefit.

Keywords

β-mannanase, Grow-finishing pigs, Net energy reduction, Equal performance, Economic benefit

Introduction

All vegetable feed ingredients commonly used in swine diets contain polysaccharides, which are polymers of monosaccharides linked by glycosidic bonds. Starch, a polymer of glucose units linked by α-(1-4) with a few α-(1-6) bonds, is digested in the small intestine of pigs through endogenous enzyme activity. Non-starch polysaccharides (NSPs) are fibrous materials found in the plant cell wall, including celluloses, hemicelluloses, pectins, and oligosaccharides. Monogastric animals like pigs lack the endogenous enzymes required to digest β-linked NSPs like β-mannans [1]. β-mannans – an antinutritive factor present in many common feed ingredients [2]– have gained increasing attention in recent years. β-Mannans are linear polysaccharides composed of repeating units of β-1,4-mannose and α-1,6-galactose and/or glucose attached to the β-mannan backbone [3,4]. In monogastric diets, high concentrations of these β-mannans are considered unsuitable due to their antinutritive properties, mainly due to stimulation of an innate immune response at the level of the intestinal lining. The innate immune cells recognize pathogens through distinct molecules, called pathogen-associated molecular patterns (PAMPs), which are expressed on the pathogen surface [5]. The binding of PAMPs to pathogen recognition receptors (PRR) present on innate immune cells, results in the release of innate defense molecules such as reactive oxygen and nitrogen species, bacteriolytic enzymes, antimicrobial peptides and complement proteins [6]. These PAMPs include complex polysaccharides that resemble β-mannans [5]. Consequently, β-mannans present in the swine feed may be mistaken by the immune system in the gastrointestinal tract for invading pathogens causing an unwarranted immune activation [7,8], also known as a feed-induced immune response [9]. This misrecognition of β-mannans as invading pathogens results in a futile immune response that wastes energy and nutrients [3]. The hydrolysis of β-mannans through the inclusion of an exogenous β-mannanase enzyme can reduce and potentially eliminate their ability to induce FIIR.

Supplementation of β-mannanase to low- and high-mannan diets has the potential to improve the performance of growing pigs [10]. Other studies have concluded that β-mannanase improved growth performance in both weaning and grow-finishing pigs on corn-SBM diets [11-13] with minimal effects on nutrient digestibility [12]. Moreover, innate immune activation is accompanied by downregulation of anabolic functions [14], resulting in a reduced performance capacity. Therefore, supplementation of a β-mannanase enzyme to grow-finishing diets could reduce or eliminate the occurrence of FIIR and increase available energy and proteins for growth.

The objective of the current field study was to evaluate the effects of β-mannanase supplementation of grow-finishing diets with a reduced net energy content of 45 kcal/kg NE of feed containing a high level of β-mannans on grow-finishing pig performance, carcass quality, and economic parameters during the grow-finishing phase.

Materials and Methods

Description of Experimental Farm

The field trial was conducted on a conventional 400-pig grow-finishing unit in Belgium. The field study consisted of 7 Control batches and 2 Enzyme-treated batches that were enrolled between April 2023 and November 2024. A total of 3111 grow-finishing pigs were included in the study, of which 2335 grow-finishing pigs were assigned to the Control group and 776 grower pigs to the Enzyme-treated group. Each pen housed 11-12 grower pigs. Compartments were ventilated through mechanical ventilation with an air inlet through side walls. All pens were equipped with partially slatted concrete floors, dry feeders, and water was distributed through nipples in the feeders. Meal feed consumption was registered at group level. Both study groups were randomly allocated in time, eliminating potential seasonal effects throughout the study. No other changes were implemented during the period of the field study that might have impacted on the grow-finishing performance.

Experimental Design

Treatment Groups

At the start of the grow-finishing period, the entire batch of pigs was assigned to one of the treatment groups: Control or Enzyme-treated, respectively. A four-phase diet was distributed. The specific treatment was blinded to the farm personnel and only registered by the nutritionist at the feed mill. Grow-finishing pigs in the entire barn were considered one experimental unit and were weighed together at the start and end of the study period.

Experimental Diets

The pigs were fed a four-phase diet consisting of Phase 1 (0-30 days), Phase 2 (31-60 days), Phase 3 (61-90 days), and Phase 4 (91-130 days) in each of the treatment groups. The main difference between the diets in the Control and Enzyme-treated groups was the reduction in net energy content of 45 kcal/kg NE of feed in all phases (Table 1). The Enzyme-treated group was supplemented with a β-mannanase enzyme (Hemicell HT; Elanco, Indianapolis; IN) at an inclusion rate of 300 g per tonne of feed, according to the manufacturer’s instructions for use. All other enzymes (xylanase and phytase) in the diets remained at the same levels in both study groups.

Table 1: Feed formulation (expressed as % of total feed) of the different phases (Phase 1-2-3-4) for Control and Enzyme-treated diets with a 63 kcal/kg NE reduction in Enzyme-treated diets. All major feed ingredients in both treatment groups are mentioned. Premix composition is identical in both treatment groups. β-mannan content (expressed as %), proportion of diet that was taken into account for calculation of β-mannan content (expressed as %), and net energy content (calculation 2015; expressed as kcal/kg feed) is given for each of the feed formulations.

 

Phase 1 (day 1-30) Phase 2 (31-60) Phase 3 (day 61-90) Phase 4 (day 91-130)
Component Control Enzyme Control Enzyme Control Enzyme Control

Enzyme

Wheat

34.95

25.34 36.72 34.36 38.33 27.27 39.86 37.32

Barley

17.50 23.00 17.50 17.50 17.50 17.50 18.00

17.50

Corn

7.50

10.00 10.00 10.00 5.00 12.50 5.00

7.50

Wheat bran

6.00

6.00 6.00 6.00 6.00 8.00 6.00

8.00

Corn gluten

4.30

5.00 1.50 6.00 3.00 6.00 4.00

6.00

Biscuit by-product

4.00

4.00 4.00 4.00 3.00 4.00 4.00

4.00

Palm kernel meal

3.00

4.00 4.00 2.60 4.00 2.80 3.90

5.00

Corn by-product

3.00

3.00 . . 2.50 3.00 1.50

3.00

Swine fat

1.00

1.29 1.49 1.51 1.53 1.13 1.49

1.36

CaCO3

1.09

1.21 0.92 0.94 0.80 0.84 0.84

0.89

Piglet feed core

0.50

0.50 . . . . .

.

L-lysin HCl

0.45

0.45 . 0.47 0.46 0.50 0.46

0.45

NaCl

0.37

0.38 0.37 0.33 0.34 0.33 0.24

0.36

CaPO4

0.20

0.20 0.10 0.10 . . .

.

L-threonine

0.19

0.18 0.17 0.17 0.17 0.18 0.15

0.16

Liquid betaine

0.17

0.17 0.17 0.17 0.27 0.17 0.27

0.27

NaHCO3

0.10

0.15 0.10 0.15 0.15 0.15 0.28

0.17

Hydroxymethionine 88

0.14

0.15 0.10 0.10 0.09 0.09 0.07

0.07

L-tryptofaan 25%

0.09

0.09 0.07 0.06 0.02 0.06 0.01

0.01

Enzyme mix 10

0.05

0.05 0.05 0.05 0.05 0.05 0.05

0.05

Soy products

15.40

14.80 9.50 9.00 6.40 4.90 2.00

3.40

Rapeseed meal

.

. 3.00 3.00 6.00 5.00 6.00

3.20

Beet pulp

.

. . . . . 1.50

1.50

Sun flower pellets

.

. 3.00 3.00 2.69 5.00 3.87

3.26

Hemicell HT

.

0.03 . 0.03 . 0.03 .

0.03

β-mannan content (%)

0.511

0.581 0.552 0471 0.559 0.466 0.568

0.628

Proportion (%)

88.65

88.14 89.82 91.46 88.92 88.97 94.13

93.08

Net energy 2015 (kcal)

2,427

2,453* 2,412 2,405* 2,388 2,420* 2,389

2,412*

*Supplementation of Hemicell HT was incorporated into the feed net energy calculation for a total value of 63 kcal per kg feed.

Experimental Animals

DanBred * Belgian Piétrain grow-finishing pigs were obtained from a conventional commercial sow farm linked to the grow-finishing facility. The pigs were vaccinated to protect against Mycoplasma hyopneumoniae, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and Porcine Circovirus type 2 (PCV-2). A study batch consisted of approximately 389 (± 1) grow-finishing pigs in each consecutive trial period.

Performance Data Collection

Pig body weight was measured at group level at 0 and 130 days. Feed provision (ad libitum) was recorded at treatment group level. Average daily weight gain (ADWG; expressed as g/d), average daily feed intake (ADFI; expressed as g/d) and feed conversion rate (FCR; expressed as kg feed per kg of weight gain) were calculated for the entire study period. Mortality was recorded along with the date of death and the number of dead animals.

Slaughter Parameters

The economically important slaughter parameters were collected from the slaughterhouse carcass database and consisted of the number of underweight (small) pigs, carcass rentability (%), basic price per kg, meat percentage (%), backfat depth (mm), loin depth (mm), AFI (quality index), proportion of pig with a rentability above 82%, quality supplement, and weight supplement.

Veterinary Treatments

Individual antibiotic treatments were administered as needed due to the critical state of the piglets and in case of a broader health issue in the barn, group treatment could be administered. The same veterinary products and dosages (ml/kg) were used throughout the entire study period. Individual antibiotic treatments or group treatments were recorded daily including the date, product, dose, ID number of treated piglets, presumed cause of treatment, and the number of times the treatment was repeated.

Economic Benefit per Piglet and per Tonne of Feed

The economic benefit of β-mannanase supplementation combined with a reduction in net energy of approximately 45 kcal/kg NE feed was calculated both at the grow-finishing pig level and at the feed cost level. For the calculation of economic benefit at the grow-finishing pig level, the following parameters were considered: feed cost reduction, pig price correction (standard price for 130 kg slaughter pig), and opportunity costs of mortality. For the calculation of economic benefit at the feed cost level, the following parameters were considered: total feed cost and the total amount of feed consumed.

Data Management and Statistical Analysis

Data was hand-recorded by the farm personnel and stored in MS Excel on OneDrive at the end of each day. Following the end of the feed trial, the data were extracted from Excel into JMP 17.0 and the blinded treatments were unblinded to reveal the respective treatment groups. Calculations, exploratory data analysis and quality review, and subsequent statistical analysis were all performed in JMP 17.0. All data were presented as a means with their respective pooled standard error of the mean (SEM). All means were tested for significant differences (P < 0.05) using a T-test.

Results

Piglet Weight

Data on weight and days in fattening are given in Table 2. The pigs arrived at the grow-finishing facility at an average weight of 22.41 kg. There were no significant differences (P > 0.05) observed in the start weight (d0) between both treatment groups. At slaughter (approx. day 130) pigs in the Enzyme-treated group were significantly (P < 0.05) heavier as compared to the Control group (130.5 kg ± 0.9 kg vs. 124.5 ± 2.3 kg, respectively). However, grow-finishing pigs in the Control group were slaughtered on average 6 days earlier as compared to the pigs in the Enzyme-treated group (126 d ± 2.3 vs. 132 d ± 2.1, respectively).

Table 2: Performance parameters and slaughter data for both Control and Enzyme-treated groups with a 63 kcal/kg NE reduction in Enzyme-treated diets. Continuous data are given as mean ± SEM. P-values < 0.05 represent statistically significant differences.

Parameter

Control Hemicell HT

P-value

Descriptive parameters

 

 

 

Number of groups

6

2

Average number of pigs per group – start

389

388

Average number of pigs per group

379

374

       
Production parameters      
Mortality (#)

10 ± 2.4

14 ± 8.0

0.347

Mortality (%)

2.5 ± 0.6

3.6 ± 2.1

0.346

Days in fattening

126 ± 2.3

132 ± 2.1

0.197

Average weight at start (kg)

22.33 ± 0.34

22.50 ± 0.00

0.316

Average weight at slaughter (kg)

124.5 ± 2.3

130.5 ± 0.9

0.018

Average daily weight gain (g/d)

813 ± 14

822 ± 3

0.420

ADFI (g/d)

2,135 ± 40

2,177 ± 169

0.423

FCR (kg feed/kg growth)

2.626 ± 0.028

2.643 ± 0.093

0.441

Cost per kg growth (€)

0.877 ± 0.053

0.828 ± 0.034

0.235

Slaugher parameters      
Number of small pigs

0.33 ± 0.33

0.50 ± 0.50

0.404

Rentability (%)

80.7 ± 0.5

80.8 ± 0.6

0.456

Basic price (€/kg)

1.79 ± 0.05

1.83 ± 0.04

0.320

Meat (%)

64.2 ± 0.3

64.6 ± 0.8

0.362

Backfat depth (mm)

12.7 ± 0.2

12.1 ± 0.1

0.121

Loin depth (mm)

73.6 ± 0.6

73.7 ± 1.3

0.461

AFI

3.2 ± 0.1

3.1 ± 0.1

0.293

Supplement >82%

6.4 ± 1.0

5.8 ± 1.1

0.336

Quality supplement

0.0270 ± 0.0034

0.0298 ± 0.0034

0.301

Weight supplement

-0.0066 ± 0.0031

-0.0162 ± 0.0029

0.045

Average Daily Weight Gain, Average Daily Feed Intake and Feed Conversion Rate

Data on ADWG, ADFI, and FCR are given in Table 2. Average daily weight gain was slightly, but not significantly (P > 0.05) higher in the Enzyme-treated group as compared to the Control group (822 ± 3 g/d vs. 813 ± 14 g/d, respectively). Average daily feed intake was slightly, but not significantly (P > 0.05) higher in the Enzyme-treated group as compared to the Control group (2,177 ± 196 g/d vs. 2,135 ± 40 g/d, respectively). Feed conversion rate was slightly, but not significantly (P > 0.05) higher in the Enzyme-treated group as compared to the Control group (2.643 ± 0.043 kg feed/kg gain vs. 2.626 ± 0.028 kg feed/kg gain, respectively).

Antimicrobial Treatment

No significant differences were observed at either the level of individual treatment or at the level of group treatment between both treatment groups during both feed trials.

Mortality

Data on mortality are given in Table 2. Overall, mortality was slightly, but not significantly (P > 0.05) higher in the Enzyme-treated group as compared to the Control group (3.6 % vs. 2.5 %, respectively).

Slaughter Parameters

Overall, slaughter parameters were not significantly (P > 0.05) different between both treatment groups. Only the weight supplement was significantly (P < 0.05) lower in the Enzyme-treated group as compared to the Control group (- 0.0162 ± 0.0029 vs. – 0.0066 ± 00.31, respectively). This might be the result of the average higher slaughter weight in the Enzyme-treated group.

Economic Benefit per Piglet and per Tonne of Feed

The detailed calculation of economic benefit per grow-finishing pig is given in Table 3. Overall, supplementation of a β-mannanase enzyme combined with a reduction of net energy by 45 kcal/kg feed over the four phases resulted in an economic benefit per piglet of € 5.24. The detailed calculation of economic benefit per tonne of feed is given in Table 4. Overall, supplementation of a β-mannanase enzyme resulted in a feed cost reduction of € 2.38 per tonne of feed.

Table 3: Detailed calculation of economic benefit per piglet considering a reduction in feed cost, piglet price corrections (130 kg; % slaughter rentability) and the opportunity cost of mortality for a reduction of 63 kcal/kg NE in Enzyme-treated diets.

Parameter

Control

Hemicell HT

Feed cost per fattening pig (0-130 d)

€ 92.18

€ 88.24

Benefit feed cost reduction

+€ 3.94

Pig price corrections (130 kg, % rentability)

 € 180.44

€ 182.38

Benefit technical results

+€ 1.94

Mortality (#)

5

6

Total opportunity cost due to mortality (€)

€ 1,170

€ 1,410

Opportunity cost per marketed piglet (€/piglet)

€ 3.05

€ 3.69

Benefits mortality

– € 0.64

Overall benefit per piglet  

+€ 5.24

Table 4: Detailed calculation of economic benefit of feed cost per tonne of feed considering total feed costs and total amount of feed consumed for a reduction of 63 kcal/kg NE in Enzyme-treated diets.

Parameter

Control

Hemicell HT

Total feed costs (0-130 d)

€ 30,843

€ 27,667

Total amount of feed consumed (tonne)

110.244

99.742

Feed cost per unit (€/tonne)

€ 279.77

€ 277.39

Overall benefit per tonne of feed

– € 2.38

Discussion

In current field trial, involving the 45 kcal/kg NE reduction, the β-mannan content ranged from 0.466% to 0.628% in all four phases. This β-mannan content was sufficiently high to maintain the standard feed composition without the need for additional protein substitutions as previously reported [15]. Since high levels of β-mannans are known to be an antinutritive factor [2], this may stimulate an innate immune response mainly due to their resemblance with PAMPs [5]. This innate immune response, known as FIIR [9] can lead to unnecessary immune activation, causing energy and nutrients to be wasted [3]. Therefore, 300 g/tonne of an exogenous β-mannanase enzyme (Hemicell HT; Elanco, Greenfield, IA) was added to the diet to hydrolyze these antinutritive β-mannans. The overall results demonstrated no significant differences between treatment groups in the measured (pig weight, feed provision) or calculated (ADWG, ADFI, FCR) performance parameters. Despite some minor numerical differences, the overall result confirmed that the addition of an exogenous β-mannanase to adapted formulations, with a reduction in net energy content of 45 kcal/kg of feed and the presence of a sufficient level of β-mannans, allowed grow-finishing pigs to perform equally to those fed the standard Control diets. These results are consistent with other recent studies in low- and high-mannan diets [10,15-18]. Slaughter data, including carcass quality parameters and economically important traits, did not differ significantly between treatment groups. These observations are in accordance with another study with inclusion of β-mannanase [16].

In addition to similar results in production performance, a substantial economic benefit of supplementation of a β-mannanase enzyme could be calculated. Based on the actual feed prices and measured feed intake, we obtained a 4.27% reduction in the feed cost (€ 88.24 vs. € 92.18 in the Enzyme-treated vs. the Control group, respectively) per grow-finishing pig produced and a 0.85% reduction in feed cost per tonne of feed (€ 277.39 vs. € 279.77, in Enzyme-treated vs. Control group, respectively). Considering all costs (feed cost, pig price correction at 130 kg, and opportunity costs for mortality) the income per produced piglet was € 5.24 higher for the Enzyme-treated group. Others concluded that β-mannanase improved growth performance in both weanling and grow-finishing pigs on corn-SBM diets [11-13]. A diet with a 150 kcal/kg reduction in digestible energy supplemented with β-mannanase outperformed in weight gain and feed efficiency [11].

Overall, the results from the current grow-finishing trial demonstrated that in the presence of a sufficient amount of β-mannans in the diet formulations, the addition of a β-mannanase enzyme (Hemicell HT; Elanco) could support grow-finishing pig performances under field conditions with formulations adapted towards net energy reduction. The adapted diet formulation resulted in improved economic benefits at the individual grow-finishing pig level and the cost per tonne of feed level.

Conclusions

The current trial demonstrated that the inclusion of Hemicell HT in reformulated diets with a lower energy content (45 kcal /kg NE) was able to retain production performance in grow-finishing pigs with an economic benefit. The inclusion of Hemicell HT provided an overall benefit of € 5.24 per piglet and € 2.38 per tonne of feed attributable to the 45 kcal/kg NE reduction. There was no significant difference in the overall quality of the slaughtered pigs.

Declarations

Ethics Approval and Consent to Participate

Field trial with an EFSA approved feed supplement for use in swine. No additional ethical approval was needed. Consent to participate was obtained following full information of the farmer on the protocol to be carried out.

Availability of Data and Material

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Competing Interests

The authors declare that they have no other competing interests.

Funding

The study was funded by Elanco Animal Health.

Author’s Contributions

FV and AdB were both involved in study design, data collection, data analysis and manuscript preparation. Both authors read and approved the final manuscript.

Acknowledgements

The authors greatly acknowledge the swine farmer for his participation in the study.

Author’s Information

FV is currently a Principal Technical Advisor Swine & Nutritional Health for Benelux / UK&ROI within Elanco Animal Health. He holds a DVM, a Master in Veterinary Public Health and Food Safety, a PhD in Veterinary Sciences, a PhD in Applied Biological Sciences, an EBVSTM European Specialist in Porcine Health Management and a Diplomate of the American Board of Veterinary Practitioners – Swine Health Management. He has an interest in swine intestinal health and specific approaches to improve intestinal health through non-antibiotic solutions.

Abbreviations

ADFI: Average Daily Feed Intake; ADWG: Average Daily Weight Gain; FCR: Feed Conversion Rate; NSP: Non-Starch Polysaccharide; PCV-2: Porcine Circo Virus – type 2; PRRSV: Porcine Reproductive and Respiratory Syndrome Virus; SBM: Soybean Meal

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Real Time, Sequential and Semantic-Neuropsychological Analysis of Verb Fluency

DOI: 10.31038/PSYJ.2025724

Abstract

A real time semantic neuropsychological approach is proposed for the study and classification of verbs, during fluency testing. The Audio-recording of 100 young university students (equal male-female ratio) has been analyzed. A Second-by-second performance has been registered and analyzed for each participant. The database consisted of more than 2000 verbs. Results: the verb output of this sample was organized in four semantic dimensions motor actions verbs (MAV), psychological, emotional, and abstract. The real time sequential analysis indicated an initial/high production of MAV (particularly whole-body actions). Overall, MAV were the most produced, followed by psychological verbs. Specific analysis for time range (each 10 seconds) indicates several semantic-neuropsychological organization characteristics along the 60 seconds’ range. This approach provides not only more parameters, but most important, more precise parameters for neuropsychological analysis; in addition, it has the advantage of being easy to replicate in clinical conditions.

Introduction

Verb fluency tests (VFT) have been increasingly used during the past years, not only because verbs are harder to retrieve than names [1]; but mainly because verbs provide more complex psycholinguistic and neuropsychological properties [2,3]. Although positive and significant correlation may be found on the performance at different fluency tests (i.e., phonological, semantic, verbs), low to moderate association indicates that the performance depends on different cognitive abilities [4].

Linguistic and psycholinguistic analysis has produced different approaches to verbs analysis and classification criteria, however the highly complex features of verbs and the syntaxes template in which verbs are contextualized, have produced a significant debate [5,6]. Based on a neuropsychological point of view, a semantic approach has been proposed by several authors [7-10]. In particular, the embodied cognition framework makes emphasis on motor and tool-use verbs [11]. According to our review of the literature, four semantic dimensions may be properly identified: motor-action, psychological, abstract, and emotional.

Motor-Action Verbs

Initial studies with functional magnetic resonance imaging (fMRI) and healthy participants, found that different brain networks were activated in the motor and premotor areas when specific motor-verbs are processed: “leg/foot” areas for verbs like Kick, “arm/hand” areas for verbs like Pick and “mouth” areas for Lick [12]. More recent studies have found a higher specificity within motor/premotor regions activation, for example: grasping vs reaching [13-15]. Semantic similarity judgements also elicit specific activations: running verbs preferentially activates the dorsal precentral gyrus (bilaterally); speaking verbs preferentially activates Brodmann area 45; hitting verbs activates bilaterally the dorsal precentral gyrus Péran et al. [16] found that action-verbs and motor representation of the same actions activate a common fronto-parietal network, and in general an increment in activation for concrete (motor) verbs versus concrete nouns, at the central and precentral motor cortex is present [17]. A difference between low specific (whole-body) versus highly specific verbs (actions performed by a specific part of the body), has been proposed and used by Herrera et al. and Roberts et al. [18] in Parkinson disease studies.

A particular subtype of MAV is the instrumental subtype; these verbs describe the utilization of a tool/object as an instrument. In recent years, the study of tools utilization phenomena has been identified as action semantics [19,20]. Manipulable man-made-objects elicit significant activation in the parietal cortex, particularly the supramarginal gyrus [21]. Tool use verbs activate a distributed network along parietal, temporal and frontal regions. Yang et al., [22] found that, in contrast to hand actions per se, tool-hand actions elicit stronger activity in left superior parietal lobule, left middle frontal gyrus and left posterior middle temporal gyrus and greater connectivity among the same brain areas. At present time, the instrumental dimension of actions represents one of the most important fields in cognitive neuroscience [23].

Psych Verbs

Refers to mental states [24], providing an important link between theory of mind and language processing [25]. Psychological process and mental states are the core of human psychology: thinking, planning, learning, etc. are every day cognitive actions. Despite that psych verbs present a wide variety in argument structure patterns, many of them allow the alternation between the position of the inanimate and agent-like animate stimuli: upset, fascinate, surprise. These verbs may express the experiencer argument either as a subject or as an object [26]. Brennan and Pylkkänen studied in healthy participants the coercion effect (a cognitive mechanism that enriches or specifies the meaning), and the lexical semantic complexity for psych verbs: distrusted, annoyed. The authors reported a significant ventral-medial prefrontal cortex and anterior temporal lobe activation.

Emotional Verbs

Several limbic (anterior cingulate gyrus, insula and basal ganglia) anterior temporal lobe and orbito-frontal areas are involved in emotion word processing in healthy population [27,28]. Emotion vs motion verbs elicit more activity in the anterior middle temporal lobe [29]. Interestingly frontal-motor areas present greater activation when processing action-words with emotional content; consequently, a close relation between limbic and motor-expression of emotions has been proposed conceptualizing them as “emotional-actions” -kiss, cuddle- [30].

By EEG studies it’s been found that access to emotional content is faster than lexical (neutral content) access [31], one of the main lexical signatures is the early posterior negativity (EPN). This effect is earlier for nouns and adjectives than for verbs; also, verbs produce longer latencies of EPN [32]. Palazova et al. [33] found an EPN onset at 250 milliseconds post-stimulus for concrete emotional verbs; for abstract emotional verbs the onset was 50 milliseconds later. The emotional valance is processed previously to the semantic features.

Abstract versus Concrete Dimension

Initial studies in this arena have explored the abstract vs concrete property of verbs. Main findings indicate that highly concrete verbs preferentially activate sensorimotor networks (left lateral precentral gyrus, and inferior parietal cortex), while purely-abstracts verbs do not. In general, literature reports a tendency to more sensorimotor activation for more concrete verbs, and less sensorimotor activation for more abstract verbs [34,35]. More recent findings indicate that the anterior temporal lobe (mainly left hemisphere) is highly active when processing abstract verbs [36]

Real Time Analysis

For most fluency tests the main scoring criteria is the number of items (verbs) produced in one minute; in addition, in most procedures the registry is made by pencil (the examiner writes –in hurry- the subject’s production). Some testing procedures divide the registry every 15 seconds [37]. This procedure allows a higher sensibility to neuropsychological dysfunction in several pathologies such as Alzheimer [38]. However theoretical justification for the 15 seconds division has not been provided and usually the main score is the total number of verbs produced in the 15 second range (accumulative criteria), nor the type of verbs produced, nor the item-sequence (type of item) of the production. In addition, the great majority of VFT studies do not use audio-recording; the main limitation for writing to dictation is that, during the first 10-15 seconds subjects produces many items in a very fast manner. Consequently, the examiner has no time enough to write the production appropriately, thus, most examiners even prompt the subjects to “slow down” so he/she can write properly the production; some participant slow-down the pace of production to wait for the examiner to write.

Two general phases have been identified: 1) Selection (In the firsts 15-20 seconds) when items are highly available, automatically activated and abundantly produced; 2) Retrieval (seconds 20 to 60) when items are scarcely available, a more active/effortful search is required to produce each item [39,40]. At the present time, no real time registry (e.g. audio recording) validated to identify each phase, as the main testing procedure

Semantic Competition

When the phenomenon of co-activation is present: when subjects prepare to talk, and more specifically to perform VFT, semantic representations (names, verbs, etc.) are abundantly pre-activated and ready to be selected and produced, making the neurocognitive process efficient [41]; this phenomenon creates a competition between the multiple automatically activated representations [42]. In healthy participants retrieval always occurs after the initial abundant item production. What types of verbs are initially available? Is there any type of sequential (real time) semantic production during verbs fluency? These questions guided this research.

Here we propose that, additional to the semantic neuropsychological approach, by using audio-recording of VFT at least three four new neuropsychological criteria may be obtained:

  1. Semantic organization of the overall verb production
  2. Semantic analysis for the selectivity phase (abundant production of verbs)
  3. Semantic analysis for the retrieval phase (paced production of items)
  4. Sequential real time (second by second) semantic analysis of the output

Method

Participants. A hundred young healthy university students (equal male-female ratio), from three different universities/cities, with no neurological or psychiatric history, and with normal-average academic performance, participated in this study. Mean age was 21.97 (E.D. 1.74) years old, and the mean of school years: 16.10 (E.D. 2.34). Participation in the study was voluntary, and all the participants consciously agreed to be audio-recorded. Bilingual people (high proficiency) were excluded. Evaluation was done in individual form and in adequate private conditions. After audio-record setting was prepared, these instructions were given to each participant: “you must say as much verbs as you can, in one minute”. To compare the curvilineal performance (selection/retrieval), a semantic (animal) fluency task was also included; in this case, the instructions were: “you must say as much animals/verbs as you can, in one minute”. All the participants first carried out the verb fluency test and secondly the animal fluency one. A smart phone with the program audio recorder (version 5.00) was used to record the verbal production of each participant.

Verbs were analyzed considering the core meaning -the main nuclear action expressed- and classified according to the semantic dimensions described in the introduction:

a) Body actions: require the whole body -or most of it-: (run, jump, walk) b) Specific actions: require only parts of the body (eat, drink, chew) c) Instrumental actions: use-manipulation of an object-instrument (drive, clean, sweep) d) Psych verbs: cognitive/psychological actions (think, learn, listen) e) Emotional actions: emotional states or emotional components (love, hate)

Abstract Dimension

Additionally, we also explored the possibility that verbs-core meaning was abstract (evolve, grant). GAP (general all-purpose) verbs were considered in this category). Verbs that do not fitted within any of these previous categories (e.g. kill, steal), were considered as not classifiable.

Data Analysis

Inter-rate agreement was established at 95%, two different analyses were already performed from our group at two different sample-analysis [43]. Complimentary to descriptive and basic statistics, a non-linear regression analysis of the performance in both verb and semantic tests was conducted. A detailed description of this procedure is presented in appendix A.

Results

To explore the executive competence (item production), we modeled the real time performance at each second. Using a dichotomist approach (0= no item produced, 1= one item produced), the curves modeled in Figure 1 show the frequency of subjects that produced one item at each second. In general, a significant fall in production competence (curvilinear decay) is present in both verb and semantic tests. Selectivity-retrieval (abundant-scarce transition) is modeled in this sample. As expected, the verb-paradigm decline is more pronounced than the semantic-paradigm, producing a different curvilinear decay. The regression analysis revealed that both curves are fitted well with the model, because the values of R squared (coefficient of determination) are sufficiently high. The initial performance value (B3) and the asymptotic value of performance (B4) do not differ between the two groups. However, the performance rate (B2) is significantly higher (p = .01) in the verb performance group (see Table 1).

Figure 1: Curvilineal modeling for the verb (solid line), and the semantic (dashed line) performance.

Table 1: Results of the regression analysis.

Group

B2 B3 B4

R squared

The verb performance

0.122±0.015

0.912±0.044 0.246±,015

0.847

Significant level, p

0.01

>0.2

>0.2

 
The semantic performance

0.077±0.007

0.934±0.027 0.247±0.014

0.932

Note: Each coefficient value is given with its standard error.

General results are presented in Table 2. The average of production was 25.44 words for animals and 22.65 words for verbs; this result coincides with the literature (at different languages), indicating that verbs are harder to retrieve than names. Average performance is presented at each 10 second. The first-time range (1-10 seconds) represents the higher abundant item production phase, with seven verbs on average, on the second range productivity falls to 4 verbs, and progressively continues to fall at each time-range; in the 1-20 second range, half of the all the verbs are already produced. Regarding the speed of production, in the 1-10 second range one verb is produced each 1.35 seconds (on average), this speed continuously decrements, thus, in the last time range a verb is produced each 4.65 seconds (in average).

Table 2: Average production of verbs by time range.

Time range

Number of verbs Average ítem-second Relative percentage

Cumulative percentage

1-10

7.37 (1.45) 1.35 32.34

32.34

11-20

4.09 (1.34) 2.44 17.95 50.29
21-30 3.40 (1.20) 2.99 14.92

65.21

31-40

3.05 (1.45) 3.27 13.38 78.59
41-50 2.73 (1.34) 3.66 11.97

90.56

51-60

2.15 (1-02) 4.56 9.44

100.0

Semantic Neuropsychological Approach

A critical question for the semantic dimension criteria was if most verbs produced felt within these above-mentioned dimensions, and most importantly: what was the percentage of verbs that are not classified by these criteria? The main finding was that more than 80% of all verbs produced were classifiable in these semantic dimensions. By analyzing the not-classifiable verbs, abstract criteria (abstract non-psych verbs) represented 10.06 %; finally non-classifiable verbs represented only 8.14 % of all verbs produced. These results are presented in Table 3.

Table 3: Semantic production by times ranges.

1-10 11-20 21-30 31-40 41-50 51-60 Total

% **

Psych Freq

210

92 71 73 64 47 548

26.07

%*

28.49

24.33 23.58 25.00 26.77

23.85

   
Specific Freq

100

75 65 70 58 58 405

19.26

%*

13.56

19.84 21.59 23.97 24.26

29.44

   
Whole-Body Freq

214

47 35 24 33 15 367

17.45

%*

29.03

12.43 11.62 8.21 13.80

7.61

   
Instrum Freq

61

56 44 52 16 25 254

12.08

%*

8.27

14.81 14.61 17.80 6.69

12.69

   
Emotional Freq

58

26 14 19 15 14 146

6.94

%*

7.86

6.87 4.65 6.50 6.27

7.10

   
Abstract Freq

38

48 30 41 38 26 211

10.06

%*

5.15

12.69 9.96 14.04 15.89

13.91

   
Not-class Freq

56

34 42 13 15 12 171

8.14

%*

7.59

8.99 13.95 4.45 6.27

6.09

   

*Percentage to all verbs produced within the time range; ** Percentage to all verbs produced.

The verb production of this sample of young-university students can be properly classified in four semantic dimensions: MAV, psych verbs, abstract and emotional. Overall, the most frequently produced type of verbs was MAV (considering all three subtypes), followed by psych verbs (think, study, read); the less frequent verbs were abstract (to grant, transform, consume), and emotional (laugh, cry, love).

Results for the Selectivity (Abundant) Phase

By dividing whole-body versus specific motor-action, allowing us to identify two different types of production (see Figure 2). In the 1-10 second range, whole-body actions verbs are the dominant dimension, mainly in the first 3 seconds, then a dramatic fall is presented. Specific actions verbs were produced in more discrete form, and like psych verbs follows a wave form of availability: slowly arising until reaching the highest value. The whole-body dimension presents a particularity: immediate and maximum availability, but also an immediate dramatic fall in production.

Figure 2: Real time production for the most frequent dimensions (range: 1-10 seconds).

By analyzing the frequency of production within the first 20 seconds (see Figure 3), results indicate that during the abundant phase of the production, the semantic dimensions present different types of production. Whole-body and psych verbs are the only types of verbs that present a fast and high initial production (during the 10 seconds range), and both dimensions fall dramatically in their production. A more discreet pattern of production was presented by the instrumental verbs (clean, sweep, drive). Emotional and abstract verbs are situated at the bottom of the production.

Figure 3: All types of verbs produced by times range, a) frequency, b) relative percentage.

The dynamic semantic production for the whole 60 seconds is presented in Figure 3; results are divided in six intervals -10 seconds range-. Whole-body actions verbs continuously fall until the fourth interval (40´s seconds interval), where a plateau is reached. Psych verbs are the second (of only two) semantic abundant categories in the initial interval (mainly first five seconds); presenting a dramatic fall for the second interval, then showing a relative stable frequency of production from the third to the fifth interval. Specific actions verbs present a discrete decrement from the first to the third interval. These three principal semantic dimensions present a different productivity pattern along the six intervals.

Relative Percentage

The relative percentage within categories is presented in Table 3 and Figure 3b. This form of analysis illustrates that psych and specific actions verbs are the most frequently produced verbs along the different time ranges. The dynamic relative percentage of the production for each type of verb indicates that psych and specific-body verbs, remains on the top of the production. This value for the whole-body action verbs shows a less dramatic fall (see Figure 3a), indicating a more stable presence all through the 60 seconds. Instrumental verbs present an increment until the third interval, with a dramatic fall. Emotional verbs production presents discrete changes; the abstract dimension presents a similar behavior than the instrumental dimension, although with less dramatic changes observed.

Except for abstract verbs, all five semantic dimensions present the highest value at the first interval, two of them present a dramatic fall in the production (whole-body and psych), the other three only a discrete fall.

Item Fluency versus Semantic Fluency

Considering the relative percentage of the fluency performance -all items produced- (Figure 4); interesting results can be noticed: although overall item fluency is diminishing during the abundant-scarce transition, the relative semantic fluency is maintained on top of the relative item fluency. This is evident for Psych and specific actions verbs, and more discretely for instrumental verbs (-a- figure). If both whole and specific motor actions are considered together (-b- figure), this effect is more evident.

As a final note it is important to state that participants also produced verbs of several different semantic dimensions; however, the relative percentage of these items was lower than 2%.

Figure 4: Comparison of overall fluency versus semantic dimensions (relative percentages).

Discussion

To our knowledge, this is the first real-time semantic-neuropsychological analysis of verbs fluency. This novel approach may represent promising cognitive and neuropsychological evaluation procedures.

Curvilinear modelling confirms significant differences in performance when comparing with the lexical-semantic paradigm. The semantics networks-correlates identified in the literature by functional neuroimaging and neuropsychological evidence, allows us to classify the great majority of verbs produced; and by identifying the abstract category, only 8% of the verbs were not-classified. However, considering that neuropsychological correlations for the abstract dimension are less clear, our proposal permits us to classify up to 82% of the verbs produced, in four semantic dimensions (or semantic templates).

In the very first seconds, whole-body-movements verbs are fast and abundantly produced, in contrast, the production of specific body-part-actions verbs arises slowly; this cognitive data differentiation parallels the functional neuroimaging evidence, indicating different brain networks [44]. However, in contrast to the functional-neuroimaging evidence, our results reflect that both types of verbs became lexically available in different forms. Our data reflects (for the first time in literature) different real-time production characteristics (frequency and sequence) between whole-body and specific body action verbs.

Why Whole-body verbs are so abundantly available at the first seconds? The great majority of these verbs are intransitive: they do not require a second agent-object to be performed; this grammatical simplicity may represent an additional factor . Another (complimentary) explanation is that every day whole-body actions are highly frequent for very-young active students, and the cognitive limitation of the testing condition: “only verbs in a limited time”, may provoke that the motor-action system activates highly available representations.

In several cognitive studies participants recall verb phrases better if they perform the physical actions described; the effect is also achieved if participants are asked to order phrases by the higher frequency of performance [45]. The imageability of the action described is also a priming factor in verb processing [46]; fictive motion comprehension (figurative motion processing: “my mind runs”) activates primary visual areas and motion sensitive visual areas [47]. Future studies may indicate if the verbs run, swim, walk, etc. present also higher imageability.

Why then are these verbs not the most overall produced? There are not many whole-body actions (or verbs), then as easy as they may be to produce, the semantic bank of this type of verb has little exemplars.

If all-motor verbs are considered together, this is the dominant semantic template; Bayram and Akbostanci [48] found that a normative group with a mean of 56 years old, produced more motor actions verbs than non-action verbs. This type of data suggests that in normative samples, motor verbs are the most type of verbs produced. We are currently finishing a report with a sample close to 200 participants from two different countries (same language) that show the relevance of the MAV production in normative samples as a preclinical neuropsychological marker, due that clinical research suggests that the presence of motor verbs may reflect healthy sensorimotor (frontal-striatal) networks (Chávez-Oliveros et al., 2023; Herrera et al. 2012; Roberts, 2015).

Specific Action Verbs

Production is more discreet at the beginning, but with constant relative production throughout the 60-second period. Differential and specific network-activation has been widely documented between specific body-part actions in healthy populations and, in brain damage patients [49]. Our data provides concrete cognitive evidence for the study of normative samples that support a methodology already used to differentiate specific versus non-specific motor/actions verbs in Parkinson disease patients.

Instrumental Verbs

This type of verb requires the most distributed neuropsychological networks along the frontal, temporal and parietal cortex [50]. Due to the complexity of the semantic-instrumental cognitive networks a higher semantic competence-processing may be required [51]. We recently reported that among the MAV the instrumental subcategory was the most affected in a sample of Parkinson disease patients.

Psych Verbs

Representing the second predominant semantic template production. Object-experiencer verbs are more cognitively demanding to produce than subject-experiencer verbs; actions that refers to concrete events requires specific semantic processing, due to their shared perceptual features for example jump versus hop, (therefore higher semantic-competence), subjective actions don’t [52]. Then, cognitive retrieval of this type of verbs may be easier than other verbs that describe concrete actions; brain correlates of psych verbs are scarce, but indicative of theory of mind networks. As with the motor division, separating abstract and emotional verbs, from subject-agency verbs, shows different initial availability and overall production. Further, a more precise analysis of subtypes of psych verbs may indicate intra-dimensional characteristics, for example the different cognitive properties (factive, negative representational, fictive representational, etc.). The factive dimension indicates the degree of certainty: “know” (I know= highly certain) versus “think”; non factive verbs denote only an intention: “promise”. Up to date a clear proposal for an intra-dimension analysis of psych verbs is not available.

Emotional Verbs

Representing the lowest frequency of verbs produced, networks for these verbs are highly constrained to limbic and paralimbic regions; therefore, the production of emotional verbs is more cognitive demanding than psych verbs. Traditionally considered as “psych” verbs, the data presented here indicates different frequency and sequence in the production characteristics than psych (non-emotional) verbs. Furthermore, as with motor-action verbs a differentiation within semantic categories (emotional vs non-emotional), suggest within brain-networks differentiation during fluency testing.

Some proposals have been developed to rate the type of emotion expressed -for example: basic versus secondary emotions-, and the frequency, valence, arousal, etc. When participants are directed to produce actions words that relate to basic emotions Cry and Laugh are among the top three most frequently generated action words for each emotion [53]. In our results, Cry and Laugh were also among the most frequent emotional verbs produced. These results suggest that if induced or not, these verbs will be produced, suggesting a specific form of action-emotion semantic organization.

Abstract Verbs

It has been described that abstract-semantic associations are scarce and vague and may be difficult to perform by a semantic template. Literature is scarce in abstract-semantic verbs, the main results indicate lesser sensorial and motor areas activation , with a significant anterior-temporal participation.

Semantic Dimensions/Templates

Our data suggests that in verb-fluency performance, four neuropsychological semantic-dimensions supported in literature (brain/neuropsychological correlates) are present. These results support the “Two level theory of verb meaning,” verbs meaning have two levels: a general framework or class, representing the thematic core -for example change of state verbs-, and a more specific level representing a specific semantic meaning -melt vs shake- [54]. When facing a verb fluency test, the output is organized in semantic templates (motor, psych, emotional, abstract), these templates are mainly neuropsychological, and not cognitive or linguistic: our results suggest that in the verb-fluency paradigm, cognitive performance is driven by specific brain networks (premotor, limbic, frontal-parietal, frontal-medial), rather than by language-lexical representations.

Some categories of verbs are harder to map and select than others [55]. Our results indicate that psych verbs and specific motor action verbs are the easiest to produce, whole body-actions present a medium degree of difficulty; finally, the hardest types of verbs to produce are emotional and abstract.

Why are verbs so hard to retrieve? verbs are not taxonomically structured as names -i.e. animals- [56]. According to Earles & Kernsten , verbs exhibit context effects more like superordinate nouns; also, verbs generally have more dictionary entries and interpretations than nouns: “catch the ball versus catching a cold”. A possible explanation for the lower semantic production (instrumental, emotional and abstract) is that verbs that implicate specific dimensions are more constrained to specific actions, been lesser in number in most languages; thus, harder to find-map and indeed to be semantically produced. If the later I correct, then motor/lighter verbs will be less produced because they present conflicting semantics (“catch”); less conflicting semantic verbs (running, swim) will be easier to produce.

The relative semantic percentage across the time range indicates that the semantic production occurs in templates that are resistant to the curvilinear decay of the item production (number of items produced).

“Surfing” the semantic system? The executive participation is -only- to navigate on top of these semantic templates? The same way a surfer cannot produce a wave, and only chooses which wave to ride (selection)? Once on top of these waves, there is not much to do, but to stay in balance (inhibitory control)? Figures 3 and 4 suggest this effect, but more research on real time cognitive/neuropsychological performance (neuroimaging) is needed to confirm-expand these initial findings.

Conclusions

The results presented here indicate that a real time/semantic-neuropsychological approach is highly plausible for the neuropsychological analysis of verb fluency testing. Several new parameters represent promising future cognitive and neuropsychological criteria (that of course requires proper validation): the semantic templates/dimensions, the initial faster and higher availability of whole-body motor verbs, and the time-range relative-percentage.

A real time analysis of the VF performance is necessary to discover different neuropsychological features of executive-semantic performance. We believe that the high content of motor-action verbs may represent neuropsychological markers of a healthy brain. Disruption of these semantic-neuropsychological templates may occur in brain-impairment pathologies e.g. Parkinson, Traumatic brain injury, affecting cognitive efficiency; but of course, clinical validation is required.

The motor-action dimension for neuropsychology is fundamental, literal and figurative language is full of actions (“grasp the idea”). Literature progressively indicates the close relation of motor-language verbs/actions and the optimal functioning of fronto-striatal functioning [57-59]. Also, the high presence of psych verbs is relevant for another fundamental field on human cognition: mentalization/theory of mind, and may represent an additional neuropsychological dimension for autism disorder spectrum.

Limitations and Future Directions

A wider evaluation and replication with several hundred participants of different age-school years (from different countries-language-cultures) are required; also, different interrater agreements from different research groups are required to achieve proper validation (e.g. kappa analysis).

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Fundamental Mechanism of Qianghuo in the Treatment of Leukemia Based on Network Pharmacology and Molecular Docking Combined with Preclinical Evaluation

DOI: 10.31038/JPPR.2025812

Abstract

Background: The Qianghuo herb pair is renowned in traditional Chinese medicine (TCM) for its blood-activating and stasis-resolving properties, frequently incorporated into formulas for leukemia treatment. However, the specific relationship between the Qianghuo herb pair and leukemia, along with its underlying mechanisms, remains elusive. Based on network pharmacology and molecular docking technology, explore the relevant targets of Qianghuo and its mechanisms in treating leukemia.

Method: Diverse databases were utilized to investigate the chemical components and their respective targets of Qianghuo, alongside acquiring disease-related target information. A network relationship diagram of Qianghuo’s active ingredients, targets, and diseases was constructed using Cytoscape 3.8.2 software. Protein-protein interaction (PPI) networks were created and potential pathways were explored through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Molecular docking was utilized to assess the interactions between active ingredients and potential targets.

Result: In Qianghuo, a total of 15 active ingredients and 554 corresponding targets were obtained. The main active ingredients include Ammidin, 6’-Feruloylnodakenin, 8-geranoxy-5-methoxypsoralen, coumarin, glycoside, Demethylfuropinnarin, Bergaptin, etc. Among them, 162 targets intersect with drug-disease interactions, with AKT1, EGFR, SRC, CASP3, and HSP90AAA1 identified as core targets. Functional enrichment analysis of Gene Ontology (GO) yielded 870 entries, while Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified 154 pathways, notably including Pathways in cancer, PI3K-Akt signaling pathway, and Human papillomavirus infection. Molecular docking simulations revealed significant binding affinity between the main active ingredient of the drug and the core target proteins.

Conclusion: This study successfully elucidates the mechanism of action of Qianghuo in treating leukemia, predicts the potential targets of traditional Chinese medicine, elucidates complex signaling pathways, and demonstrates its therapeutic characteristics of multi-components, multi-targets, and multi-pathways. This provides a basis for exploring alternative approaches to researching novel leukemia drugs at the level of network pharmacology.

Keywords

Qianghuo, Leukemia, Network pharmacology, Bioinformatics, Molecular docking

Introduction

Leukemia is a malignant hematological disease with a high incidence in our country, which usually occurs in children and adolescents [1]. It is categorized into four types based on cell differentiation and disease progression: chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML). Among them, acute myeloid leukemia stands as the most prevalent, constituting approximately 70% of adult leukemias. This disease develops rapidly, with its natural course typically lasting only a few months. Studies indicate a close association between leukemia and various factors such as genetics, smoking, obesity, alcoholism, exposure to chemical agents (like benzene, formaldehyde, etc.), high-dose radiation, and contact with electromagnetic fields [2-4]. Currently, leukemia treatment methods encompass chemotherapy, immunotherapy, targeted therapy, etc. However, owing to the clonal biological traits inherent in leukemia, the cure rate remains low. Prolonged chemotherapy often induces a sharp decline in the patient’s physical resilience, potentially leading to complications such as bleeding and infection. Hence, the pursuit of novel leukemia drugs with potent efficacy at affordable costs has emerged as a focal point in drug research and development. Traditional Chinese medicine (TCM), with its notable advantages including affordability, minimal side effects, and evident therapeutic effects, has garnered attention as a promising avenue in current drug research and development [5,6].

Qianghuo documented in “Shen Nong’s Materia Medica”, consists of the dried rhizome and root of Qianghuo or Qianghuo Latifolia, belonging to the Umbelliferae family. It holds significance as one of the vital and precious medicinal resources in our country. Qianghuo is mainly composed of sugars, volatile oils, coumarin and other ingredients [7]. The synergy of these components addresses various health issues. It is reported that Qianghuo has anti-inflammatory, analgesic, antipyretic, antibacterial, anti-cancer cell proliferation, anti-arrhythmic, anti-thrombosis and promotion of brain blood circulation effects [8]. Its clinical versatility spans multiple disciplines, with applications in conditions like cancer, cardiovascular and cerebrovascular diseases, rheumatology, and immunology, among others. Despite its widespread clinical use, there remains a gap in understanding the mechanism of Qianghuo in treating leukemia.

Therefore, in order to clarify the effect and mechanism of Qianghuo in the treatment of leukemia, and to expand its clinical application, the potential targets and core active ingredients of Qianghuo anti-leukemia were screened by compound network pharmacology approach in this study. Then, the core active components and the core targets that exert anti-leukemia effects were reverse verified by molecular docking technology. Subsequently, the signaling pathway of Qianghuo in the treatment of leukemia was elucidated, and the therapeutic effect of Qianghuo in leukemia was predicted. These results provide ideas and directions for elucidating the mechanism of Qianghuo in the treatment of leukemia at the molecular level.

Materials and Methods

Software and Databases

Databases used: Drug ingredients and target acquisition database TCMSP (https://tcmspw.com/tcmsp.php), disease target acquisition database Gene Cards (https://www.genecards.org/), OMIM database (https://www.omim.org/), Therapeutic Target Database (http://db.idrblab.net/ttd); Protein information acquisition database (https://www.uniprot.org/); Gene interaction database String (https://www.string-db.org/); Intersection gene acquisition Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/index.html); Software used: Cytoscape V3.8.0 software [9], AutoDock 1.5.7 [10], Openbabel, Pymol [11].

Acquisition of Active Ingredients of Qianghuo and Screening of Targets

With the help of the TCMSP database, the corresponding active ingredients were searched using the keyword “Qianghuo”, and the two indicators of oral bioavailability (OB) ≥ 30% and drug likeness (DL) ≥ 0.18 were set as filtering conditions. Under the Related Targets column, search for target proteins related to the effective active ingredients respectively; then use the Uniprot database and Perl language to standardize the human gene names corresponding to the target proteins.

Obtaining Leukemia Target Proteins

Use OMIM, Genecards, TTD, DisGNET database and other databases, search with “leukemia” as the keyword, select the species as Homo sapiens, and obtain leukemia-related targets. The targets of the database were merged and duplicate targets were removed and corrected by Uniport as the final result of leukemia-related targets for subsequent analysis. Use the WeChat website to draw a Venn diagram of compounds and disease targets.

Construct a Nodule-active Ingredient-Target-Disease Interaction Network and Active Ingredients-Target-Pathway Network Diagram

First, we’ll match the target genes of the active ingredients of Qianghuo obtained from the TCMSP database with leukemia-related target genes. Then, using the WeChat website, we’ll draw a compound-disease target network. The intersection targets will be visualized through a Venn diagram to identify key targets specific to Qianghuo in treating leukemia. Next, we’ll submit the obtained intersection targets to the String11.0 database, setting the biological species as “Homo sapiens” to construct a protein interaction network. Finally, we’ll import the data into Cytoscape 3.7.1 for Network analysis and then screen the core targets based on degree values.

GO Function and KEGG Function Enrichment Analysis

DAVID 6.8 database was used for GO analysis, and the key targets were analyzed in the four modules of Biological process (BP), Cellular component (CC) and Molecular function (MF), and Kyot Encyclopedia of genes and genomes (KEGG). Analysis to identify key targets and enriched pathways.

Molecular Docking of Important Components and Core Targets of Qianghuo

Use the PubChem database to obtain the small molecule structure, save it in sdf format, convert the format in Openbable software, and save it in mol2 format. Download the protein structure of the relevant target through the PDB database, use Pymol software to remove water and ligands, and save it in pdb format. Finally, the database Auto Dock Vina is used to preprocess the small molecule ligands and protein receptors, and its plug-in is used to obtain docking active sites and perform molecular docking to obtain the lowest binding energy for docking between the target protein and the compound. Pymol software is used for visualize the docking results [12].

Results and Discussion

Traditional Chinese herbal medicine serves as a vital reservoir for the development of effective anticancer drugs, offering numerous active molecules with unique structures that can inspire the creation of novel therapeutics. Moreover, the rapid advancements in network pharmacology provide robust support for understanding the mechanisms underlying Chinese herbal medicine’s efficacy in cancer treatment [13]. Qianghuo, as a traditional Chinese medicinal material, has emerged as a research hotspot in recent years, and its compound preparations, action targets, pharmacological activities and other aspects have been continuously explored. Research has demonstrated that Qianghuo contains compounds with varying degrees of inhibitory effects on the proliferation of various cancer cells. This highlights its potential as a promising candidate for further exploration in anticancer drug development. Yang and co-works investigated the two major active ingredients in Qianghuo, nodol and isoimperatorin, and confirmed their inhibitory effects on the proliferation of rat fibroblast-like synovial cells [14]. Notably, the inhibitory effect of isoimperatorin was found to be superior to that of chanzolinol. Chang and co-works discovered the association of the SET domain-containing 2 (SETD2) gene with acute lymphoblastic leukemia [15]. Using this discovery as a starting point, they conducted sub-docking and molecular dynamics simulations. Their findings indicated that coniferin and coniferyl ferulate exhibit high affinity with the SETD 2 protein, demonstrating strong binding affinity and stable interaction. Importantly, coniferyl ferulate can be isolated from Rhizoctonia latifolia. Therefore, the traditional Chinese medicine compound coniferyl februate emerges as a promising lead compound with potential to develop into an anti-leukemia drug. While the aforementioned research findings offer valuable insights into the treatment of leukemia with Qianghuo, much remains to be explored regarding its active ingredients and pharmacological mechanisms. Therefore, further research in this area is necessary to elucidate these aspects and unlock the full therapeutic potential of Qianghuo in leukemia treatment.

Screening of Active Compounds and Targets of Qianghuo

Utilizing the TCMSP database, a total of 185 compounds contained in Qianghuo were collected. According to the ADME standard, which sets two parameter standards of OB≥30% and DL≥0.18, a total of 16 compounds were screened, primarily including Ammidin, 6′-Feruloylnodakenin, Bergaptin, sitosterol, etc., (Table 1 and Figure 1). Concurrently, we acquired a total of 554 targets associated with the drug ingredients. To standardize the protein target information, we utilized the UniProt database, specifically focusing on the “human” species. After merging the data, 297 duplicate values were removed.

Table 1: Information on the active ingredients of Qianghuo screened in TCMSP. OB, oral bioavailability; DL, drug-likeness.

MOL ID

Name Compound OB (%)

DL

MOL001941 QH1 Ammidin

34.55

0.22

MOL011962 QH2 6′-Feruloylnodakenin

32.02

0.67

MOL011963 QH10 8-Geranoxy-5-methoxypsoralen

40.97

0.5

MOL011968 QH11 coumarin

33.07

0.78

MOL011969 QH3 Demethylfuropinnarin

41.31

0.21

MOL011971 QH12 Diversoside

67.57

0.31

MOL011975 QH13 Notoptol

62.97

0.48

MOL001951 QH4 Bergaptin

41.73

0.42

MOL001956 QH5 Cnidilin

32.69

0.28

MOL000359 QH6 Sitosterol

36.91

0.75

MOL004792 QH14 Nodakenin

57.12

0.69

MOL000358 QH15 beta-Sitosterol

36.91

0.75

MOL001942 QH7 Isoimperatorin

45.46

0.23

MOL002644 QH8 Phellopterin

40.19

0.28

MOL002881 QH9 Diosmetin

31.14

0.27

Figure 1: Compound structural formula.

Leukemia-related Targets

Through the Genecards, TTD, and DisGNET databases, we conducted a keyword search for “leukemia”. We obtained 31 targets from the TTD database, 3507 targets from the Genecards database based on empirical screening with correlations greater than 1.5, and 2111 targets from the DisGeNET database. The targets obtained from the two databases were merged, deduplicated, corrected, and integrated using UniProt. These results constitute the final set of targets related to leukemia for subsequent analysis, totaling 4301 targets (Figure 2).

Figure 2: Venn diagram of the intersection targets between Qianghuo and leukemia.

Construction of PPI Network and Screening of Core Proteins

The intersection of the target genes related to the chemical components of Qianghuo and the leukemia target genes was obtained through Venny 2.1.0 and a venn diagram was generated. In the diagram, blue represents the target points of the active ingredients in Qianghuo, totaling 297, while red represents the targets of leukemia, totalling 4301. The overlapping area in the middle of blue and red represents the intersection target, totalling 162, as illustrated in Figure 2. The resulting 162 intersection genes were submitted to the STRING database to analyze the PPI network, as depicted in the Figure 3. The graph comprises 161 nodes and 1982 edges, with an average node degree of 24.6 and a local clustering coefficient of 0.565. Among them, according to the degree value, the top five targets are AKT1, EGFR, SRC, CASP3 and HSP90AA1, in Table 2. Similarly, the top five active ingredients in Qianghuo are Diosmetin, Cnidilin, Bergaptin, and Ammidin and Phellopterin in Table 3. The AKT1, EGFR, SRC, and CASP3 are the hub nodes of the network and can be regarded as potential key targets for the treatment of leukemia by Qianghuo. Akt, a serine/threonine kinase expressed in three isoforms (Akt1, Akt2, and Akt3), acts as the main effector of the PI3K signalling pathway. Its numerous substrates regulate various cellular processes such as protein synthesis, cell cycle progression, cell survival, apoptosis, angiogenesis [16].

Figure 3: Protein interaction map.

Table 2: The top five targets in Drug-ingredient-intersection target-disease network.

Gene

Degree

AKT1

100

EGFR

89

SRC

89

CASP3

84

HSP90AA1

81

Table 3: The top five compounds in Drug-ingredient-intersection target-disease network.

Compounds name

Degree

Diosmetin

58

Cnidilin

52

Bergaptin

43

Ammidin

41

Phellopterin

38

The PI3K-Akt-mTOR pathway, aberrantly upregulated in cancers including AML, plays a critical role in intracellular signalling [17]. Daniela Bres Sanin et al. [18] demonstrated that targeting the PI3K/Akt/mTOR pathway at different levels can effectively combat cancer cells, leading to the development of dual inhibitors that show efficacy against T-ALL cell lines and primary patient samples. The strong cytotoxic effect of targeting the PI3K/Akt/mTOR pathway demonstrates promising potential for the treatment of T-cell acute lymphoblastic leukemia (T-ALL). Epidermal growth factor receptor (EGFR), a member of the receptor tyrosine kinase (TK) family, is widely expressed in various cancers, including breast cancer, lung cancer, esophageal cancer, and head and neck cancer. EGFR and its family members play pivotal roles in complex signaling cascades that regulate tumor cell growth, differentiation, adhesion, migration, and survival. Consequently, they have emerged as promising candidates for cancer treatment [19]. SRC, a proto-oncogene tyrosine protein kinase, belongs to the non-receptor protein tyrosine kinase family [20]. This family is involved in a plethora of crucial cellular functions essential for maintaining cellular homeostasis, including cell cycle progression, motility, proliferation, differentiation, and survival. Small tyrosine kinase inhibitors have been indicated for the treatment of certain blood malignancies, including CML [21]. Zhang He et al. [22] discovered that SRC tyrosine kinase inhibitors exert an anti-tumor cell role by regulating EGFR and its downstream signaling pathways, which significantly inhibits tumor cell proliferation and infiltration. This finding provides valuable insights for the development of drugs for leukemia treatment. Based on previous research, it has demonstrated potential in leukemia treatment by influencing multiple proven effective targets. These findings suggest that these ingredients play a crucial role in the treatment of leukemia.

Construct Traditional Chinese Medicine-Active Ingredients-Target-Disease and Active Ingredients-Target-Pathway Network Diagram

Import the intersection genes into CytoScape 3.8.0 software to construct a drug-component-target-disease network diagram, as shown in Figure 4. Each goal in the diagram can correspond to one or more components, and the same component can be associated with multiple goals. This shows that Qianghuo has multi-component and multi-target properties in treating leukemia. At the same time, we also constructed a component-target-pathway network diagram, as shown in Figure 5. The top five genes by degree are shown in Table 4. The top five pathways are PI3K-Akt signaling pathway, Human papillomavirus infection, MicroRNAs in cancer and Chemical carcinog, Enesis – reactive oxygen species, Proteoglycans in cancer (Table 5). The above results indicate that the PI3K-Akt signaling pathway is one of the important ways to explore the mechanism of proteoglycans in the treatment of leukemia.

Figure 4: Construction of “herb-active ingredient-target-disease” network.

Figure 5: Construct a network diagram of Qianghuo’s active ingredients-genes-pathway.  In the figure, orange ovals represent genes, light green squares represent signaling pathways, and blue squares represent active components.

Table 4: The top five targets in Drug-ingredient-intersection target pathway network.

Gene

Degree

PIK3CA

15

PIK3CB

15

PIK3CD

15

MTOR

12

MAP2K1

11

Table 5: The top five compounds in Drug-ingredient-intersection target pathway network.

Pathway

Degree

PI3K-Akt signaling pathway

34

Human papillomavirus infection

29

MicroRNAs in cancer

26

Chemical carcinogenesis – reactive oxygen species

25

Proteoglycans in cancer

24

In the figure, the red inverted triangle represents the disease, the blue square represents the intersection target, the green ellipse represents the active ingredient of traditional Chinese medicine, and the pink triangle represents traditional Chinese medicine.

GO and KEGG Enrichment Analysis Results

To elucidate the potential mechanism of action of Qianghuo in the treatment of leukemia, the 162 common targets were imported into the DAVID database for KEGG and GO analysis. The KEGG pathway analysis revealed enrichment in 154 pathways, with prominent pathways including Pathways in cancer, PI3K-Akt signalling pathway (Figure 6), and Human papillomavirus infection (Figure 7A).

Figure 6: The PI3K-AKT signaling pathway that is the target of Qianghuo treatment of leukemia.

Concurrently, 870 GO biological processes were identified, with 152 related to molecular functions (MF). The top three significantly enriched MF terms were “protein binding,” “ATP binding,” and “protein serine/threonine/tyrosine kinase activity.” Additionally, 634 GO biological processes were linked to biological processes (BP), with the top three enriched terms being “signal transduction,” “protein autophosphorylation,” and “negative regulation of apoptotic process.” Furthermore, 84 GO terms were associated with cellular components (CC), with the top three enriched terms being “cytosol,” “cytoplasm,” and “plasma membrane” (Figure 7B). Among them, the PI3K-AKT pathway has been proven to play an important role in the occurrence and development of leukemia and is a key target for the treatment of leukemia [23]. The PI3K/AKT pathway is involved in cell growth, proliferation, and differentiation, and is often activated in cancer due to various mechanisms such as dysregulation of intrinsic components, mutations in upstream receptors, and/or abnormalities in other interacting signaling molecules [24,25]. Gan and others [26] found that overexpression of miR-217 can inhibit the abnormal activation of the PI3K/Akt pathway by targeting the expression of Akt3, thereby inhibiting cell proliferation, promoting tumor cell apoptosis, and enhancing the sensitivity of doxorubicin to AML. Veronika Ecker et al. [27] reported that the PI3K/AKT pathway can promote the oxidative metabolism pathway in CLL through mar AKTI overexpression, genetic phosphatase SH2-containing-inositol-5′- phosphatase-1 (SHIP1) deletion, or through SHIPI inhibition are activated, leading to the formation of toxic ROS levels. These events ultimately lead to the accumulation of toxic reactive oxygen species (ROS). These findings suggest that the anti-leukemia effect of Qianghuo may be related to multiple signaling pathways, including the PI3K/AKT pathway and oxidative metabolism pathway. Understanding these molecular mechanisms could provide valuable insights into Qianghuo’s efficacy in leukemia treatment.

Figure 7: GO and KEGG enrichment analysis in overlapping targets. (A) KEGG enrichment was consist of the top 10 pathways following the gene ratio. (B) The three sections from left to right were the top five main pathway bubble plots of BP, CC, MF. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; BP, biological process; CC, cellular component; MF, molecular function.

Molecular Docking Results

Molecular docking was performed for the hub active ingredients Diosmetin, Cnidilin, Bergaptin, and Ammidin with the core targets AKT1, EGFR, SRC, and CASP3 respectively. Part of the molecular docking results are shown in the Table 7, while the detailed docking results for ligands and receptors are provided in Table 6. In molecular docking, a lower the binding energy indicates a more stable conformation, and a binding energy less than negative five indicates that the ligand and receptor can spontaneously combine. The molecular docking results were consistent with the network pharmacology-related screening results, thereby further validating the reliability of the predicted targets from network pharmacology through molecular docking.

Table 6: Molecular docking visualization of the hub ingredients and hub targets of Qianghuo tablets for the treatment of leukemia.

Table 7: Molecular docking results of core targets.

Conclusion

In summary, this study used network pharmacology methods to analyze the mechanism of action of Qianghuo in the treatment of leukemia. It was found that the core active ingredients of Qianghuo in the treatment of leukemia are Cnidilin, Bergaptin, Ammidin, and Diosmetin. By stimulating the cancer signalling pathway, PI3K-Akt signalling pathway, Human cytomegalovirus infection and other pathways, it affects AKT1, EGFR, SRC, and CASP3, and other targets. It plays a role in complex signalling cascades that resist cancer cell proliferation and regulate tumor cell growth, differentiation, adhesion, and survival, thereby effectively inhibiting the infiltration of cancer cells and promoting the recovery of leukemia patients. At the same time, The targeted disease network shows that Qianghuo not only has a good therapeutic effect on cancer, but also has a good effect on other diseases such as rheumatoid, nutritional and metabolic diseases, indicating that different diseases can be treated with the same prescription. Compared with western medicine, which has severe side effects and high cost in treating leukemia, Qianghuo has absolute advantages in treating leukemia with low side effects and low price. In short, we provide an entry point into the mechanism of action of Qianghuo in the treatment of leukemia, and also provide a scientific basis for the treatment of leukemia by Qianghuo. In the next step, in vitro and in vivo experimental verification will be conducted to clarify the specific mechanism of Qianghuo in treating leukemia.

Funding

This work was supported by the Hubei Science and Technology Program (2022CFB781), the Hubei University of Science and Technology (Doctoral Startup Fund Project 170562), and Hubei Provincial Department of Education scientific research guidance project (B2022189).

Conflict of Interest

The authors have no conflicts of interest to declare.

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Effect of Moringa oleifera Aqueous Seed Extract on Growth and Nutrient Digestion in Awassi Lambs

DOI: 10.31038/IJVB.2025921

Abstract

Background: Recently, there has been increased interest in studying extracts of medicinal and aromatic plants and their use in improving animal production. Therefore, this research aimed to study the effect of aqueous extract of Moringa oleifera seeds (MSE) on growth and nutrient digestion in Awassi lambs. Sixty lambs of similar weight (23 ± 2 kg) and age (3 months ± 4 days) were divided into four groups (G1 control, G2, G3 and G4), each containing 15 lambs. They were fed a standardized concentrated diet for all groups. MSE was added at a rate of 0, 20, 40 and 60 ml/L of water.

Results: The results showed a significant improvement (P<0.05) in the final weight of the lambs, the daily and total weight gain rate, the feed conversion ratio, and the digestibility coefficients of nutrients in the two groups G3 and G4, which were given drinking water containing 40 and 60 ml/liter, compared to the control groups G1 and G2, which were treated with 20 ml/liter, and which also outperformed the control group G1.

Conclusions: Addition of moringa seed aqueous extract to drinking water improved growth rates, feed conversion, and nutrient digestibility in Awassi lambs.

Keywords

Moringa oleifera, Growth, Nutrient digestion, Awassi lambs

Introduction

Moringa oleifera (Moringa oleifera L.) is a fast-growing tree native to tropical and subtropical regions, but it can also be cultivated in arid regions, where it contributes to improving ecological balance and providing food for many organisms [1]. It is known as the “tree of life” and is sometimes called the “miracle tree” due to its high value in nutrition and alternative medicine [2]. Its leaves are rich in vitamins and minerals [3] and are an excellent source of iron, calcium, and vitamin C. They play a role in increasing energy levels and strengthening the immune system [4]. They are also an excellent source of biologically active compounds, such as polyphenols (tannins or flavonoids), in addition to containing small amounts of anti-nutritional factors, such as tannins and saponins. [5,6]. In recent years, the use of Moringa plant parts (leaves, seeds, flowers, and roots) or their extracts as feed additives in animal nutrition has been gaining popularity, improving health and productivity. An experiment replacing cottonseed meal in the diet of growing sheep with M. oleifera leaf powder (dry matter 20%) resulted in improved dry matter digestibility and increased growth rate (20%) [7]. Feeding lambs defatted M. oleifera seeds at rates of 2, 4, and 6 g of dry matter/lamb/day had no effect on feed intake, nutrient digestibility, or nitrogen levels; however, the highest daily weight gain of lambs was achieved [8]. In addition, adding moringa to the diets of Najd ewes led to a higher average daily weight gain and milk production compared to those fed alfalfa diets [9]. Some studies also found that using moringa leaves in diets led to an improvement in the feed conversion factor and growth [10], and the results of feeding lambs at 0 and 25 And 50 g/kg of moringa roots showed improvements in feed efficiency and nitrogen absorption in the body, which led to a higher average daily weight gain for these lambs [11]. Adding 4 g of moringa seeds daily to the diets of male Barki sheep resulted in increased final body weight and significantly improved daily weight gain [12]. Adding 25% Moringa oleifera leaves to sheep diets increased nutrient digestibility [13]. Studies have shown that M. oleifera extract improves the digestibility of organic matter (OM), dry matter, and neutral detergent fiber (NDF) in Nubian goats [14]. Therefore, this study aimed to investigate the effect of adding aqueous extract of Moringa oleifera seeds to the drinking water of Awassi lambs on growth and nutrient digestibility.

Materials and Methods

Animals, Treatments and Experimental Design

The study was conducted using 60 male Awassi lambs, similar in age (3 months ± 4 days) and weight (23 ± 2 kg, mean ± standard deviation), in a private sheep farm in the south of Hama city. The experimental lambs were fed a standardized concentrated ration twice daily at 7 a.m. and 4 p.m. It consisted of barley, bran, and cottonseed cake, in addition to salt, vitamins, minerals, calcium carbonate, and an antifungal as shown in Table 1.

Table 1: Ingredients and chemical composition of diet.

  Item

Feed ingredient ratios %

 
  Ingredients    
  Barley

64

 
  Bran

14

 
  Urea

0

 
  Decorticated cottonseed

18

 
  Salt

1

 
  Calcium carbonate

1

 
  Vitamins and minerals1

1.75

 
  Antifungal2

0.25

 
  Chemical composition  

Wheat straw

  Dry Matter

89.13

91

  Organic Matter

95.85

93.3

  Crude Protein

15.23

4.2

  Ether Extract

3.09

1.4

  Crude Fiber

7.39

41.5

  Ash

4.15

6.7

  Nitrogen Free Extract

66.23

46.2

  Metabolic Energy Kcal/Kg

2576

997

G1: Control group: water devoid of MSE. G2: drinking water containing 20 ml of MSE.
G3: drinking water containing 40 ml of MSE. G4: drinking water containing 60 ml of MSE
1 Each kg contained: vitamin A (2000000 IU), vitamin D3 (500000 IU), vitamin E (2500 mg), iron sulphate (5000 mg), manganese sulphate (1000 mg), copper sulphate (3000 mg), potassium iodide (100 mg), zinc sulphate (5000 mg), cobalt sulphate (50 mg), slinat sodium (100 mg), sodium chloride (25000 mg).
2Contains: Calcium propionate, Benzoic acid, Calcium formate, Citric acid, Calcium lactate, Plant essential oil extracts.

The ration was formulated to meet the nutritional requirements of lambs according to guidelines of the national research council NRC [15]. Wheat straw was offered ad libitum to the experimental groups. The experimental period lasted for 3 months, during which the lambs were divided using a completely randomized design into four treatment groups as follows = 15 lambs/treatment: The first group (G1): was given drinking water devoid of Moringa oleifera seed extract (MSE) (control group); the second group (G2): was given drinking water containing 20 ml of MSE /L of water; The third group (G3) was given drinking water containing 40 ml of MSE /L of water, and the fourth group (G4) was given drinking water containing 60 ml of MSE /L of water. The MSE -containing drinking water was provided ad libitum after the morning and evening feeds were provided.

During the experimental period, all lambs were housed in pens that met health care requirements and were numbered for easy monitoring and recording of results. They received preventive vaccinations and were treated for internal and external parasites before the start of the experiment.

Moringa Oleifera Seed Extract

Moringa seeds were obtained from private herbal shops in Hama Governorate. They were cleaned, removed from leaves and foreign matter, dried, and ground using a special mill for medicinal plants until a fine powder was obtained. 100 g of the powder was then mixed with 1000 ml of distilled water (ratio 1: 10) using an electric mixer. The mixture was then left to stand for 24 hours at room temperature. The mixture was then filtered using several layers of medical gauze to remove suspended particles. The mixture was then placed in a centrifuge (Bio-Rad, USA) at 3000 rpm for 10 minutes. The extract was then filtered using Whatman No. 101 filter paper to obtain a clear solution. The extract was then diluted with clean drinking water to obtain the daily doses given to the lambs [16].

Growth Performance

The growth performance was recorded during the period from the beginning of March until the end of June 2024, where the initial weight of the lambs was taken at the beginning of the experiment, and at the end of the experiment the final weights of the lambs were taken. During the experimental period, the amounts of feed offered and orts were recorded daily, to calculate the daily dry matter intake (DMI) by the difference between the offered and orts. The feed conversion ratio as was also calculated. as follows:

Feed conversion ratio = amount of feed consumed (g) / rate of weight gain (g)

Digestion Experiment

After the fattening experiment was completed, on the first day of July, the digestion experiment began. 16 male Awassi lambs were selected for use in the fattening experiment, with an average age of (6 months ± 4 days) and a weight of (44.5 ± 3 kg). were selected for easy collection of dung and urine. They were divided into four groups, each group containing 4 male lambs, and placed in metabolic cages measuring (1.2 × 0.75) meter. Bags dedicated to collecting dung and urine were attached with special ties, after each meal provided in order to calculate the digestibility coefficients of dry matter, crude protein, ether extract, crude fiber, organic matter, and total digestible nutrients (TDN). The offerd and refusals of the diets manure and urine samples were collected and recorded daily and for a 10-day period to calculate the digestibility coefficients for dry and organic matter, crude protein, crude fat, and crude fiber [17]. Chemical analyzes of the feed, urine and feces were conducted following the methods described in reference [17].

Statistical Analysis

All data were analyzed statistically using SPSS 26 software through One Way ANOVA at a significance level of 5%, and performed the Duncan multiple range test to assess the significance of differences between the means [18].

The mathematical model was as follows: Yij = μ + Ti + eij

Where:

Yij = Individual observation.

μ = The overall mean for the trial under consideration.

Ti = The effect of the ith treatment.

eij = Random residual error.

Results

Growth, Feed Intake and Feed Conversion Ratio

The results in Table 2 indicated a significant increase (P<0.05) in the final weight, average daily weight gain, and total weight gain of lambs in groups G3 and G4. The final live weight of lambs was 46.74 and 47.04 kg, the average total weight gain was 22.88 and 23.48 kg, and the average daily weight gain was 254.23 and 260.89 g, respectively, compared to group G2, where the final weight of lambs was 44.24 kg, the average total weight gain was 20.6 kg, and the average daily weight gain was 228.89 g/day. The control group G1, where the values ​​were 43.45 kg, 19.39 kg, and 219.22 g, respectively.

Table 2: The effect of Moringa oleifera aqueous seed extract on growth performance of lambs.

Growth performance

Experimental groups (Mean ± SD)

G1

G2 G3 G4

P-value

Initial weight (kg)

23.72 ± 0.45

23.64 ± 0.21 23.86 ± 0.24 23.56 ± 0.14

Final weight (kg)

43.45 ± 2.13c

44.24 ± 1.43b 46.74 ± 1.61a 47.04 ± 1.23a

0.03

Total weight gain (kg)

19.73 ± 6.47c

20.6 ± 8.58b 22.88 ± 7.59a 23.48 ± 7.13a

0.02

Average daily gain (g)

219.22 ± 6.32c

228.89 ± 9.89b 254.23 ± 13.40a 260.89 ± 12.44a

0.04

Feed intake (g/d)

1103 ± 51ns

1113 ± 75ns 1115 ± 63ns 1122 ± 60ns

0.03

Feed conversion ratio

5.03 ± 0.28c

4.86 ± 0.38b 4.38 ± 0.40a 4.30 ± 0.35a

0.01

G1: Control group: water devoid of MSE. G2: drinking water containing 20 ml of MSE.
G3: drinking water containing 40 ml of MSE. G4: drinking water containing 60 ml of MSE
Different letters a, b, c within the same line indicates significant differences between groups at a level of (P≤0.05).

The results also showed that there was no significant effect (P>0.05) of the aqueous extract of Moringa oleifera seeds on the amount of feed intake, which amounted to 1103, 1113, 1115, and 1122 g/day for groups G1, G2, G3, and G4, respectively. However, the results showed a significant improvement (P<0.05) in the feed conversion ratio in groups G3 and G4, which amounted to 4.38 and 4.30 g/g, respectively, compared to group G2, whose feed conversion ratio amounted to 4.86 g/g, and the control group G1, which averaged 5.03 g/g per day.

Nutrient Digestibility Coefficient and Nutritional Value of the Feed

The results in Table 3 show a significant improvement (P<0.05) in the digestibility coefficients of nutrients in the treatment groups G2, G3 and G4 compared with the control group G1, as the dry matter digestibility coefficient reached 75.42, 77.99, 80.98 and 81.38% for the groups, respectively, the organic matter digestibility coefficient reached 76.85, 78.85, 82.01 and 82.31%, respectively, the crude protein digestibility coefficient reached 77.50, 79.01, 81.51 and 81.79%, respectively, and the crude fiber digestibility coefficient reached 59.54, 62.30, 64.24 and 64.71%, respectively. The crude fat digestibility coefficient was 78.23, 82.81, 84.69 and 85.06% for the groups, respectively. The results also showed a significant improvement (P<0.05) in the percentage of total digestible nutrients (TDN) for the groups, 74.63, 76.7, 78.69 and 78.92%, respectively.

Table 3: The effect of Moringa oleifera aqueous seed extract on the nutrient’s digestibility and the diet’s nutritional

Digestibility coefficient (%)

Experimental groups (Mean ± SD)
G1 G2 G3 G4

P-value

Dry Matter

75.42 ± 2.3c

77.99 ± 1.5b 80.98 ± 2.72a 81.38 ± 3.32a

0.04

Organic Matter

76.85 ± 2.34c

78.85 ± 1.19b 82.01 ± 1.61a 82.31 ± 1.21a

0.02

Crude Protein

77.50 ± 1.9c

79.01 ± 1.73b 81.51 ± 2.62a 81.79 ± 1.65a

0.03

Crude Fiber

59.54 ± 1.06c

62.30 ± 1.82b 64.24 ± 1.45a 64.71 ± 1.55a

0.04

Ether Extract

78.23 ± 1.87c

82.81 ± 1.95b 84.69 ± 1.68a 85.06 ± 2.28a

0.01

Total Digestible Nutrients

74.63 ± 2.10c

76.7 ± 2.21b 78.69 ± 1.52a 78.92 ± 2.51a

0.03

G1: Control group: water devoid of MSE. G2: drinking water containing 20 ml of MSE.
G3: drinking water containing 40 ml of MSE. G4: drinking water containing 60 ml of MSE.
Different letters a, b, c within the same line indicates significant differences between groups at a level of (P≤0.05).
ns indicates no significant differences within the same line between the experimental groups (P>0.05).

Discussion

The results of the study show the important role of the aqueous extract of Moringa oleifera seeds in improving the growth rates and daily and total weight gain of Awassi lambs. This may be due to the richness of Moringa seeds in proteins rich in sulfur amino acids and their high content of oil and beneficial unsaturated fatty acids [19]. The results are consistent with the findings of [10] when using Moringa leaves in lamb rations, and with [11] when feeding lambs Moringa roots at rates of 25 and 50 g/kg.

The results also showed no significant effect of Moringa seed extract on the amount of feed consumed, which is consistent with the results of [20] when using Moringa oleifera seed meal as a protein source in the diets of Rahmani lambs.

The results also show a positive effect of the aqueous extract of moringa seeds in improving the feed conversion ratio of lambs in the treatment groups. This may be due to the effect of moringa in stimulating rumen fermentation and improving the digestibility of feed nutrients. [21]. This is consistent with the results of [20] when using Moringa seed meal in feeding Rahmani lambs, and also consistent with the results of [10] when using Moringa leaves in lamb rations.

The results indicate a significant improvement in nutrient digestibility (dry matter, organic matter, crude protein, crude fat, crude fiber, and TDN) in the groups treated with moringa seed extract. This may be attributed to moringa’s richness in digestive enzymes, which may help break down complex substances in the feed, facilitating their absorption in the digestive tract. Additionally, moringa may contribute to improving the rumen environment by promoting the growth of beneficial bacteria, which improves digestion and absorption. The study results are consistent with those obtained by [20] when using moringa seed meal in feeding Rahmani lambs. They are also consistent with the findings of [13] when using moringa leaves at a 25% rate in sheep rations. They are also consistent with the findings of [14] when using Moringa oleifera seed extract in Nubian goats.

Conclusions

The study concluded that the use of aqueous extract of Moringa seeds in the drinking water of Awassi lambs contributed to improving growth rates, weight gain, and feed conversion ratio, in addition to increasing the digestibility of feed nutrients.

Author Contributions

Researcher dr. Mohamed Alrez wrote the research, conducted the experiments, statistically analyzed the results, tabulated them, reviewed the research, and prepared it for publication.

Declarations

Ethics Approval and Consent to Participate

Approval was obtained from the Institutional Animal Care and Use Committee (IACUC) and informed consent was obtained from the animal owner for the experiments and publication of the results, with a commitment to applying the best veterinary practices for animal care

Consent for Publication

Not applicable.

Availability of Data and Materials

The datasets analyzed during the current study are available from the corresponding author upon reasonable request due to their sensitivity and are available upon request on the web site. https: //orcid.org/0009-0003-0735-1807.

Competing Interests

The authors declare no competing interests.

Funding

The research was funded with support from Hama University.

Corresponding Authors

Correspondence to Mohamed Alrez.

References

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Predicting Antimicrobial Resistance in Surgical ICU Patients: A Short Commentary

DOI: 10.31038/IGOJ.2025811

 
 

Infections are a significant cause of mortality and morbidity worldwide. Antimicrobial resistance (AMR) represents a mounting global health crisis, particularly within high-risk clinical settings such as intensive care units (ICUs), where patients are often immune compromised, frequently exposed to invasive procedures, and heavily reliant on broad-spectrum antibiotics. In the surgical ICUs, patients undergo a variety of surgical interventions and are prone to postoperative complications, which could possibly increase infection risk and antibiotic exposure [1-3].

In the resource constraint settings, antimicrobial stewardship continues to be challenging due to varied clinical practices and infrastructure gaps, the need for early prediction tools for AMR is essentially critical [4].

A very recent study published in the International Journal of Risk & Safety in Medicine [5] has reported the development and comparison of two machine learning-based predictive models— logistic regression (LR) and artificial neural networks (ANN)—to forecast AMR in surgical ICU patients using real-world clinical data from India. The study leverages a robust, real-world dataset drawn from ICU admissions in a private tertiary care hospital in India. Following screening of 8,010 patients, 4,542 surgical ICU patients were included, and over 104 clinical, demographical, microbiological, and pharmacological variables were initially assessed, reflecting that the dataset used is both comprehensive and contextually rich. The comprehensive nature of the data enabled systematic feature selection, resulting in a streamlined model with 38 significant predictors, which balanced performance with interpretability. The inclusion of diverse variables, such as surgical prophylaxis appropriateness, comorbidity burden, device usage, and specific antimicrobial exposures, demonstrates an insightful understanding of the multifactorial nature of AMR in critical care settings.

The results indicated that the ANN model demonstrated superior predictive performance over LR in multiple key metrics, including specificity (91.2% vs. 86%) and area under the receiver operating characteristic curve (94% vs. 93%), underscoring its potential for capturing complex nonlinear interactions often missed by traditional regression methods. The strength of the study lies not only in its large cohort (n=4542) but also in the breadth of clinical, demographic, and microbiological features, which allowed for robust model development. By integrating clinical parameters like device usage, comorbidity burden, surgical prophylaxis timing, and antibiotic history, the models reflect real-world clinical complexity.

Notably, this study represents one of the first applications of ANN to predict broad-spectrum antimicrobial resistance across multiple pathogens in a post-surgical ICU setting. By enabling the early identification of high-risk patients, these models have the potential to support personalized empiric therapy, strengthen antimicrobial stewardship, and mitigate the impact of resistant infections.

The absence of external validation and reliance on data from a single healthcare facility limits the generalizability across other healthcare settings. To optimize the clinical utility, future research should focus on multicentre validation across healthcare systems and incorporation into real-time electronic health record systems. Moreover, evaluating the influence of model outputs on clinical decision-making and patient outcomes following the implementation will be vital to understanding their long-term effectiveness.

In an era demanding precision and prudence in antimicrobial use, this study offers a compelling framework for integrating predictive analytics into ICU care and highlights the promise of machine learning tools in confronting AMR. Predictive tools like ANN can potentially be integrated into electronic health record systems to flag high-risk patients in real time, personalize empiric therapy, and avoid unnecessary antibiotic use. By enabling early risk identification, this approach may curb the emergence of resistant infections and provide timely support to clinicians navigating complex cases in ICU settings.

In conclusion, this study highlights the promising role of ANN in predicting antimicrobial resistance among surgical ICU patients, offering a valuable step toward personalized infection management. The findings support a data-driven shift in antimicrobial stewardship and ICU clinical decision-making. With increased precision and external validation, such models may become integral to the future infection control strategies.

References

  1. Schlapbach LJ, Kissoon N, Alhawsawi A, Aljuaid MH, Daniels R, Gorordo-Delsol LA, et (2020) World Sepsis Day: a global agenda to target a leading cause of morbidity and mortality. Am J Physiol-Lung Cell Mol Physiol 319(3): L518-L522. [crossref]
  2. Ferrara F, Castagna T, Pantolini B, Campanardi MC, Roperti M, Grotto A, et (2024) The challenge of antimicrobial resistance (AMR): Current status and future prospects. Naunyn Schmiedebergs Arch Pharmacol 397(12): 9603-9615. [crossref]
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  4. Singh C, Pandey A, Singh L (2024) Health Policy Analysis on the Containment of Antimicrobial Resistance (AMR) in India: A Mixed Methods Study of Antimicrobial Stewardship as a Pivotal Intervention in Tackling Anti-Infect Agents 22(4): 8-22.
  5. Deshwal PR, Tiwari P (2025) A comparative analysis of logistic regression (LR) and artificial neural network (ANN) models for predicting antimicrobial resistance in surgical ICU patients: Insights from real-world evidence in India. Int J Risk Saf Med 09246479251337933.

Depositional Process and Palaeogeography of the Ogasa Group, Pleistocene Series on the Pacific Coast of Central Japan

DOI: 10.31038/GEMS.2025742

Abstract

Quaternary crustal movement of the Japanese islands caused the rapid uplift of mountain ranges and the relative subsidence of coastal and inland basins. In particular, from the Early Pleistocene, there was rapid uplift of the major mountain ranges, and in coastal areas, fan-deltas caused the burial of land shelf slopes and fan expansion. As one example, this paper presents the depositional processes of the Ogasa Group, which consists of gravelly fan-delta deposits on the Pacific coast in central Japan. The Ogasa Group divided into the Soga, Osuga, Kasui and Fukuroi Formations in ascending order. The depositional ages of these formations are estimated to be 1.78–1.19 Ma (Soga Formation), 1.19–0.91 Ma (Osuga Formation), 0.91–0.77 Ma (Kasui Formation), and after 0.77 Ma (Fukuroi Formation). The depositional processes and paleogeographical changes of the Ogasa Group can be divided into four stages as follows. Stage A: during the depositional stage of the Soga Formation, sand and mud were deposited from the shoreface to the continental slope, and at the late of this stage, the northwestward submarine channel was buried by coarse-grained sediments from the Tenryu River. Stage B: during the depositional stage of the Osuga Formation, the formation of a large fan-delta by the Oi River from the northeast, which successively advanced to the southwest, expanding the fan and extending offshore. Therefore, an inner bay was formed on its northwestern side, and sand and gravel from the Tenryu River were deposited. Stage C: during the depositional stage of the Kasui Formation, a lake was formed by sandbars formed offshore duo to the transgression. Stage D: during the subsequent deposition of the Fukuroi Formation, the fan formed by the Tenryu and Oi Rivers was thought to have expanded to the south.

Keywords

Middle Pleistocene, Ogasa Group, Fan-delta, Akaishi Mountains, Oi River, Tenryu River

Introduction

In central Honshu, Japan, there was rapid uplift of the Akaishi Mountains and other major mountain ranges from the late Pleistocene (Calabrian), and the land area expanded due to the supply of gravelly sediments in the surrounding areas [1]. Based on detailed stratigraphic and chronostratigraphic data of the Pliocene to Pleistocene in the central Honshu area, Shiba [2,3] divided the stratigraphic processes into four stages, placing the late early Pleistocene to early middle Pleistocene (Calabrian to early Chibanian), when the entire area became terrestrial with large-scale uplift of the Akaishi Mountains and other mountain ranges, in the Stage 2. During this stage, the island arc underwent a general uplift, and the subsidence area of the inland basin expanded, while in the coastal areas, the fan- delta caused the burial and landwardization of the shelf slopes and the expansion of fan areas. The Ogasa Group [4] is distributed in the coastal area of the Pacific Ocean between the Tenryu and Oi Rivers in western Shizuoka Prefecture (Figure 1) and consists mainly of gravelly fan-delta deposits from the late Early to early Middle Pleistocene. The large amount of sand and gravel comprising the Ogasa Group is supplied by the Tenryu and Oi Rivers, which originate from the Akaishi Mountains in the northern part of the distribution area, and the fan-deltas were formed when the Akaishi Mountains began to uplift on a large-scale [5]. Therefore, clarifying the details of the stratigraphy of the Ogasa Group and its depositional processes will help to better characterise the Stage 2 [2,3] of the Quaternary crastal tectonics. Shiba et al. [6] established the stratigraphy and depositional age of the Ogasa Group and inferred the depositional process based on the reconstruction of the depositional environment and its changes. This paper introduces the depositional processes of the Ogasa Group based on that paper [6].

Figure 1: Location map showing the study area indicated by the red box. R, River; Mts, Mountains; Mt. Mountain.

Stratigraphic Overview of the Ogasa Group

The Ogasa Group is Pleistocene series, mainly in Kakegawa and Fukuroi cities, and is exposed northwest to southeast on the Iwatahara Plateau, Enden Hills, Kasui Hills, Ogasa Hills and Minamiyama Hills (Figure 2). Many stratigraphic and palaeontological studies [4,7,8] have been carried out on the Ogasa Group. However, the lithology of the Ogasa Group is composed of thick gravels interbedded with sand and mud beds, which repeat from the lower to the upper part of the Group many times. The Ogasa Group is distributed far apart on the Ogasa Hills and other Hills, with different lithologies on each Hills, it was difficult to correlate the stratigraphy between hills.

Figure 2: Geological map of the Ogasa Group. M., Member.

The Ogasa Group consists of fan-delta deposits, mainly gravels, and unconformably overlies the uppermost part of the Kakegawa Group, the Hijikata Formation. The Ogasa Group is subdivided into the Soga, Osuga, Kasui and Fukuroi Formations (Figure 3), northwest-southeast running, inclined 5–10 degree to the southwest. The Kakegawa Group is a marine formation consisting of sand, mud, alternating sand and mud, and gravel beds, and is mostly composed of submarine fan deposits such as turbidite, except for shallow-water shelf deposits of the transgressive period in the upper part of this Group. The strata of the Kakegawa Group strike northwest-southeast and dip 15–30 degree to the southwest.

Figure 3: Stratigraphy and geological age of the Ogasa Group. F., Formation; M., Member; Strat., Stratigraphy. Cobb Mtn., Cobb Mountain. Wave line shows erosional surface.

The Soga Formation consists of the Aburayama sand Member in the Iwatahara Plateau, Enda Hills and Kasui Hills. In the Ogasa Hills, the Soga Formation consists of the Kogosho sand, Itasawa sand, Kamihijikata mud, Gansyoji gravel and Shinonba sand Members, while in the Minamiyama Hills, the Minamiyama gravel Member is distributed. The Osuga Formation is subdivided into the Takatenjin gravel, Ogasayama gravel, Oshiro sand, Hirano gravel, Obuchi gravel, Kamiishino sand, Higashioya sand and Mitsutoge gravel Membersin the Ogasa Hills. On the other hills, it consists of the Muramatsu gravel and mud Member. The Kasui Formation consists of the Kuno mud Member in the Kasui Hills and the Hattasan mud Member in the Ogasa Hills. The Fukuroi Formation consists of the Mukasa gravel Member in the Iwatahara Plateau, the Horikoshi gravel and mud Member in the Kasui Hills and the Yokosuka gravel Member on the Ogasa Hills.

Depositional Age of the Ogasa Group

The basement of the Ogasa Group is directly above the upper limit of the Olduvai Subchron [9], which corresponds to the Calabrian basement (approximately 1.8 Ma). Kameo [10] recognised the occurrence of the calcareous nannofossil Gephyrocapsa caribbeanica near the horizon of the Haruoka volcanic ash layer in the lower part of the Soga Formation at the eastern foot of the Ogasa Hills, and Gephyrocapsa oceanica slightly above the volcanic ash layer. According to Takayama et al. [11], the Haruoka volcanic ash layer was deposited at 1.72–1.65 Ma. Kameo [10] recognised the extinction of a large Gephyrocapsa genus in the uppermost horizon of the Soga Formation at the eastern foot of the Ogasa Hills, and assigned a base date of 1.2 Ma according to Takayama et al [11]. Ishida et al. [8] found normal magneticepoch in the uppermost part of the Soga Formation northwest of Takatenjin and in the basal part of the Gansyoji gravel Member. This is contrasted with the Cobb Mountain Subchron when combined with data from Kameo [10]. Therefore, the Soga Formation at the eastern foot of the Ogasa Hills is estimated to have been deposited between 1.78 and 1.19 Ma. In contrast, the Soga Formation of the Kasui Hills is not distributed strata in the Soga Formation above 1.65 Ma because its uppermost stratigraphic horizon is the Haruoka volcanic ash layer. The Soga Formation was deposited between 1.78 and 1.19 Ma, which means that the Osuga Formation was deposited after 1.19 Ma. Ishida et al. [8] recognised that the Kamiishino sand Member and the lower part of the Muramatsu gravel and mud Member of the Kasui Hills were deposited during the normal magnetic epoch, and compared their horizons to the Jaramillo Subchron. The Jaramillo Subchron is 1.07–0.99 Ma according to Gibbard and Head [12]. Therefore, in the Kasui Hills, the Muramatsu gravel and mud Member may have been deposited after 1.07 Ma, the base of the Jaramillo Subchron, while the upper part of this Member is reverse magnetic epoch, suggesting that it was deposited after 0.99 Ma. The upper part of the Osuga Formation in the Iwatahara Plateau is intercalated with the Kamikanzo volcanic ash layer. This ash layer is contrasted with the U8 volcanic ash layer of the Umegase Formation of the Kazusa Group, whose eruption age is estimated to be 0.92–0.91 Ma (MIS 23–24) according to Suzuki et al. [13].

The Kasui Formation of the Kasui Hills is intercalated with the Kunovolcanic ash layer, which is correlate to the Azuki volcanic ash layer, which is intercalated with the Ma 3 Member of the Osaka Group; Satoguchi and Nagahashi [14] estimated the age of this ash layer to be about 0.85 Ma. The Kasui Formation of the Ogasa and Kasui Hills was deposited in the reverse magnetic epoch [8], which corresponds to the horizon between the Jaramillo Subchron and the Brunhes Chron. The Kasui Formation is considered to have been deposited between 0.91–0.77 Ma based on the absence of Metasequoia and the inferred age of the lower Osuga Formation.

According to Ishida et al. [8], the four mud beds interbedded near the base of the Fukuroi Formation (the Yokosuka gravel Member) in the Ogasa Hills and the mud beds of the Horikoshi gravel and mud Member in the Kasui Hills are all normal magnetic epoch. This suggests that the base of the Fukuroi Formation was deposited after 0.77 Ma at the Brunhes-Matuyama Chron boundary, i.e. the base of Chibanian. The age of the uppermost part of the Ogasa Group cannot be specifically estimated because the uppermost horizon of the Ogasa Group could not be determined in this study. However, if the Ogasa Group is contrasted with the Osaka and Kazusa Groups, the uppermost part of the Ogasa Group is probably dated at around 0.4 Ma, which is considered to be the upper limit of both groups.

Depositional Environment of the Ogasa Group

The Soga Formation, the lowermost member of the Ogasa Group, is distributed in the Iwatahara-Kasui area by the Aburayama sand Member, which was deposited in the upper to lower shoreface. On the Ogasa Hills, on the other hand, the Kamihijikata mud Member was deposited on the continental slope from the outer continental shelf, while the Kogosho sand and the Itasawa sand Members were deposited on the inner shelf during the storm. The submarine channel deposits, the Gansyoji gravel and the Minamiyama gravel Members, were deposited above them, and the Shinonba sand Member, which have been deposited on the upper shoreface to inner shelf, accumulated. The sands of the Soga Formation include biotite and the gravels contain quartz porphyry and diorite, all of which are presumed to have been supplied by the Tenryu River system. In the Iwatahara-Kasui area, the Osuga Formation consists of the Muramatsu gravel and mud Formation, which is presumed to have been formed by several repetitions of the depositional environment of the inner bay or lagoon and upper shoreface or the diverging sandbar near the river mouth. From the sand and gravel of this member, it is assumed that these sediments were supplied by the Tenryu River system. On the other hand, the Osuga Formation at the eastern foot of the Ogasa Hills consists of the Takatenjin gravel Member, which was deposited near the mouth of the river from a sandbar on the river margin, followed by large-scale fan-delta front gravels (the Ogasayama gravel and Obuchi gravel Members) and fan braided river deposits (the Hirano gravel and Mitsutoge gravel Members). They consist of delta to fan deposits and are thought to have buried continental slopes. The absence of Tenryu River derived elements in the gravels of the Takatenjin gravel Member suggests that the sediments were supplied from the Oi River system and that the fan-delta of the Oi River system was first formed in the Ogasa Hills area during the Osuga Formation depositional period. The Ogasayama gravel Member is thought to have been supplied by the Oi River system, and this fan-delta expanded by advancing from north-east to south-west in the direction of the tilted stratification. The Oshiro sand Member overlying the Ogasayama gravel Member is thought to have been deposited on the upper shoreface, and the Hirano gravel Member of fan deposits and the Obuchi gravel Member deposited on the fan-delta front to the south of it were deposited above it. These gravel beds are also composed of sediments from the Oi River system, and this horizontal and vertical succession of lithologies corresponds to the progressive delta of Reading and Collinson [15], suggesting that the fan-delta developed in a more southerly offshore area at this time. The fan-delta is thought to have developed in a more southerly offshore extent at this period.

The Hiranogravel and the Obuchi gravel Members are overlain by the Kamiishino sand Member, the Higashioyasand and the Mitsutogegavel Members. These Members are deposits of lower shoreface, lower shoreface to backshore and fan, respectively. The presence of the Kamiishino sand and the Higashioya sand Members suggests that the transgression occurred prior to the deposition of these Members. The subsequent relative sea-level stagnation or falling of the sea-level formed the Kamiishino sand and Higashioyasand Members, and the fan sediments, the Mitsutoge gravel Member, was deposited above them, suggesting that an advancing fan-delta developed further to the south, extending the fan. The deposition of the Osuga Formation in the Ogasa Hills is thought to have completely buried and terrestrialised the continental slopes. In the sediments of the Osuga Formation, no sediments such as biotite sands or quartz porphyry gravels suggest that they were supplied from the Tenryu River system, except for the Kamiishino sand Member, and these sediments are thought to have been supplied from the Oi River system. The Kuno mud Member distributed in the Kasui Hills are presumed to have been deposited on the bottom of a freshwater lake with little influence from waves, based on the mud beds with well-developed laminae and shell fossils. The Hattasanmud Member in the Ogasa Hills is also presumed to have been deposited on a water bed based on its lithology. The Kasui Formation is considered to have been deposited during a transgressive period, because the depositional period of the Kasui Formation correlates with that of the Ma 3 Member, a transgressive deposit of the Osaka Group, and the mud beds of the Kasui Formation are widely distributed continuously from the Ogasa Hills to the Kasui Hills. Regarding the depositional environment of the Fukuroi Formation, the Mukasa gravel Member of the Iwatahara Plateau and the Yokosuka gravel Member of the Ogasa Hills are similar to the braided river depositional facies of Miall [16] and are considered braided river deposits on two different large fans. The Horikoshi gravel and mud Member of the Kasui Hills has a recurring environment of lake mud deposition based on the freshwater shell fossils produced, and a gravel layer near the river mouth. The Fukuroi Formation consists of similar fan deposits on the Iwatahara Plateau and Ogasa Hills, while the Kasui Hills are thought to be lake deposits derived from the Kasui Formation. The gravel beds on the Iwatahara Plateau and Kasui Hills contain quartz porphyry, which was supplied by the Tenryu River system, whereas the gravel beds on the Ogasa Hills lack such elements, suggesting that they were supplied by the Oi River system.

Depositional Processes and Palaeogeography

The Ogasa Group consists mainly of coarse-grained sediments formed ravelly fan-delta that started in Calabrian, which was formed when the Akaishi Mountains began to uplift on a large-scale and the Tenryu River and Oi River became the supply rivers of coarse-grained sediments [5]. According to Moriyama and Mitsuno [17], the Ina-Akaishi tectonic crastal movement, which uplifted a mountain range with a specific height of 2,500 m from Ina Valley to the Akaishi Mountains, started after about 1.8 Ma in the early Pleistocene. Suganuma et al. [18] suggest that full-scale uplift of the Akaishi Mountains began between 1.4 and 1.0 Ma, based on the contrast of widespread volcanic ash layers interbedded in fan deposits in the Ina Basin. In this study area, it is estimated that after about 1.8 Ma in the early Pleistocene after the deposition of the Kakegawa Group, the area was generally uplifted, the Iwatahara-Kasui area became a land area, the north-eastern to eastern foot of the Ogasa Hills area became shallow water and a fan-delta was formed between the mouth of the Tenryu River system to the coast. During the late depositional stage of the Soga Formation at 1.2 Ma, a submarine channel-filled gravel bed was formed at the northeastern foot of the Ogasa Hills from the Tenryu River system, and a full-scale fan-delta of coarse-grained sediment was formed in the eastern Ogasa Hills during the depositional stage of the Osuga Formation that started after 1.19 Ma, receiving gravel supply from the Oi River system on its northeastern side. This period roughly coincides with the start of the full-scale, rapid uplift of the Akaishi Mountainsrange. Based on the depositional ages and depositional environments of the various Formations of the Ogasa Group described so far, the depositional processes and palaeogeography of the Group are shown in the following four stages, A to D (Figure4).

Stage A (The Soga Formation: 1.78–1.19 Ma)

In the Iwatahara-Kasui area to the Ogasa Hills area facing the Pacific Ocean, there was a sea area where the continental slope spread offshore from the shoreface during the Soga Formation depositional period, and there was a fan-delta of the Tenryu River system and a coast continuous with it (Figure 4A). At the end of this period, at 1.2 Ma, there was an inflow of coarse-grained sediments from the Tenryu River system from the northwest direction, resulting in the formation of submarine channels and their burial (Figure 4A(a)).

Stage B (The Osuga Formation: 1.19–0.91 Ma)

Two depositional areas, the Ogasa Hills area and the nearby Iwatahara-Kasui area, resulted in contrastingly different depositional environments: fan-deltas and inner bays respectively. The formation of a large fan-delta of the Oi River, which juts offshore into the Ogasa Hills area, has resulted in the development of different depositional environments in the two neighbouring east-west areas. An inner bay or lagoon was formed in the Iwatahara-Kasui area from 1.07 Ma onwards (Figure 4B). In the Ogasa Hills area, a large fan-delta was formed at the beginning of the Osuga Formation depositional stage by the Oi River system flowing into the Takatenjin area (Figure 4B(a)), followed by the Oi River system flowing in from the northeast side of the Kakegawa. Subsequently, the fan-delta progressively advanced to the southwest, fan expanding to offshore (Figure 4B(b)). An inner bay or lagoon (Figure 4B(c)) was formed in the Iwatahara-Kasui area on its northwestern side, and mud, sand and gravel beds were deposited in the inner bay from the Tenryu River system. During this period, a large fan-delta is assumed to have formed in the lower reaches of the present-day Tenryu River (Figure 4B(d)), and the Iwatahara-Kasui area is estimated to have been a deep inner bay environment due to fan-deltas extending on both east and west sides.

Stage C (The Kasui Formation: 0.91–0.77 Ma)

The fan area extending offshore from the Iwatahara-Kasui and Ogasa areas was submerged by the transgression, but the inner part of the fan area was closed by offshore sandbars formed by wave erosion of the fan area, forming a freshwater lake. The lake then expanded to the north due to the transgression (Figure 4C).

Stage D (Fukuroi Formation: after 0.77 Ma)

Large fan-deltas were formed by the Tenryu River and the Oi River in both the Iwatahara Plateau and the Ogasa Hills, respectively, and fan areas spread out. Each of them moved southwards and buried the offshore continental slopes. However, a lake remained in the southern part of the Kasui Hills in this early stage (Figure 4D).

Figure 4: Depositional processes and paleogeographic evolution of the Ogasa Group. The dotted shapes in the background represent the current topography, and the solid lines indicate isobaths at 100 m intervals. The dotted areas represent depths shallower than 100 m. A, The depositional stage of the Soga Formation (1.78–1.19 Ma), (a) The Gansyoji submarine channel; B, The depositional stage of the Osuga Formation (1.19–0.91 Ma), (a) The Takatenjin delta plain, (b) Fan-delta of the Oi River during the depositional stage of the Ogasayama gravel to the Mitsutoge gravel Members, (c) The inner bay between the Oi and Tenryu River fans, (d) Fan-delta of the Tenryu River; C, During the depositional stage of the Kasui Formation (0.91–0.77 Ma), a lake appeared on the Ogasa and Kasui Hills; D, The depositional stage of the Fukuroi Formation (0.77 Ma~), The fans of the Tenryu and Oi Rivers developed to the south again.

Acknowledgment

The author would like to thank Ms. Kate Mariana, Geology, Managing Editor of Earth & Marine Sciences, for the publication of this paper.

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