Toxicity Study In Rats Research Articles

Nose to brain delivery of radiolabeled chemotherapeutic micelles: Meeting the unmet needs of brain tumors

The blood brain barrier postures colossal challenges to the CNS delivery of drugs administered via the oral/parenteral route. In conditions like glioblastoma where mean patient survival rate is mere 15 months from diagnosis, outwitting the blood brain barrier pathway could be a boon to the patient treatment with added compliance. The presented work aims at formulating a chemotherapeutic aid for CNS tumors to be delivered through the non-invasive nose-to-brain pathway. We also propose intranasal delivery of a chemotherapeutic agent amalgamated with radiotherapy nuclide to be used against CNS tumors owing to the synergistic anti-cancer potential of the both. For demonstration purposes, methotrexate was conjugated with a bifunctional chelating agent, radiolabeled with a model radionuclide 99mTc and delivered to the CNS upon intranasal administration. The invented micelles were found to be safe for nasal administration when subjected to in vivo nasal toxicity studies in rats. The fabricated micelles due to their mucoadhesivity, dimension, geometry and improved permeation were found to have an increased nose-to-brain uptake when evaluated for in vivo organ biodistribution studies in mice. The in-vitro cytotoxicity analysis in U87-MG glioblastoma cell lines showed a threefold enhanced activity of the drug-conjugate loaded micelles when compared to the drug solution. Upon SPECT imaging of rabbits administered intranasally with radiolabeled micelles, it was observed that the micellar formulation presented an improved uptake in the animal brain. The authors are of the view that the presented formulation could potentially conglomerate the chemotherapy with radiotherapy towards a synergistic anti-cancer conclusion resulting in an effective GBM treatment.

Safety evaluation of the food enzyme pectinesterase from the genetically modified Aspergillus niger strain PME.

The food enzyme pectinesterase (pectin pectylhydrolase; EC 3.1.1.11) is produced with the genetically modified Aspergillus niger strain PME by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its recombinant DNA. It is intended to be used in fruit and vegetable processing, for juice production and fruit and vegetable processing for products other than juices. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.095 mg TOS/kg body weight (bw) per day in European populations. The toxicity studies were carried out with a xylanase obtained from A. niger strain XEA. The Panel considered this food enzyme as a suitable substitute for the pectinesterase to be used in the toxicological studies, because both production strains are derived from the same recipient strain, the location of the inserts is comparable, no partial inserts were present and the production methods are essentially the same. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. ThePanel identified a no observed adverse effect level (NOAEL) of 1,852 mg TOS/kg bw per day, the highest dose tested, resulting in a margin of exposure of at least 19,495. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches with pollen allergens were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to pollen allergens, cannot be excluded. The Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme inulinase from the genetically modified Aspergillus oryzae strain MUCL 44346.

The food enzyme inulinase (1-β-d-fructan fructanohydrolase; EC 3.2.1.7) is produced with the genetically modified Aspergillus oryzae strain MUCL 44346 by PURATOS NV. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in the production of fructo-oligosaccharides (FOS) from inulin extracted from chicory roots. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.01 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) of 100 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 10,000. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and two matches were found with tomato allergens. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme, particularly in individuals sensitised to tomato, cannot be excluded. However, the likelihood of allergic reactions is expected not to exceed the likelihood of allergic reactions to tomato. As the prevalence of allergic reactions to tomato is low, also the likelihood of such reactions to occur to the food enzyme is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of a food enzyme containing endo-polygalacturonase and pectin lyase activities from the non-genetically modified Aspergillus tubingensis strain NZYM-PE.

The food enzyme with the declared activities endo-polygalacturonase ((1-4)-α-D-galacturonan glycanohydrolase; EC 3.2.1.15) and pectin lyase ((1-4)-6-O-methyl-α-D-galacturonan lyase; EC 4.2.2.10) is produced with the non-genetically modified Aspergillus tubingensis strain NZYM-PE by Novozymes A/S. It is intended to be used in four food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices, refined olive oil production and wine and wine vinegar production. Since residual amounts of total organic solids (TOS) are removed during production, dietary exposure was not calculated for refined olive oil production. For the remaining three food processes, it was estimated to be up to 0.132 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level (NOAEL) of 1,430 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure above 10,833. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and 13 matches were found, including one food allergen (papaya). The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, in particular for individuals sensitised to papaya, but that the risk will not exceed that of consumption of papaya. In addition, oral allergy reactions cannot be excluded in pollen-sensitised individuals. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Non-clinical pharmacology and toxicology studies of LPM6690061, a novel 5-hydroxytryptamine (5-HT)2A receptor inverse agonist.

LPM6690061 is a novel compound with 5-HT2A receptor antagonist and inverse agonist activities. To support the clinical trial and marketing application of LPM6690061, a series of pharmacology and toxicology studies have been conducted. In vitro and in vivo pharmacology studies showed that LPM6690061 had high inverse agonism and antagonism activities against human 5-HT2A receptors, and demonstrated significant antipsychotic-like effects in two rat models: the DOI-induced head-twitch model and the MK-801-induced hyperactivity model, which was more effective than the control drug pimavanserin. LPM6690061 did not have detectable side effects on the neurobehavioral activities and respiratory function in rats, or on the ECG or blood pressure in dogs at the doses of 2 and 6mg/kg. The half maximal inhibitory concentration (IC50) of LPM6690061 for inhibiting hERG current was 1.02μM. Three in vivo toxicology studies were conducted. In the single dose toxicity study in rats and dogs, the maximum tolerated dose of LPM6690061 was 100mg/kg. In the 4-week repeat dose toxicity study in rats, the main detectable toxic reactions of LPM6690061 included moderate artery wall hypertrophy, minimal to mild mixed cell inflammation and increased macrophages in the lung, which generally recovered after a 4-week drug withdrawal period. In the 4-week repeat dose toxicity study in dogs, no detectable toxicity was observed. The doses of no-observed-adverse-effect-level (NOAEL) in rats and dogs were 10mg/kg and 20mg/kg, respectively. In conclusion, both in vitro and in vivo pharmacological and toxicological studies showed that LPM6690061 was a safe and efficacious 5-HT2A receptor antagonist/inverse agonist which supports the clinical development as a novel antipsychotic drug.

Safety evaluation of the food enzyme endo-polygalacturonase from the genetically modified Trichoderma reesei strain RF6197.

The food enzyme endo-polygalacturonase ((1-4)-α-d-galacturonan glycanohydrolase; EC 3.2.1.15) is produced with the genetically modified Trichoderma reesei strain RF6197 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices, production of wine and wine vinegar, coffee demucilation and production of plant extracts as flavouring preparations. Since residual amounts of the total organic solids (TOS) are removed during the coffee demucilation and the production of flavouring extracts, dietary exposure was calculated only for the remaining three food processes. It was estimated to be up to 0.156 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 6,410. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and matches were found with a number of pollen allergens. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure, particularly in individuals sensitised to pollen cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety and efficacy of a feed additive consisting of β-mannanase produced by Aspergillus nigerCBS 120604 (Nutrixtend Optim) for use in all poultry for fattening (Kerry Ingredients & Flavours Ltd).

Following a request from the European Commission, the EFSA Panelon Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of a product containing β-mannanase produced by a non-genetically modified strain of Aspergillus niger (CBS 120604). The commercial name is Nutrixtend Optim and it is intended to be used as a zootechnical feed additive for all poultry for fattening. Based on a tolerance trial in chickens for fattening and the no observed adverse effect level identified in a subchronic oral toxicity study in rats, the additive was considered safe for all poultry for fattening. The Panelconcluded that the use of the product as a feed additive does not give rise to concerns for consumers and the environment. The additive is considered an irritant to skin and eyes and a dermal sensitiser. Due to the proteinaceous nature of the active substance, it is also considered a respiratory sensitiser. The Panelconcludes that the additive has the potential to be efficacious as a zootechnical additive at the level of inclusion in feed of chickens for fattening of 30 U β-mannanase/kg complete feed. This conclusion was extrapolated to all poultry for fattening.

Safety Evaluation of Arsenicum album in Acute and Sub-Acute Toxicity Studies in Rats

Arsenic album is frequently prescribed in homoeopathy for many diseases. However, its safety data is not available. Thus, the study’s purpose is to evaluate the oral toxicity of Arsenic album 6C, 30C, and 200C in rats. Arsenic album (6C, 30C, and 200C) was given at 2000 μl/kg for acute toxicity and observed for up to 14 days. For subacute toxicity, it was given for 28 days and observed for clinical signs, change in body weight and Mortality. Hematological, biochemical, organ weight and histopathological analyses were assessed. Results indicate no mortality of arsenic album in acute toxicity and LD50 is >2000 μl/kg. In the subacute toxicity study, arsenic album (200 μl/kg) did not show any significant changes in above parameters. It may be concluded that the arsenic album (6C, 30C, and 200C) is safe and produces no toxicity when administered orally for a prolonged duration at 200 μl/kg in rats.

A 28-Day Repeated Dose Toxicity Evaluation of Creatinine Monohydrate and β-Hydroxy β-Methyl Butyrate Combination in Rodent Model

Substance abuse is a global concern, having a significant influence on society, healthcare, and the economy. Athletes are increasingly using substances to improve their performance or to cope with stress. Therefore, having a thorough awareness of the often misused substances, their effects, and potential toxicities is essential. One such chemical is the mixture of Creatinine Monohydrate (CM) and -Hydroxy Methyl Butyrate (HMB), which is widely used by athletes to increase strength and performance. According to studies, the combination can boost athletic performance, build muscle, and offer several health advantages. To determine the chemicals’ long-term impacts on human health, more research is required because their safety and toxicity are not well-established. Using TOPKAT(Computer-Aided Toxicity Prediction) and Discovery Studio Version 4.0, this study examined the toxicity and pharmacokinetic profiles of CM and HMB. The interaction of HMB and CM may significantly reduce cell viability compared to either drug alone. There were no negative effects seen in the animal models used for the acute oral toxicity study, but additional research is needed to confirm these results and determine the compounds’ long-term safety for human use. The study also performed cytotoxicity studies of individual substances and combinations on VERO and HEP-G2 cell lines and a 28-day repeated dose toxicity study in Wistar albino rats. The study results provide valuable information for healthcare professionals to understand the physiological complications of exposure to such drugs. Overall, the article emphasizes the importance of understanding the potential toxicity of chemical compounds, particularly those used as nutritional supplements.

Unexpected neurotoxicity in chronic toxicity studies with a HBV viral expression inhibitor

The non-clinical safety profile of the small molecule hepatitis B virus viral expression inhibitor RG7834 was studied in a package consisting of safety pharmacology, genotoxicity, repeat dose toxicity and reproductive toxicity studies. The chronic monkey toxicity study identified dose- and time-dependent symptoms of polyneuropathy, with correlating nerve conduction velocity reductions and axonal degeneration in peripheral nerves and spinal cord, in all compound treatment groups with no evidence of reversibility after approximately 3 months of treatment cessation. Similar histopathological findings were observed in the chronic rat toxicity study. Subsequent in vitro neurotoxicity investigations and ion channel electrophysiology did not elucidate a potential mechanism for the late toxicity. However, based on similar findings observed with a structurally different molecule, an inhibition of their common pharmacological targets, PAPD5 & PAPD 7, was considered as a possible mechanism of toxicity. In conclusion, the marked neuropathies, only observed after chronic dosing, did not support further clinical development of RG7834 because of its foreseen clinical treatment duration of up to 48 weeks in chronic HBV patients.

Prenatal Developmental Oral Toxicity Evaluation of Defatted Fenugreek Seed Flakes (FenuflakesTM) in Laboratory Rats

Fenugreek seed-based ingredients showed potential health benefits towards female-specific conditions. The present work is aimed to assess the prenatal oral toxicity of fibers and protein rich defatted fenugreek seed flakes (Fenuflakes™). The acute oral toxicity and dose range-finding studies in non-pregnant and pregnant rats were conducted before the main study. The selected doses of Fenuflakes (500, 1000, and 2000 mg/kg) were orally gavaged to rats daily from day 0 to day 19 (one day before the expected day of parturition) post-conception with the concurrent vehicle control (VC) group. On the 20th day of gestation, the maternal and embryo-fetal toxicity parameters were recorded after the cesarean sections of dams. Results: Fenuflakes in tested doses exposure did not show significant toxicological changes in maternal (body weights, food intake, anogenital distance, or clinical observations) and embryo-fetal evaluations (number of corpora lutea, resorptions, and implantations, or fetus weights, sex ratio or incidence of anomalies) compared with VC. Conclusion: Oral prenatal exposure to Fenuflakes was found safe with no significant maternal and embryo-fetal toxicities. The "No Observed Adverse Effect Level” (NOAEL) of Fenuflakes (> 2000 mg/kg/day) can be used for risk assessment before human consumption in pregnant female population.

Abstract CT249: A study to evaluate the safety and tolerability of the covalent phosphoinositide-3-kinase (PI3K)-alpha Inhibitor, TOS-358, in adult subjects with select solid tumors

Abstract Background: TOS-358 is an orally available, highly selective covalent inhibitor of PI3Kα capable of achieving a deep and durable inhibition of PI3K-AKT signaling with virtually no off-target effects. In patient-derived xenograft models, TOS-358 has shown anti-cancer activity in several different tumor types including breast, colorectal, gastric, head and neck, bladder, and esophageal cancer, and its’ activity was superior to non-covalent compounds targeting PI3Kα. Administered at the equivalent dose, TOS-358 has shown a good safety profile in 28-day repeat dose toxicity studies in rats and dogs. Methods: The TOS-358-001 study is a multicenter dose escalation (phase 1) and dose expansion (phase 1b) clinical study evaluating the safety, tolerability, and preliminary efficacy of TOS-358. Eligible subjects are adults with one of protocol-defined tumor types, known PIK3CA mutation or amplification, adequate organ function, ECOG performance status 0 or 1, available archived or fresh tumor tissue for PIK3CA mutation detection and no prior treatment with PI3K, AKT or mTOR inhibitors (except subjects with breast cancer). The phase 1 portion of the study follows a 3+3 dose escalation design and is intended to determine the minimum effective dose (MED) and the maximum tolerated dose (MTD) of TOS-358 when administered orally on a once daily (QD) or a twice daily (BID) schedule, and the recommended phase 2 dose (RP2D). Cohort expansions at the MTD and MED to support the RP2D selection are planned. The phase 1b portion of the study will enroll subjects with tumor PIK3CA mutations or amplifications in several tumor-specific cohorts (colorectal, gastric, non-small cell lung, breast, squamous cell carcinoma of the head and neck, urothelial and select gynecological cancers) to further assess the safety and tolerability of TOS-358 and to evaluate the initial efficacy of the investigational agent. Enrolment to the study commences in US with European sites planned to open later in 2023. Clinical trial information: NCT05683418. Citation Format: Marwan Fakih, Davendra P. Sohal, Navid Hafez, Jordan D. Berlin, Kimberly Hari, Ovid C. Trifan. A study to evaluate the safety and tolerability of the covalent phosphoinositide-3-kinase (PI3K)-alpha Inhibitor, TOS-358, in adult subjects with select solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT249.

Abstract LB221: Novel peptide linker-based nectin-4 targeting ADC shows improved tolerability with long-lasting anti-tumor efficacy at low doses

Abstract The Araris site-specific and one-step peptide linker conjugation technology generates stable, safe and highly potent ADCs without the need for antibody engineering prior to payload conjugation. We generated an anti-Nectin-4 ADC that shows superior anti-tumor activity and tolerability compared to enfortumab-vedotin (EV) in head-to-head in vitro and in vivo studies. The Araris ADC is based on enfortumab as the targeting antibody and monomethyl auristatin E (MMAE) as payload. Using a peptide linker and site-specific enzymatic conjugation approach, we generated a pure ADC with a drug-to-antibody-ratio (DAR) of approximately 2 and above 98 percent monomeric content. The Araris ADC demonstrated potent cell cytotoxicity similar to the approved enfortumab-vedotin which has a DAR of 4, excellent stability in mouse, cynomolgus and human sera exemplified by the absence of payload deconjugation or linker cleavage while EV showed significant payload deconjugation. Despite high stability, the Araris ADC releases the free active MMAE metabolite at comparable rate to EV in human Cathepsin B or human liver-lysosome (HLL) enzyme cleavage assays. The ADC was also shown to be extremely stable in circulation in pharmacokinetic studies in rodents, leading to an intact ADC exposure profile comparable to the unmodified enfortumab parent antibody. No free payload was detectable in circulation during the 3 week study by LCMS-MRM. In efficacy studies using a SUM-190PT established breast cancer model, a single injection at a dose of 10 ug/kg normalized by payload induced a complete tumor regression lasting for more than 100 days (i.e. a very durable response or tumor eradication). EV administered at the same payload dose showed only a short and transient (until day 20 only) tumor regression with no animal (0/6) reaching a complete response. Despite the higher in vivo exposure and extremely efficient anti-tumor response at low payload doses, there was no increased toxicity but in contrast, overall tolerability was improved, i.e., less neutropenia, skin involvement and signs of toxicity - the skin toxicity being the dose-limiting toxicity of Enfortumab vedotin in humans and rats. Overall, the highest non-severely toxic dose (HNSTD) in 4-week repeat dose rat toxicity studies for the Araris ADC (25 mg/kg) was 5-fold higher compared to the HNSTD (5mg/kg) reported for Enfortumab vedotin. Our data impressively show that the Araris ADC has superior efficacy and durable anti-tumor response even at 3-fold lower payload dose compared to EV. The improved efficacy in mice and tolerability in rates resulted in a 8-fold better TI for the Araris ADC and offers the opportunity to develop a highly efficacious ADC having potentially lower dose-limiting toxicities such as peripheral neuropathy, rashes or neutropenia. Citation Format: Isabella Attinger-Toller, Philipp Probst, Romain Bertrand, Emma Renard, Ramona Stark, Roger Santimaria, Dragan Grabulovski, Bernd Schlereth, Philipp Rene Spycher. Novel peptide linker-based nectin-4 targeting ADC shows improved tolerability with long-lasting anti-tumor efficacy at low doses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB221.

Abstract 3448: Discovery of HYP-2A,a 2nd- generation KRASG12C inhibitor exhibits potent in vivo efficacy in drug resistant tumor models

Abstract Although activating mutations in KRAS are well recognized as a hallmark of cancer, KRAS was considered an undruggable target for over 30 years after its discovery, due to the intrinsic characteristics of KRAS proteins. The pocket of KRAS is small and has a considerably smooth and shallow surface, resulting in difficulty for small molecule to bind to the protein. Recent accelerated approval of Sotorasib (AMG 510) by FDA marked the first marketed KRASG12C inhibitor for the treatment of 2nd line KRASG12C mutation-positive NSCLC patients. Although great clinical benefits were delivered by this breakthrough medicine, intrinsic or acquired drug resistances were developed in a large portion of patients treated. HYP-2A is a new chemical entity (NCE) being developed by Sichuan Huiyu Pharmaceuticals for the treatment of KRASG12C inhibitor resistant tumors. It is an orally bioavailable, potent KRASG12C inhibitor that shows very high anti-proliferation activities in NCI-H358 cell line and various Sotorasib and Adagrasib (MRTX 849) resistant tumor cell lines in vitro and demonstrates strong in vivo efficacy in the corresponding xenograft mouse models. It inhibits NCI-H358, H358 AMGR, Mia PaCa-2, PaCa-2 AMGR, KYSE-410, and SW1573 cell lines with IC50 values range from low double-digit to single-digit nanomolar. When dosing at 10 mpk via PO, HYP-2A demonstrates significant efficacy (TGI > 75%) in SW1573 Xenograph mouse model, whereas neither Sotorasib nor Adagrasib shows any obvious efficacy at 30 mpk with TGIs less than 20% in the same experiment. Similarly, when dosing at 10 mpk via PO, HYP-2A exhibits strong efficacy (TGI > 90%) in KYSE-410 Xenograph mouse model, while neither Sotorasib nor Adagrasib shows any significant efficacy (TGI < 50%) at 30 mpk via PO dosing in the same experiment. In an acute toxicity study in rats, the maximum tolerated dose (MTD) of HYP-2A is found higher than 30 mpk. In a two-week toxicity studies in rodents, rats are found well tolerated when treated with HYP-2A via PO at 3-10mpk (QD) for 14 consecutive days. The MTD of HYP-2A is determined higher than 10 mpk. In this presentation, we will highlight the research program that leads to the discovery of HYP-2A, which is currently under IND-enabling studies. Citation Format: Shoujun Chen, Xiaoming Qiang, Haibo Wang, Yong Xiong, Ke Liu, Dengming Liao, Nan Zhong, Mingdeng Liu, Xuemei Mu, Rudan Huang, Min Li, Dengwei Gui, Meilin Huang, Yuting Chen, Yuanfu Pan, Wengang Yao, Yingte Song, Zhongbo Wang, Wenbin Wang, Jun Liu, Xingchi Yu, Zhen Liang, Yike Yuan, Zhao Ding. Discovery of HYP-2A,a 2nd- generation KRASG12C inhibitor exhibits potent in vivo efficacy in drug resistant tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3448.

Abstract 475: Preclinical development of ABM-168, a novel MEK Inhibitor to treat cancer with brain tumors

Abstract The Mitogen-Activated Extracellular Signal-Regulated Kinase (MEK) is a critical member of the MAPK signaling pathway family. In cancer cells, the abnormality in MAPK pathway associates with the mutation occurred in the upstream KARS or BRAF. Blockage of the pathway with MEK1/2 inhibitors result in inhibiting cell proliferation and inducing apoptosis. It hence has the potential for clinical benefits for treating cancers with RAS/RAF dysfunctions.ABM-168 is a novel small molecule, allosteric, highly selective MEK inhibitor with high water solubility, cell permeability and brain penetration. We have demonstrated its anti-cancer efficacy in vitro in multiple cancer cell lines, and in vivo in several xenograft, subcutaneous, intracardiac and intracranial, animal models. In-vitro data showed the similar on-target enzyme activity of ABM-168 to marketed MEK inhibitors, as well as high anti-proliferation activities (IC50 <30nM) in multiple cancer cell lines with BRAF or RAS or NF1 mutation like A375, Colo-829, HT-29, MiaPaca-2, LN-229 etc. In vivo pharmacology studies demonstrated that ABM-168 had good potencies of tumor growth inhibitions with oral dose alone or combo with other drugs or compounds in multiple xenograft cancer models: In an A375-luc intracardiac melanoma metastatic model, ABM-168 at 2 mg/kg PO BID demonstrated an antitumor activity comparable to ABM-1310 (A highly BBB-permeable BRAF inhibitor developed by ABM Therapeutics) at different dose levels and frequencies. On Day 28, the BLI% decreased 95.5%, 93.2%, 99.44% and 99.7% for ABM-168 (2 mg/kg), ABM-1310 at 1 mg/kg PO QD, 5 mg/kg PO QD and 2.5 mg/kg PO BID, respectively. In a LN229 glioblastoma orthotopic model in BALB/c nude mice, ABM-168 at 5-10 mg/kg PO QD showed a significant activity, the BLI (%) decreased in the brain 68 % on Day 49. These results indicate that the BBB-permeable ABM-168 may be efficacious as a single agent or combo with ABM-1310 to treat BRAF-mutant, advanced cancers with or without brain metastases or primary CNS tumors with an abnormal up-regulated MAPK signal. Non-clinical pharmacokinetics studies also showed that ABM-168 had a favorable ADME profile both in vitro and in vivo, and good brain penetration in animals. Single-dose, seven-day repeat dose non-GLP studies and four-week GLP toxicity studies in SD rats and beagle dogs were all completed. Based on these supportive preclinical study results, the IND of ABM-168 was submitted in 2022 Q3 to investigate its safety in human, which will be followed by further clinic development as a single agent, or in combination with other molecules to treat advanced cancers resulted from the abnormal MAPK signal pathway, particularly with brain tumors. Citation Format: Chen Chen, Charles Huang, Min Xu, YouQin Chen, Lanjiao Zhao, Yang Chen, Yong Hu. Preclinical development of ABM-168, a novel MEK Inhibitor to treat cancer with brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 475.

Safety evaluation of the food enzyme asparaginase from the genetically modified Aspergillus oryzae strain NZYM-OA.

The food enzyme asparaginase (l-asparagine amidohydrolase; EC 3.5.1.1) is produced with the genetically modified microorganism Aspergillus oryzae strain NZYM-OA by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in various food manufacturing processes to prevent acrylamide formation. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.051 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,182 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 23,176. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Preclinical toxicological screening of Siddha medicine Ganthi Mezhugu by acute toxicity studies in Wistar albino rats

Introduction and Aim: Siddha formulations of drugs had been used for hundreds of years in international conventional drugs for his or her capacity fitness benefits. Ganthi Mezhugu (GM) has a wide range of therapeutic properties. The objective of the present research was to establish the safety profile of the experimental drug GM in a rat model by acute and sub-acute oral toxicity, as per OECD regulatory guidelines. Materials and Methods: Acute toxicity testing is done for an animal species to determine the effects of a single dose on that species. In this investigation, acute toxicity was assessed using single oral administrations of 50 mg/kg, 300 mg/kg, and 2000 mg/kg. In the subacute investigation, the test substance GM was repeatedly dosed (4.68, 23.4, and 46.8 mg/kg/day) for 28 days followed by these hematological and biochemical parameters were assessed. Results: The study's findings showed that after administering the test substance GM to experimental animals once or repeatedly, there was no evidence of toxicity and no deaths. The subacute toxicity study's GM treated groups' normal hematological and serological profiles provided additional evidence that the formulation is not harmful. Conclusion: The finding of the study supported the notion that Siddha formulation GM is non-toxic and has a broad spectrum of safety.

Safety evaluation of the food enzyme triacylglycerol lipase from the non-genetically modified Burkholderia stagnalis strain PL266-QLM.

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase EC 3.1.1.3) is produced with the non-genetically modified Burkholderia stagnalis strain PL266-QLM by Meito Sangyo CO., LTD. The production strain harbours genes conferring resistance to highly important antimicrobials for human and veterinary medicine. The food enzyme is free from viable cells of the production organism, but not of its DNA. Therefore, the food enzyme poses a risk of promoting the spread of antimicrobial resistance (AMR) genes. It is intended to be used in milk processing for cheese production and modification of fats and oils by interesterification. Since residual amounts of total organic solids (TOS) are removed in the downstream processing of the oils, dietary exposure was calculated only for the milk processing for cheese production. Dietary exposure to the food enzyme-TOS was estimated to be up to 0.663mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 2,756mg TOS/kg bw per day in males, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 4,157. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. As there is a risk of spreading AMR genes, the use of this food enzyme could not be considered safe.

Safety evaluation of the food enzyme endo-polygalacturonase from the genetically modified Aspergillus niger strain EPG.

The food enzyme endo-polygalacturonase ((1-4)-α-d-galacturonan glycanohydrolase; EC 3.2.1.15) is produced with the genetically modified Aspergillus niger strain EPG by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in fruit and vegetable processing for juice production. The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.122mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panelidentified a no observed adverse effect level of 1,014mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure at least 8,311. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and 38 matches were found, two of which are food allergens. The Panelconsidered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, in particular for individuals sensitised to papaya or maize, but that the risk will not exceed that of consumption of papaya or maize. In addition, oral allergy reactions cannot be excluded in pollen-sensitised individuals. Based on the data provided, the Panelconcluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Safety evaluation of the food enzyme glucan 1,4-α-glucosidase from the non-genetically modified Aspergillus niger strain NZYM-BO.

The food enzyme glucan 1,4-α-glucosidase (4-α-d-glucan α-glucohydrolase; EC 3.2.1.3) is produced with the non-genetically modified Aspergillus niger strain NZYM-BO by Novozymes A/S. It was considered free from viable cells of the production organism. It is intended to be used in seven food manufacturing processes: baking processes, brewing processes, cereal-based processes, distilled alcohol production, fruit and vegetable processing for juice production, production of dairy analogues and starch processing for the production of glucose syrups and other starch hydrolysates. Since residual amounts of total organic solids (TOS) are removed by distillation and during starch processing, dietary exposure was not calculated for these two food manufacturing processes. For the remaining five food manufacturing processes, dietary exposure to the food enzyme-TOS was estimated to be up to 2.97 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,920 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 646. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and one match with a respiratory allergen was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure to this food enzyme cannot be excluded (except for distilled alcohol production), but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.