Chronic (180-day) and sub-chronic (90-Day) oral toxicity studies of a novel polyethyleneglycol (PEG)-carbohydrate-lipid conjugate in Wistar rats and beagle dogs

Abstract

The toxicity of a new synthetic PEG-carbohydrate-lipid conjugate; 3,3-oleoyl-monomethoxy-polyethyleneglycol (n = 12 mean) ether-propanediamino-lactobionate or Oleoylpropane-diaminododecaethyleneglycolmonomethoxy ether-lactobionate (“DOPS-F02”) has been evaluated in formal GLP (Good Laboratory Practice) chronic and sub-chronic toxicity studies. The oral toxicity study describes, for the first time, the unique safety features and characteristics of a PEG-saccharide-lipid conjugate. The oral toxicity studies of DOPS-F02 in male and female beagle dogs and Wistar rats documented no deaths or treatment-related signs at high doses. DOPS-F02 was individually administered (by gavage) to male and female Wistar rats at concentrations of 750, 1250, and 2500 mg/kg bodyweight for 180 consecutive days. For the sub-chronic study, juvenile beagle dogs were administered oral doses of DOPS-F02 at 600, 800 and 1000 mg/kg bodyweight, once daily for 90 consecutive days. All animals survived the duration of the studies, the No Observed Effect Level (NOEL) / No Observed Adverse Effect Level (NOAEL) were at the highest doses tested that did not produce any significant changes in clinical signs, hematological parameters, organ weights, ophthalmology evaluations, or histopathological findings. These studies establish that DOPS-F02 is non-toxic in rats and dogs following oral administration. The no-observed-adverse-effect level was determined at 2500 mg/kg for rats and 1000 mg/kg for dogs following oral administration. DOPS-F02 has been developed as a solubilizer with potential to improve formulation of poorly soluble drugs for oral administration. DOPS-F02 demonstrated a great potential for use as a safe solubilizer to improve the pharmacology profile and solubility of lipophilic drugs in aqueous systems by the absence of severe adverse events. Results from these studies are presented to support the future use of this new material in early clinical trials.

Graphical abstract

Highlights

• •The Novel polyethyleneglycol-saccharide-lipid conjugates have the capacity to improve the pharmacology profile and solubility of lipophilic drugs in aqueous systems.
• •The chronic and sub-chronic repeated dose oral toxicity study describes, for the first time, the unique safety features and characteristics of a PEG-saccharide-lipid conjugate.
• •The study demonstrated a favorable safety profile DOPS-F02 by the absence of severe adverse events; NOAEL is 2500 mg/kg/day for rats and 1000 mg kg/day for dogs

1. Introduction

While there is a considerable amount of innovation occurring with emerging therapeutics, modes of drug delivery and other areas, etc., novel excipients should also be considered to address the current and future challenges associated with these new technologies. For example, consider the solubility challenges facing drug pharmaceutical formulators. Examining the pipelines of most pharma companies, reveals that the percentage of drugs with poor solubility is far greater than it was 20–30 years ago. In fact, up to 90 % of small molecules are poorly soluble [1]. Such intractable issues may be overcome with enabling excipients that can play a central role in the drug development process. For instance, polyoxyethylene-derived polymers have been successfully used as pharmaceutical delivery vehicles for many decades. These materials offer several benefits, including enhancement of solubility, but also have several issues based on their tendency to form peroxides [2] that can have deleterious effects on various actives [3]. In addition, like other surface-active compounds, the polyoxyethylene polymers may cause hemolysis when they come in contact with red blood cells [4]. Results in a published study [5] indicated that the hemolytic activity of polyoxyethylene polymer may be ascribed to the tendency to form peroxides during the synthesis that involves free (3) radical reactions. It should, however, be emphasized that hemolysis is only one form of cytotoxicity in these reported studies.

A significant problem for product development for new pharmaceuticals is that many of the advancing NCE drug entities are insoluble in water. In some cases, a promising drug candidate may be discontinued due to insufficient water solubility. Solubilizing excipients can resolve some of these challenges for various routes of delivery provided they maintain product efficacy and safety. In some cases, these materials may cause adverse effects that can be severe, and which may limit their use, depending on the specific intended use. For example, despite the adverse effects caused by polysorbates, e.g., polysorbate 80 and polyethoxylated castor oil (sold under the trade name “Cremophor”), these two leading regulatory approved intravenous excipients have been accepted in products for cancer treatment since the drugs are effective and not allowing use of the solubilizer could prevent patient access to important treatments [6].

This novel conjugate (DOPS-F02) is suitable for both parenteral and oral applications; the oral route of drug administration is generally preferred because of its versatility, safety, and ease of administration. DOPS-F02 is therefore being developed as a solubility enhancer as an alternative to NP or liposomes, that is stable through extended shelf life, has acceptable toxicity when administered orally. Substitution of polysorbates and Cremophor by PEG-carbohydrate-lipid conjugates offers a potential opportunity to create formulations that improve stability, reduce immunogenicity, and extend shelf life.

In this study, a synthetic PEG-carbohydrate-lipid conjugate has been developed for use as a delivery carrier and solubilizer. These lipids have the general structure shown in Fig. 1 and DOPS-F02 has a PEG-12 head group, a long hydrocarbon chain (oleoyl) and a disaccharide function. This conjugate spontaneously forms micelles upon hydration. All components in DOPS-F02 are less biologically active compared to the starting materials. Unlike peptides or proteins, the amide bonds in DOPS-F02 are “locked” and inactive (no free amine).

Fig. 1.

Chemical structure of DOPS-F02 (n = 12); CAS registration number:2918283–09–5.

This PEG-based excipient has never been used in clinical settings to the authors’ knowledge and no evaluation has been conducted to assess the oral toxicity of any PEG-carbohydrate-lipid conjugate. Given the formulation potential of this novel excipient as a unique solubilizer, conducting a safety evaluation program to facilitate its adoption and use is both critical and timely.

A rigorous safety evaluation of DOPS-F02 would support use as a solubility enhancer for pharmaceutic actives and nutritional supplements in oral formulations. Results are provided from a GLP sub-chronic toxicity study conducted in dogs, and a GLP chronic study in rats, achieved by feeding DOPS-F02 individually at various levels for a period of 90–180 days respectively, these no-observed-adverse-effect-level (NOAEL) results from the studies are provided in this report.

2. Materials and methods

2.1. Test material

The conjugate used in these studies was 3,3-oleoylmonomethoxypolyethyleneglycol (n = 12 mean) ether-propanediaminolactobionate (CAS 2918283–09–5) or DOPS-F02 was synthesized by LipoSeuticals (Monmouth Junction, New Jersey). It is a solid or wax at room temperature that becomes a sticky gel with moisture levels of 5 % or more. Synthesis involved one mole of mPEG reacted with 1 mol each of oleic acid and lactobionic acid, conjugated via a central backbone of a diamine to give DOPS-F02 (Fig. 1). Total synthesis for the conjugation comprised of alkylation, etherification, esterification or amidation. The purity of DOPS-F02 was then determined by HPLC. Detailed information on the chemistry and assay methods, including HPLC or mass spectrometry, and long-term stability of DOPS-F02 are available elsewhere [7].

DOPS-F02 was dissolved in sterile water at the targeted concentration of 200 mg/mL. The dose formulations were prepared weekly or bi-weekly based on the dosing volumes required, and the concentrations were determined according to the predefined schedules by a validated HPLC assay method. All dose formulation solutions were filtered with 0.45 µm membrane filters and transferred into pre-cleaned glass bottles for the storage at 2–8 ºC. The typical purity of DOPS-F02 used in the studies was in the range of 90–96 %.

Materials made and used in this study focusing on reducing waste, responsible water management, and phasing out hazardous substances to meet the environmental standards.

2.2. Animals

2.2.1. Beagle dogs

Naïve and healthy Beagle dogs (aged at 9–10 weeks) were purchased from Marshall Biotechnology Co., Ltd. (Beijing, China). Animals were housed in a room with the following environment parameters: temperature ranged from 20.08 to 25.48 °C, daily temperature fluctuation was ≤ 3.56 °C, relative humidity was 40–85 %, 12 h artificial light (07:30–19:30) and 12 h darkness (19:30–07:30) per day. Illumination was provided during the darkness period based on the study needs. Animals were supplied with dog canine growth and reproduction formula feed ad libitum.

2.2.2. Wistar rats

Naïve and healthy Wistar (Rattus norvegicus) Rats were sourced from Palamur Biosciences Pvt Ltd. (Mahabubnagar, India). 124 (62 Male + 62 Female) healthy Wistar rats (aged 4–5 weeks) with weights ranging from 116.30 to 150.02 g (Male) and 118.54–147.11 g (Female) at time of dosing. Animals were housed in groups (2–3 animals of the same sex per cage) in standard rat cages with feed holders, polycarbonate water bottles with sippers, and autoclaved corn cob bedding. Environmental conditions were maintained at a temperature of 22°C ± 2°C and a relative humidity of 46–65 %. 12-hour light/dark cycles were employed and there were 12–15 air exchanges per hour. Pelleted rodent feed and RO water was provided ad libitum.

2.3. Experimental design and conduct

Ethical approval: All studies were conducted at a third party CRO (contract research organization) facilities with pre-approval by the Institutional Animals Ethics Committee (IAEC) of the test facilities, e.g., IACUC-A2022095-T012–01 (for the dog study) or PAL/IAEC/2022/09/02/17 (for the rat study).

Study protocols: Studies were performed in accordance with OECD principles (Organisation for Economic Co-operation and Development, a quality system for non-clinical safety studies) on Good Laboratory Practice [8] and the protocol was in accordance with the U.S. Food and Drug Administration (FDA) Good Laboratory Practice (GLP) regulations, standards, and guidelines (21 CFR Part 58) and complied with the U.S. Department of Agriculture’s (USDA) guidelines for animal care and handling. Both animal experimentation sites for dog and rat study were accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International, AAALAC and key national or international GLP regulatory authorities (OECD document, 1998). Furthermore, the studies were conducted in accordance with ICH or OECD guidance for chronic toxicity studies [9].

2.4. Part I. beagle dogs

This part of the safety evaluation program was intended as a “Bridge” study to extend applications of DOPS-F02 to oral administration by building on previously completed intravenous dosing studies (Non-GLP and GLP studies in rodent and non-rodent species) [7]. Based on earlier Non-GLP studies in rodents, the bioavailability of DOPS-F02 was considered as very low, i.e., less than 0.1 %; therefore, substantial toxicity was not anticipated.

2.4.1. Dose level selection

In a preliminary study, after beagle dogs were orally administered with DOPS-F02 at 1000 and 2000 mg/kg for 5 consecutive days, occasional mild gastrointestinal responses were observed. This symptom may result from the large dosing volume (10 mL/kg) for juveniles and their inability to properly digest or process a polymer at younger ages. Therefore it was decided that using a smaller dosing volume was more appropriate, e.g., 5 mL/kg or less.

The dose levels of 1000, 800 and 600 mg/kg/day were selected also based on the available information on oral toxicity studies from other marketed PEG-lipid based polymers including Soluplus® (polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer), Kolliphor® RH40 (Polyoxyl 40 Hydrogenated Castor Oil), and Kolliphor® EL (Polyoxyl 35 Castor Oil). In addition, a vehicle control group (0 mg/kg/day) was used. The frequency and duration of administration was selected to maximize the probability of achieving the objective of the experiment using a minimum number of animals.

2.4.2. 90-day repeated dose toxicity study in beagle dogs

32 pre-examined and healthy dogs were included in the study; 16 females and 16 males of Naïve Beagle dogs (aged at 9–10 weeks) were purchased from Marshall Biotechnology Co., Ltd. (Beijing, China). An acclimatization period of 14 days was allowed before the start of treatment. Beagle dogs weighing 3–4 kg were divided into four (4) groups, DOPS-F02-treated group and Control (untreated) group as follows

Group	Number of animals		Necropsy
	F	M
Control*	5	5	End of dosing phase
			End of recovery phase
DOPS-F02 low dose	3	3	End of dosing phase
			End of recovery phase
DOPS-F02 middle dose	3	3	End of dosing phase
			End of recovery phase
DOPS-F02 high dose*	5	5	End of dosing phase
			End of recovery phase

Notes: Number at the 1st position in animal ID represents group (1, 2, 3 and 4 for control group, DOPS-F02 low, middle and high dose groups, respectively). Letter at the 2nd position represents sex: M for male and F for female. The last three numbers represent animal serial number in the specific group.

*Additional 14-day observation for recovery groups after end of the dosing phase.

The high (1000 mg/kg) dose group and Control group, each contained five animals/sex and the low (600 mg/kg) and middle (800 mg/kg) groups each contained three animals/sex. DOPS-F02 solution or sterile water was administered via gavage daily for 90 consecutive days

Detailed clinical observation was conducted at least once every week for items including but not limited to, appearance, movement, neurological signs, gland secretions, skin and mucosa color, respiration, genitals, and other signs of toxicity. Body weight of all surviving animals was recorded once before the first dosing, plus once a week in the dosing phase and recovery phase. Terminal body weight was recorded before necropsy and was only used to calculate organ weight ratios.

All surviving animals were examined once at approximately 2.0–3.0 h post-dose on Days 46, 89 and the day before the recovery phase necropsy. Dogs were placed in a hammock and lead-II ECG was evaluated with a multi-channel physiological recorder, with arterial pressure being measured with a non-invasive blood pressure meter. A stable single ECG waveform within 30 s was selected for measurement, and the corrected QT interval was calculated. A ECG segment containing at least 5 consecutive cardiac cycles was used to evaluate the presence of arrhythmia.

A Multi-channel Physiological Recorder, MP150 (Biopac Systems, Goleta, CA) was used to monitor or measure neurological signs by collecting physiological data, e.g., EEG, nerve conduction, and muscle activity.

Food intake within 24 h was calculated by substracting surplus food weight from the weights before dosing twice during a day.

Body temperature of all surviving animals was measured once on Days 7, 46, 89 and the day before the recovery phase necropsy. A thermistor thermometer was used to record the rectal temperature of dogs, once per animal.

Ophthalmic examination was conducted on all surviving animals once on recovery day 2 and on recovery day 13. Mydriasis assessment was conducted using Mydrin-P (Compound Tropicamide Eye Drops) with instillation of 1 drop/eye before examination. A binocular indirect ophthalmoscope was used for the examination of the conjunctiva, cornea, anterior chamber, iris, lens, posterior chamber and fundus.

At the end of the observation period, blood samples were collected from the jugular vein of each beagle dog and the animals were sacrificed, dissected, and examined for macroscopically visible changes.

2.4.3. Hematological investigation

Hematology for all surviving dogs was conducted at predose on Day 45,90 and before necropsy on recovery day 14. Animals were fasted for at least 12 h before sampling but given ad libitum access to water. 1.9 mL of blood was collected via the veins of limbs.

Blood samples for hematology testing (1 mL) were collected into tubes containing EDTA-K₂ as the anticoagulant, and then the whole blood was directly assayed. The analysis was completed within 24 h of collection, with samples being stored at 15–25 °C.

Samples for the coagulation test (0.9 mL) were collected into tubes with sodium citrate as the anticoagulant, then centrifuged at 15–25 °C and 1800 ×g for 10 min to harvest plasma. The analysis was completed within 8 h upon collection, with samples stored at 2–8 °C. The hematology parameters evaluated were: red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte count (RET#), reticulocyte percentage (RET%), white blood cell count (WBC), neutrophil (NEUT) absolute count and percentage, lymphocyte (LYMPH) absolute count and percentage, monocyte (MONO) absolute count and percentage, eosinophil (EOS) absolute count and percentage, basophil (BASO) absolute count and percentage, platelet count (PLT), prothrombin time (PT), and activated partial thromboplastin time (APTT).

2.4.4. Clinical chemistry

Clinical chemistry for all surviving dogs was conducted at predose on Day 45, 90 and before necropsy on recovery day 14. Animals were fasted for at least 12 h before sampling but given ad libitum access to water. Approximately 3 mL of blood was collected via veins of limbs.

Blood samples (without anticoagulant) were centrifuged at 1800 ×g and 15–25 °C for 10 min to obtain serum. The analysis was completed within 3 days and samples were stored at 2–8 °C. Any remaining sera were stored for backup. The following parameters were determined:: aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyltransferase (GGT), creatine kinase (CK), lactic dehydrogenase (LDH), alkaline phosphatase (ALP), urea (UREA), creatinine (CREA), total protein (TP), albumin (ALB), globulin (GLB), albumin/Globulin ratio (A/G), glucose (GLU), total bilirubin (TBIL), cholesterol (CHOL), triglycerides (TG), sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca), phosphorus (PHOS), a-pancreatic amylase (AMYP) and lipase (LIP).

2.4.5. Bone marrow smears

Bone marrow smears of animals for sacrifice were prepared on the scheduled necropsy days of dosing phase and recovery phase. After animals were anesthetized with pentobarbital sodium (i.v., 30 mg/kg, which was adjusted according to the health status of the animals). A myeloid puncture needle was used to puncture through the ilium to aspirate the bone marrow for smear preparation after which Wright-Giemsa staining was performed, and smears were examined microscopically. Bone marrow smears were assessed for granulocytic series (%), erythroid series (%), lymphocyte (%), monocyte (%), other cell (%), G/E ratio, nucleated cell proliferation and cell morphology.

2.4.6. Gross necropsy and histopathology

• (a)Gross Necropsy

  At the end of the treatment period, the animals were sacrificed and dissected. In beagle dogs, scheduled necropsy was conducted on 3 dogs/sex in each group at the end of dosing phase (two days after the last dosing), then on the remaining dogs at the end of recovery phase (recovery day 15).

  Animals were fasted for at least 12 h before terminal sacrifice but given ad libitum access to water. Animals were weighed and anesthetized with pentobarbital sodium (i.v., approximately 30 mg/kg) followed by external observation, femoral artery exsanguination and gross necropsy. The weight of heart, liver, lung, kidney, brain, spleen, pancreas, bladder, testis, uterine and thymus were measured and recorded

• (b)Histopathology

  All tissues and organs were preserved in 10 % neutral phosphate buffered formalin except for bilateral testes, epididymides, eyes and optic nerves, which were fixed in Modified Davidson′s Solution.

The specimens prepared from each group were further subjected to histopathology examination. They were trimmed, embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E), and examined microscopically. The results were verified and peerreviewed by board-certified veterinary pathologists.

2.4.7. TK sampling and analysis

Blood samples of animals in DOPS-F02 treated groups were collected at pre-dose and post-dose 0.5 h (±5 min), 2 h (±10 min), 4 h (±10 min), 12 h (±20 min), 24 h (±30 min), 36 h (±30 min) post-dose of the first and last dosing. Control animals were sampled at predose and 0.5 h (±1 min) post-dose of the first and last dosing. Whole blood samples (with EDTA-K₂ as anticoagulant) wiere temporarily placed in an ice box, then centrifuged at 1800 ×g and 2–8 °C for 10 min. The harvested plasma was divided into 2 tubes (50 µL in each one) and stored below −66 °C prior to analysis. The concentrations of DOPS-F02 in dog plasma were analyzed using a validated bioanalytical LC-MS/MS method. TK (Toxicokinetics) parameters, e.g., AUC, T_max, C_max and Cl were calculated using WinNonlin ver. 8.2 (Certara, Princeton, NJ).

2.4.8. Statistical analysis

Quantitative data such as body weight, food consumption, body temperature, ECG, blood pressure, hematology, clinical chemistry, urinary volume, bone marrow smear cell percentage, organ weight and ratios were presented as group mean ± standard deviation ((X̄ ± SD). Qualitative data (binomial category, unordered multi-category and ordinal multi-category) of urinalysis (excluding urinary volume) and nucleated cell proliferation on bone marrow smears were presented as observed counts (frequency). When the sample size was less than three, raw data of that group was directly presented without statistical comparison.

LEVENE’s test was used to analyze the variance homogeneity of quantitative data. In the case of homogeneity of variance (p > 0.05), they were evaluated using one-way analysis of variance (ANOVA); in the case of heterogeneity of variance (p ≤ 0.05), and small sample sizes (e.g., n ≥ 3 per group), the Kruskal-Wallis (K-W) H test was used for the analysis. In addition, if the ANOVA showed a significant difference (p ≤ 0.05), Dunnett’s t-test (Dunnett) was then used for pairwise comparisons; if ANOVA was not significant (p > 0.05), the statistical analysis was completed.

2.5. Part II. Wistar rats

2.5.1. Group assignemts

Pre-examined healthy rats were assigned to ten (10) groups including treatment and vehicle control (G1), low dose (G2), intermediate dose (G3), high dose (G4), vehicle control recovery (G5), high dose recovery (G6) as follows:

Group	Dose (mg/kg b.wt)	Dose volume (mL/kg b.wt)	Total number of animals
			Male	Female
G1 (Vehicle Control)	0	12.5	10	10
G2 (Low Dose)	750	3.75	10	10
G3 (Intermediate Dose)	1250	6.25	10	10
G4 (High Dose)	2500	12.5	10	10
G5 (Vehicle Control Recovery)1	0	12.5	5	5
G6 (High Dose Recovery)1	2500	12.5	5	5

¹additional 14 days observation after the180-day dosing phase

In addition, there were four Toxicokinetic (TK) groups:G7 (vehicle control-TK), G8 (low dose-TK), G9 (intermediate dose -TK) and G10 (high dose-TK). The test item was formulated using sterile water as the vehicle. Dose formulations were administered at dose levels of 750 mg/kg (G2, G8), 1250 mg/kg (G3, G9) and 2500 (G4, G6 and G10) mg/kg/day for 180 days. Animals were acclimatized to laboratory conditions at least 5 days before the start of the treatment. During acclimatization animals were observed for any abnormalities. They were randomly assigned to cages and the individual animal was fur marked with picric acid. Animals were selected and grouped based on stratified randomization by using body weights one day before dosing. Randomization details were included in raw data with a minimum additional 10 % of animals being taken for randomization. The DOPS-F02 solutions were administered by the oral route using an oral gavage needle, e.g., 16–18 gauge feeding tubes about 2–3 in. in length, at the desired dose level daily for up to 180 days.

2.5.2. 180-day repeated dose toxicity study in Wistar rats

In this study, the test article, DOPS-F02, was administered by the oral (gavage) route for extended periods to evaluate potential toxicity after longer term exposure. The dose levels of 2500, 1250 and 750 mg/kg/day were selected based on the available information on oral toxicity from other safety studies conducted using the marketed PEG-polymers including Kolliphor® RH40 and Kolliphor® EL. In addition, a vehicle control group (0 mg/kg/day) was used. The frequency and duration of administration was selected to maximize the probability of achieving the objective of the experiment using a minimum number of animals.

Clinical Observations. The animals were observed for morbidity/mortality twice daily throughout the observation period. All animals were observed for general clinical signs once daily during acclimatization, treatment and the recovery period. Detailed clinical observations were made once before the start of treatment, and weekly thereafter during the treatment and recovery period. Detailed clinical observations included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, and unusual respiratory pattern), Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backwards) were recorded.

Ophthalmological examination was performed using a direct ophthalmoscope once before the treatment period for all the group animals and at the end of treatment period and recovery period for vehicle control (G1, G5) and high dose (G4, G6) group animals. No abnormality was detected in the eyes of these animals; hence, ophthalmological examinations were not performed for the other groups.

Body weights of the animals were recorded on the day of randomization, on the day of dosing, and weekly thereafter for treatment groups. Terminal (fasting) body weights were taken on the day of scheduled necropsy. The body weight changes for all the animals were calculated and reported along with the body weight data.

Feed consumption was recorded weekly once and reported as grams/animal per day (averaged value/per cage, e.g., 2 animals same sex per cage).

2.5.3. Hematological and clinical chemistry investigation

Blood samples were collected at the end of the treatment period (Day 181) from main groups and day 195 (G4 & G6 Groups) from recovery groups for hematology, coagulation, and clinical biochemistry evaluations. The animals were fasted overnight prior to blood collection and water was provided ad libitum. Blood was collected from the retro-orbital plexus under light isoflurane anesthesia. Blood samples were centrifuged for separation of plasma for clinical biochemistry analysis and coagulation analysis. The following parameters were evaluated using an ADVIA 2120i analyzer (Siemens, Germany): red blood cell count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean platelet volume, reticulocytes counts, white blood corpuscles, differential leukocyte count, platelets. The following clinical biochemistry parameters were determined: glucose (GLU), creatinine (CREAT), total cholesterol (T.Chol), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT), total plasma protein (T. Pro), albumin (ALB), total bilirubin (T.BIL), HDL cholesterol (HDL-C), LDL cholesterol (LDL-C), lactate dehydrogenase (LDH), creatinine kinase (CK), triglycerides (Trig), calcium (Ca), inorganic phosphorus (Pi), sodium (Na), potassium (K), blood urea nitrogen (BUN), serum thyroxine (T4), triiodothyronine (T3), thyroid stimulating hormone (TSH).

2.5.4. Necropsy and gross pathology

At the end of the treatment period (Day 181) for the main groups and day 195 (G5 & G6 Groups) from recovery groups, all the animals were euthanized in stratified order with CO₂ and subjected to gross pathological examination. The following organs were collected and preserved, as applicable, from males and females: adrenal glands, femur, brain (cerebrum, cerebellum, and pons), cecum, colon, duodenum, epididymides, heart, ileum with Peyer's patches, jejunum, kidneys, liver, lungs, lymph nodes (one from the route of administration and another from distant site of administration), pancreas, prostate + seminal vesicles with coagulating glands, eyes (if changes were observed), parathyroid, ovaries, rectum, peripheral nerve (sciatic), spinal cord (cervical, mid thoracic, lumbar), spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus with cervix, vagina, sternum (bone marrow), skeletal muscle, skin (if changes observed), mammary glands, aorta, pituitary gland, bone marrow smear, esophagus, salivary glands. Adherent adipose tissue from the organs was trimmed off and the wet weight of organs was recorded. Organs were preserved in 10 % neutral buffered formalin except testes, which, were preserved in modified Davidson‟s fixative and eyes were preserved in Davidson’s fixative for 48 h and then preserved in 10 % neutral buffered formalin. The following organ weights: brain,thymus, spleen, testes or ovaries, heart, kidneys, liver, adrenals, uterus with cervix/ epididymides, seminal vesicles with coagulating gland and prostate (after fixation), thyroid with parathyroid and pituitary gland, thyroid with parathyroid (after fixation), pituitary (after fixation), were recorded on the scheduled dates of necropsy. Paired organs were weighed together. Terminal body weight on the day of necropsy was used to calculate organ weights relative to body weight. Wet weight was taken as soon as possible. Post fixation organ weights were recorded for seminal vesicles with coagulating gland, prostate, thyroid with parathyroid and pituitary gland.

2.5.5. Histopathology

All organ and tissue samples were processed, embedded and cut at an approximate thickness of 3–5 micrometers, and stained with hematoxylin and eosin. Histopathology was performed on all preserved organs of the vehicle control (G1) and the high dose groups (G4). The tissue sections were stained by haematoxylin and eosin (H & E) and examined under the light microscope by the study pathologist for evaluation of histopathological lesions. Additionally, target organ/tissue (stomach) from the low dose (G2), intermediate dose (G3), and reversal control (G5), along with reversal high dose (G6) group, were processed for evaluation of histopathological lesions.

2.5.6. Toxicokinetic study

The objective of the toxicokinetic (TK) study was to determine the plasma exposures and assess the dose-exposure relationship of DOPS-F02 following Days 1 and 180. Blood samples were collected at specified times with ∼ 0.1 mL of blood being collected at each sampling time point from the retro orbital sinus plexus being put into tubes (containing K₂EDTA) and held on ice until processing and transfer to a freezer. Blood samples were centrifuged following collection at ∼3000 g for 5–10 min in 4–8 °C to obtain plasma. The plasma was stored at approximately −70 °C or colder until transporting for analysis. All samples were be labeled with detailed information such as study number, animal number, and matrix, time points of collection and date of collection. No clinical pathology and necropsy examinations were carried out for these TK study animals.

Blood samples were collected at times specified under blood sample collection for pharmacokinetics. The actual mid-point time of collection of each blood sample (to the nearest minute) was noted. The following time windows for blood collection were considered as deviations: ± 2 min for 0.5 h.–8 h and ± 5 min for 24 h. The concentrations of DOPS-F02 in rat plasma were analyzed using a validated bioanalytical method (LC-MS/MS). TK (Toxicokinetics) parameters, e.g., AUC, T_max, C_max and Cl were calculated with WinNonlin ver. 8.2 (Certara, Princeton, NJ).

2.5.7. Statistical analysis

Statistical analyses were performed on the body weight, feed consumption, organ weights as well as clinical pathology data. Data were summarized in tabular form and results were analyzed using Analysis of Variance (ANOVA) followed by Dunnett’s test for post hoc comparison (n ≥ 3 per group). Values were reported as mean (X̄) ± standard deviation (SD).

3. Results

3.1. Dog study

DOPS-F02 did not affect blood pressures and electrocardiograms in the dogs exposed to ≤ 1000 mg/kg of DOPS-F02. Overall, no adverse event or severe adverse event was reported to the oral intake of DOPS-F02 in the entire study. The excipient also did not affect laboratory parameters in the dogs exposed to ≤ 1000 mg/kg.

Clinical Observation. The animals in all groups survived to the scheduled end of the study. During the dosing phase, the animals occasionally showed soft or loose stools (yellow unformed feces) in all groups, with more incidences in the dosed groups than the control, and some also infrequently had yellow watery feces, which all reversed after the dose-withdrawal. Soft stools can be due to digestive issues with juvenile dogs, especially when fed with plain water under fasting conditions, and was probably exacerbated by PEG that is an osmotic laxative commonly used to improve bowel movements. However, animals exhibiting occasional soft or loose stools did not show weight loss and had normal appetites. No emesis was noted in any groups and no other abnormalities were noted in each group during the dosing phase and the recovery phase.

Compared with the control animals, female, and male dogs in DOPS-F02 groups showed no remarkable changes in body temperature on Days 7, 46, 89 and at the end of recovery phase. Compared with the control animals, female, and male dogs in DOPS-F02 groups showed no arrhythmia on Days 46, 89 and at the end of recovery phase, with no remarkable abnormalities in PR interval, QRS duration, P duration, RR interval, QT interval, corrected QT interval, heart rate, systolic pressure, diastolic pressure or mean artery pressure.

Body Weight and Food Comsuption Both genders in DOPS-F02 600, 800 mg/kg groups and 1000 mg/kg females had normal body weight gain during the dosing phase as compared with the concurrent control (P > 0.05). There was no abnormality in the body weight of all animals during the recovery phase (Fig. 2, Fig. 3).

Fig. 2.

Effects of DOPS-F02 on Body Weight of Male Dogs.

Fig. 3.

Effects of DOPS-F02 on Body Weight of Female Dogs.

No significant differences were noted in food consumption of DOPS-F02-treated dogs during the dosing phase as compared with the control group (P > 0.05). The food consumption was lower cross all groups of male dogs, especially in untreated animals and no abnormality in body weight was observed. There were no weight abnormalities during the recovery phase (Fig. 2, Fig. 3). Three females in the 1000 mg/kg dose group showed occasional decreased food consumption, but with no change in their body weight, which was considered to be related to the eating habits of animals, from the overall qualitative evaluation of food consumption.

3.1.1. Hematology

Compared with the control animals, female and male dogs in DOPS-F02 groups showed no remarkable abnormalities in Hematology including WBC, WBC differentials, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, RET, RET%, APTT or PT on Days 45, 90 and at the end of recovery phase (Table 1).

Table 1.

(1) Effect of DOPS-F02 on hematology of male dogs at end of the treatment (Day-90).

Parameters (X̄ ± SD)		WBC (109/L)	NEUT% (%)	LYMPH% (%)	MONO% (%)	EOS% (%)	BASO% (%)	NEUT (109/L)	LYMPH (109/L)	MONO (109/L)	EOS (109/L)	BASO (109/L)
Control	N = 5	8.71 ± 1.370	47.9 ± 3.92	41.5 ± 4.03	5.9 ± 0.51	2.1 ± 0.88	1.0 ± 0.21	4.14 ± 0.446	3.65 ± 0.885	0.51 ± 0.073	0.19 ± 0.074	0.08 ± 0.019
600 mg/kg	N = 3	9.83 ± 1.956	52.8 ± 6.64	37.4 ± 5.01	5.6 ± 1.64	2.3 ± 0.95	0.8 ± 0.46	5.27 ± 1.646	3.62 ± 0.303	0.53 ± 0.061	0.22 ± 0.110	0.07 ± 0.044
800 mg/kg	N = 3	10.93 ± 1.461	48.6 ± 5.92	42.4 ± 5.47	5.8 ± 0.32	1.6 ± 0.68	0.5 ± 0.10	5.37 ± 1.313	4.58 ± 0.067	0.64 ± 0.106	0.17 ± 0.046	0.05 ± 0.010
1000 mg/kg	N = 5	10.46 ± 1.722	52.0 ± 7.03	38.3 ± 5.87	6.1 ± 0.20	1.8 ± 0.98	0.6 ± 0.04	5.51 ± 1.515	3.95 ± 0.573	0.64 ± 0.106	0.18 ± 0.091	0.06 ± 0.007
Parameters (X̄ ± SD)		RBC (1012/L)	HGB (g/L)	HCT (%)	MCV(fL)	MCH (pg)	MCHC (g/L)	PLT (109/L)	RET% (%)	RET# (1012/L)	APTT (sec)	PT (sec)
Control	N = 5	6.46 ± 0.247	149 ± 4.2	47.6 ± 1.39	73.6 ± 0.81	23.1 ± 0.31	314 ± 1.5	411 ± 62.0	1.40 ± 0.154	0.090 ± 0.0086	16.0 ± 0.49	5.7 ± 0.20
600 mg/kg	N = 3	6.60 ± 0.193	152 ± 4.9	48.1 ± 1.28	72.9 ± 0.20	23.0 ± 0.17	316 ± 2.1	374 ± 79.7	1.77 ± 0.482	0.117 ± 0.0325	15.4 ± 0.15	5.5 ± 0.32
800 mg/kg	N = 3	6.45 ± 0.070	150 ± 3.6	47.5 ± 1.01	73.6 ± 0.79	23.2 ± 0.35	316 ± 3.1	398 ± 70.8	1.61 ± 0.334	0.104 ± 0.0217	15.5 ± 0.21	5.4 ± 0.10
1000 mg/kg	N = 5	6.19 ± 0.301	144 ± 4.6	45.8 ± 1.63	73.9 ± 1.45	23.2 ± 0.54	313 ± 3.0	397 ± 94.2	0.88 ± 0.166 *	0.054 ± 0.0083 *	15.4 ± 1.22	5.5 ± 0.25
N = number of animals, mg/kg – milligram/kilogram, b.w – Body weight*Statistical significance was noted in the difference of mean value when compared with the control (P ≤ 0.05).

(2) Effects of DOPS-F02 on Hematology of Female Dogs at End of the Treatment (Day - 90)
Parameters (X̄ ± SD)		WBC (109/L)	NEUT% (%)	LYMPH% (%)	MONO% (%)	EOS% (%)	BASO% (%)	NEUT (109/L)	LYMPH (109/L)	MONO (109/L)	EOS (109/L)	BASO (109/L)
Control	N = 5	11.87 ± 1.498	53.4 ± 4.32	37.5 ± 4.67	5.6 ± 0.53	1.6 ± 0.59	0.7 ± 0.20	6.38 ± 1.262	4.42 ± 0.515	0.66 ± 0.045	0.20 ± 0.095	0.08 ± 0.024
600 mg/kg	N = 3	8.79 ± 0.885 *	54.3 ± 5.40	36.3 ± 5.33	5.8 ± 0.51	1.8 ± 1.07	0.6 ± 0.20	4.80 ± 0.981	3.16 ± 0.258	0.51 ± 0.050 *	0.16 ± 0.093	0.05 ± 0.010
800 mg/kg	N = 3	10.63 ± 1.305	49.1 ± 4.67	42.0 ± 4.88	4.7 ± 0.40	2.0 ± 0.81	0.8 ± 0.06	5.18 ± 0.307	4.51 ± 1.071	0.49 ± 0.053 *	0.21 ± 0.066	0.09 ± 0.015
1000 mg/kg	N = 5	9.50 ± 1.460 *	52.4 ± 9.24	38.6 ± 8.92	5.8 ± 0.82	1.3 ± 0.21	0.6 ± 0.15	5.00 ± 1.347	3.65 ± 0.912	0.55 ± 0.064 *	0.13 ± 0.033	0.05 ± 0.011
Parameters (X̄ ± SD)		RBC (1012/L)	HGB (g/L)	HCT (%)	MCV(fL)	MCH (pg)	MCHC (g/L)	PLT (109/L)	RET% (%)	RET# (1012/L)	APTT (sec)	PT (sec)
Control	N = 5	6.78 ± 0.392	159 ± 7.2	50.6 ± 2.10	74.8 ± 1.45	23.4 ± 0.44	313 ± 2.9	629 ± 166.8	1.57 ± 0.383	0.106 ± 0.0453	15.8 ± 0.70	5.5 ± 0.23
600 mg/kg	N = 3	6.38 ± 0.677	147 ± 15.1	46.7 ± 4.60	73.1 ± 0.59	23.0 ± 0.15	315 ± 2.0	525 ± 99.7	1.42 ± 0.251	0.155 ± 0.0751	16.7 ± 0.82	5.7 ± 0.17
800 mg/kg	N = 3	6.67 ± 0.074	159 ± 4.0	50.1 ± 1.35	75.2 ± 1.36	23.9 ± 0.44	318 ± 0.6	531 ± 104.6	1.56 ± 0.147	0.130 ± 0.0240	15.4 ± 0.40	5.6 ± 0.23
1000 mg/kg	N = 5	6.35 ± 0.360	146 ± 5.9	46.3 ± 1.31	73.0 ± 2.47	23.0 ± 0.79	315 ± 4.4	608 ± 120.0	1.07 ± 0.496	0.124 ± 0.0420	15.4 ± 0.84	5.4 ± 0.26

N = number of animals

* Statistical significance was noted in the difference of mean value when compared with the control (P ≤ 0.05).

3.1.2. Clinical chemistry

The results of Clinical Chemistry in comparison with the control animals, the DOPS-F02 dosed female and male dogs showed no remarkable abnormalities in ALB, TP, AST, ALT, TBIL, CK, GGT, CHOL, TG, Crea, GLU, Urea, ALP, LDH, K⁺, Na⁺, Cl^-, A/G, GLB, Ca, P, AMYP or LIPC on Days 45, 90 and at the end of recovery phase (Table 2).

Table 2.

(1) Effect of DOPS-F02 on clinical chemistry of male dogs at end of treatment (Day-90).

Parameters (X̄ ± SD)		ALB (g/L)	TP (g/L)	AST (U/L)	ALT (U/L)	TBIL (μmol/L)	CK (U/L)	CHOL (mmol/L)	Ca (mmolL)	TG (mmol/L)	CREA (μmol/L)	GLU (mmol/L)	UREA (mmol/L)
Control	N = 5	34.8 ± 1.10	53.9 ± 3.36	31.0 ± 3.98	21.8 ± 3.96	1.0 ± 0.24	295 ± 46.6	5.35 ± 0.560	2.93 ± 0.055	0.37 ± 0.073	47.5 ± 5.71	5.18 ± 0.219	3.94 ± 0.645
600 mg/kg	N = 3	33.9 ± 1.61	55.1 ± 1.21	32.3 ± 6.16	24.0 ± 3.61	0.6 ± 0.26	327 ± 79.7	4.63 ± 0.637	2.87 ± 0.040	0.49 ± 0.127	45.0 ± 0.46	5.64 ± 0.339	3.55 ± 0.344
800 mg/kg	N = 3	32.6 ± 1.22	52.8 ± 0.47	30.8 ± 4.03	26.0 ± 4.36	0.5 ± 0.15	370 ± 75.3	4.25 ± 0.344	2.81 ± 0.075	0.47 ± 0.080	44.4 ± 2.29	5.34 ± 0.529	3.69 ± 0.310
1000 mg/kg	N = 5	34.1 ± 1.89	52.2 ± 4.67	28.4 ± 3.39	25.0 ± 7.87	0.9 ± 0.22	291 ± 50.5	5.37 ± 0.997	2.87 ± 0.048	0.44 ± 0.063	44.9 ± 4.54	5.86 ± 0.234 *	3.49 ± 0.356
Parameters (X̄ ± SD)		GGT (U/L)	PHOS (mmol/L)	ALP (U/L)	LDH (U/L)	AMYP (U/L)	LIPC (U/L)	K+ (mmol/L)	Na + (mmol/L)	Cl- (mmol/L)	A/G	GLB (g/L)
Control	N = 5	2.5 ± 0.56	2.07 ± 0.187	120.6 ± 20.75	138 ± 34.0	352.1 ± 99.97	48.2 ± 36.24	5.29 ± 0.232	145.8 ± 0.84	110.1 ± 0.43	1.85 ± 0.262	19.1 ± 2.78
600 mg/kg	N = 3	2.4 ± 0.21	2.18 ± 0.085	122.3 ± 55.67	175 ± 77.0	310.7 ± 54.00	30.6 ± 14.95	5.55 ± 0.273	146.3 ± 1.15	110.5 ± 1.35	1.60 ± 0.108	21.2 ± 0.44
800 mg/kg	N = 3	2.3 ± 0.70	2.15 ± 0.046	112.0 ± 31.89	132 ± 46.5	294.2 ± 24.96	31.3 ± 11.91	5.52 ± 0.335	144.7 ± 1.15	109.9 ± 0.61	1.62 ± 0.150	20.1 ± 1.12
1000 mg/kg	N = 5	2.5 ± 0.66	2.06 ± 0.106	113.0 ± 18.69	123 ± 44.2	327.5 ± 34.88	41.3 ± 20.25	5.19 ± 0.271	146.2 ± 1.30	111.5 ± 1.92	1.93 ± 0.324	18.1 ± 3.55
N = number of animals *Statistical significance was noted in the difference of mean value when compared with the control (P ≤ 0.05).

(2) Effects of DOPS-F02 on Clinical Chemistry of Female Dogs at End of Treatment (Day-90)
Parameters (X̄ ± SD)			ALB (g/L)		TP (g/L)		AST (U/L)		ALT (U/L)	TBIL (μmol/L)		CK (U/L)	CHOL (mmol/L)	Ca (mmol/L)	TG (mmol/L)	CREA (μmol/L)	GLU (mmol/L)	UREA (mmol/L)
Control		N = 5	34.8 ± 1.10		53.9 ± 3.36		31.0 ± 3.98		21.8 ± 3.96	1.0 ± 0.24		295 ± 46.6	5.35 ± 0.560	2.93 ± 0.055	0.37 ± 0.073	47.5 ± 5.71	5.18 ± 0.219	3.94 ± 0.645
600 mg/kg		N = 3	33.9 ± 1.61		55.1 ± 1.21		32.3 ± 6.16		24.0 ± 3.61	0.6 ± 0.26		327 ± 79.7	4.63 ± 0.637	2.87 ± 0.040	0.49 ± 0.127	45.0 ± 0.46	5.64 ± 0.339	3.55 ± 0.344
800 mg/kg		N = 3	32.6 ± 1.22		52.8 ± 0.47		30.8 ± 4.03		26.0 ± 4.36	0.5 ± 0.15		370 ± 75.3	4.25 ± 0.344	2.81 ± 0.075	0.47 ± 0.080	44.4 ± 2.29	5.34 ± 0.529	3.69 ± 0.310
1000 mg/kg		N = 5	34.1 ± 1.89		52.2 ± 4.67		28.4 ± 3.39		25.0 ± 7.87	0.9 ± 0.22		291 ± 50.5	5.37 ± 0.997	2.87 ± 0.048	0.44 ± 0.063	44.9 ± 4.54	5.86 ± 0.234 *	3.49 ± 0.356
Parameters (X̄ ± SD)				GGT (U/L)		PHOS (mmol/L)		ALP (U/L)			LDH (U/L)	AMYP (U/L)	LIPC (U/L)	K+ (mmol/L)	Na + (mmol/L)	Cl- (mmol/L)	A/G	GLB (g/L)
Control	N = 5			2.5 ± 0.56		2.07 ± 0.187		120.6 ± 20.75			138 ± 34.0	352.1 ± 99.97	48.2 ± 36.24	5.29 ± 0.232	145.8 ± 0.84	110.1 ± 0.43	1.85 ± 0.262	19.1 ± 2.78
600 mg/kg	N = 3			2.4 ± 0.21		2.18 ± 0.085		122.3 ± 55.67			175 ± 77.0	310.7 ± 54.00	30.6 ± 14.95	5.55 ± 0.273	146.3 ± 1.15	110.5 ± 1.35	1.60 ± 0.108	21.2 ± 0.44
800 mg/kg	N = 3			2.3 ± 0.70		2.15 ± 0.046		112.0 ± 31.89			132 ± 46.5	294.2 ± 24.96	31.3 ± 11.91	5.52 ± 0.335	144.7 ± 1.15	109.9 ± 0.61	1.62 ± 0.150	20.1 ± 1.12
1000 mg/kg	N = 5			2.5 ± 0.66		2.06 ± 0.106		113.0 ± 18.69			123 ± 44.2	327.5 ± 34.88	41.3 ± 20.25	5.19 ± 0.271	146.2 ± 1.30	111.5 ± 1.92	1.93 ± 0.324	18.1 ± 3.55

N = number of animals

*Statistical significance was noted in the difference of mean value when compared with the control (P ≤ 0.05).

3.1.3. Urinalysis

Compared with the control animals, female and male dogs in DOPS-F02 groups showed no remarkable abnormalities in Urinalysis, normal values obtained in COL, CLA, pH, GLU, BIL, KET, BLO, PRO, URO, NIT, WBC, SG, urinary sediment (crystal, cast, epithelial cells) or urinary volume on Day 89 and at the end of recovery phase.

3.1.4. Ophthalmic examination

In ophthalmic examination, dogs in DOPS-F02 treated groups had clear fundus vessels with no hemorrhage or exudation on Recovery day 2 and at the end of recovery phase. The optic disc showed no edema. The diameter of arteries and veins was normal, and veins had no tortuosity. No abnormalities were noted in conjunctiva, cornea, anterior chamber, iris, lens or posterior chamber.

3.1.5. Toxicologic pathology

Bone marrow parameters

Compared with the control animals, bone marrow smears in DOPS-F02 dosed female and male dogs showed no remarkable abnormalities in granulocytic series (%), erythroid series (%), lymphocyte (%), monocyte (%), other cell (%), G/E ratio, nucleated cell proliferation or cell morphology at the end of dosing phase and recovery phase.

Absolute organ weights & Relative organ weights

No remarkable abnormalities in comparison with the control group were noted in the organ weight and ratios of brain, heart, thymus, liver, spleen, kidney, adrenal gland, uterus/cervix, ovary, testis, epididymis, thyroid and parathyroid glands of dogs in DOPS-F02 groups at the end of dosing phase and recovery phase.

No abnormalities in gross necropsy as compared to the control group regarding size, morphology, color and texture were noted in main organs, such as brain, heart, liver, spleen, lung, kidney, adrenal gland, thymus, gastrointestinal tract and reproductive organ, of all necropsied dogs in DOPS-F02 groups at the end of dosing phase and recovery phase.

No DOPS-F02-related microscopic findings were noted in examined organs or tissues, such as brain, heart, liver, spleen, lung, kidney, adrenal gland, gastrointestinal tract, uterus, ovary, testis and epididymis, of dogs in each group at the end of dosing phase and recovery phase. Microscopic observations for ovary tissues showed immature presented in all female dogs due to a juvenile or pre-pubertal stage

Compared with the control animals, in DOPS-F02 dosed female and male dogs showed no remarkable abnormalities in granulocytic series (%), erythroid series (%), lymphocyte (%), monocyte (%), other cell (%), G/E ratio, nucleated cell proliferation or cell morphology at the end of dosing phase and recovery phase. No remarkable abnormalities in comparison with the control group were noted in the organ weight and ratios of brain, heart, thymus, liver, spleen, kidney, adrenal gland, uterus/cervix, ovary, testis, epididymis, thyroid and parathyroid glands of dogs in DOPS-F02 groups at the end of dosing phase and recovery phase (Fig. 6, Fig. 7).

Fig. 4.

Effects of DOPS-F02 on Food Consumption of Male Dogs.

Fig. 5.

Effects of DOPS-F02 on Food Consumption of Female Dogs.

Fig. 6.

1 Mean organ-to-body weight ratio (%) at the end of treatment– male (0 mg/kg = control). 2 Mean organ-to-brain weight ratio (%) at the end of treatment –male.

Fig. 7.

1Mean organ-to-body weight ratio (%) at the end of treatment – female. 2 Mean organ-to-brain weight ratio (%) at the end of treatment – female.

No abnormalities in comparison with the control group regarding size, morphology, color and texture were noted in main organs, such as brain, heart, liver, spleen, lung, kidney, adrenal gland, thymus, gastrointestinal tract and reproductive organ, of all necropsied dogs in DOPS-F02 groups at the end of dosing phase or recovery phase.

3.1.6. Toxicokinetics

Following daily oral doses of DOPS-F02 at 600, 800 and 1000 mg/kg in juvenile Beagle dogs for 90 consecutive days, the mean AUC_last (calculated based on the available data at 24 hr or 36 hr) of plasma DOPS-F02 showed no obvious gender differences after the first and last doses. The mean AUC_last generally increased with increased dose levels over the range from 600 to 1000 mg/kg. Certain accumulation of DOPS-F02 was observed in juvenile Beagle dogs after the 90-day repeated dosing (Fig. 8, Fig. 9). Comparing the results from Day 90 dose with the Day 1 dose, the mean individual AUC_last ratios of plasma DOPS-F02 were 2.1, 1.9, 1.3 for females and 1.5, 1.7, 1.9 for males in each group, respectively. The half-life (t_1/2) was calculated using data from selected 800 mg/kg and 1000 mg/kg dosed female and male animals. There was some variability due to the low bioavailability, and this also prevented measurement of some TK parameters such AUC_last. The t_1/2 was 5.5 hr on Day 1 and averaged 6.9 hr. (range from 6.7 to 7.1 hr.) on Day 90 for 800 mg/kg dosed male animals, the t_1/2 averaged 6.6 hr. (range from 5.1 to 8.1 hr.) on Day 90 for 800 mg/kg dosed females, respectively. The t_1/2 averaged 4.2 hr (range from 4.1 to 4.3 hr) on Day 1 and averaged 6.4 hr (range from 4.7 to 8.6 hr.) on Day 90 for 1000 mg/kg dosed females and 5.4 hr. on Day 90 for 1000 mg/kg dosed males (Table 3).

Fig. 8.

Mean Plasma DOPS-F02 Concentration-Time Curve in Juvenile Beagle Dogs after the first Oral Administration of DOPS-F02.

Fig. 9.

Mean Plasma DOPS-F02 Concentration-Time Curve in Juvenile Beagle Dogs after the Last Oral Administration of DOPS-F02.

Table 3.

Mean TK parameters for plasma DOPS-F02 in juvenile beagle dogs orally administered with DOPS-F02.

Dosage (mg/kg)	Sex	Animal	Day 1					Day 90
		ID	AUClast	Cmax	Tmax	CL/F	T1/2	AUClast	AUClast	Cmax	Tmax	CL/F	T1/2
			(h*ng/mL)	(ng/mL)	(h)	(mL/h/kg)	(h)	(h*ng/mL)*	(h*ng/mL)**	(ng/mL)*	(h)*	(mL/h/kg)**	(h)**
600	F	Mean (n = 3)	10500	1250	2	28200	12	19600	20100	2130	3.3	33900	9.4
		SD	4090	279	0	/	/	6380	5930	781	1.2	/	/
	M	Mean	12100	1690	3.3	/	/	17700	17700	2060	4	/	/
		SD	4050	250	1.2	/	/	3300	3300	221	0	/	/
800	F	Mean (n = 3)	10600	1750	2.7	/	/	19700	19700	2220	2.7	43800	6.6
		SD	1220	274	1.2	/	/	5390	5390	590	1.2	4910	2.1
	M	Mean	12900	1650	2.7	53000	5.5	22000	22800	2630	2.7	38200	6.9
		SD	1280	247	1.2	/	/	5590	5840	668	1.2	9580	0.31
1000	F	Mean (n = 5)	18400	2070	2.8	40100	4.2	23200	23900	2470	2.8	42700	6.4
		SD	6490	593	1.1	1060	0.15	4520	5250	517	1.1	14200	2
	M	Mean (n = 5)	12700	1640	3.2	/	/	22500	22500	2370	3.6	37300	5.4
		SD	3990	345	1.1	/	/	6600	6600	580	0.89	/	/

Notes: "*" means the time period of the parameter calculation was 0–24 h, "**" means the time period of the parameter calculation was 0–36 h.

"/" means no data (due to the low bioavailability); Rsq_adjusted < 0.7 or cannot be fitted, hence the relevant parameter (CL/F, T_1/2) was not reported.

T_max of plasma DOPS-F02 concentrations generally ranged from 2.0 to 4.0 h following oral doses of DOPS-F02 at the dosing levels of 600, 800 and 1000 mg/kg (dosage ratio=1:1.3:1.7).

On Day 1, the mean AUC_last from plasma levels of DOPS-F02 were 10500, 10600 and 18400 h*ng/mL for the females corresponding to doses of 600, 800 and 1000 mg/kg, with the inter-group ratio of 1:1.0:1.8. The mean AUC_last values were 12100, 12900 and 12700 h*ng/mL for the males corresponding to doses of 600, 800 and 1000 mg/kg, with the inter-group ratio of 1:1.1:1.0. The female-to-male mean AUC_last ratios were 0.9, 0.8 and 1.4 in each of the dosed groups, respectively.

On Day 90, the mean AUC_last values from plasma levels of DOPS-F02 were 19600, 19700 and 23200 h*ng/mL for females corresponding to 600, 800 and 1000 mg/kg dose exposure, with the inter-group ratio of 1:1.0:1.2. The mean AUC_last results were 17700, 22000 and 22500 h*ng/mL for males corresponding to 600, 800 and 1000 mg/kg dose exposure, with the inter-group ratio of 1:1.2:1.3. The female-to-male mean AUC_last ratios were 1.1, 0.9 and 1.0 in each of the dosed groups, respectively.

The plasma DOPS-F02 concentrations of dogs in all dosed groups were generally below the detection limit at 36 h post the last dose. Only four dogs showed detectable levels of DOPS-F02 in their blood samples, and the detectable concentrations accounted for only ∼ 2–5 % of their individual C_max.

Due to low detectable DOPS-F02 concentrations, the half-life (t_1/2) was calculated on selected 800 mg/kg and 1000 mg/kg dosed female and male animals. While there was some variability, the t_1/2 was 5.5 hr on Day 1 and averaged 6.9 hr (range from 6.7 to 7.1 hr) on Day 90 for 800 mg/kg dosed male animals, the t_1/2 averaged 6.6 hr (range from 5.1 to 8.1 hr) on Day 90 for 800 mg/kg dosed females, respectively. The t_1/2 averaged 4.2 hr (range from 4.1 to 4.3 hr) on Day 1 and averaged 6.4 hr (range from 4.7 to 8.6 hr) on Day 90 for 1000 mg/kg dosed females and 5.4 hr on Day 90 for 1000 mg/kg dosed male.

There was no other abnormality found in the entire studying period.

3.2. Rat study results

DOPS-F02 did not affect laboratory parameters in the rats exposed to ≤ 2500 mg. Overall, no adverse event or severe adverse event was reported to the oral intake of DOPS-F02 in the entire study

3.2.1. Clinical observation

During the repeated dose phase, no clinical signs or mortality were observed in animals treated with 750, 1250, and 2500 mg/kg body weight throughout the treatment period when compared with vehicle control group. No treatment related or significant changes were observed in any of the functional observation battery parameters. The ophthalmological examinations also showed no abnormalities during the treatment period.

3.2.1.1. Body weights

There was no treatment related significant differences in body weight and body weight changes observed during the study. However, a statistically significant difference in the recovery group high dose male animals was observed on week 0, week 3, and week 7 (slightly elevated weight gains) when compared with vehicle control group (Fig. 10, Fig. 11, Fig. 12, Fig. 13).

Fig. 10.

Effects of DOPS-F02 on Body Weight of Male Rats.

Fig. 11.

Effects of DOPS-F02 on Body Weight of Female Rats.

Fig. 12.

Effects of DOPS-F02 on Body Weight of Male Rats in the Recovery Group.

Fig. 13.

Effects of DOPS-F02 on Body Weight of Female Rats in the Recovery Group.

3.2.1.2. Feed consumption

No treatment-related changes were observed in the feed consumption across different groups compared to the respective vehicle control groups,

3.2.1.3. Clinical laboratory investigations

There were no findings of treatment related changes in the hematology and coagulation parameters observed in any of the treated groups up to 2500 mg/kg when compared with vehicle control group (Table 4, Table 5). While all were within the normal ranges, some of the results fluctuated with these results considered to be due to biological variance and unrelated to treatment. This view was supported by the evidence that there were no dose dependent increase or decrease as compared (e.g., the high dosed group) to vehicle at the end of the study.

Table 4.

Summary of haematology values at the end of treatment – rodent males (high dose group).

Group &Dose		WBC	RBC	HGB	HCT	MCV	MCH	MCHC
(mg/kgb.w/Day)		109/L	1012/L	g/L	L/L	fL	Pg	g/L
G1 &	Mean	13.53	8.52	143.5	0.44	51.64	16.88	327.9
	SD	3.03	0.63	7.07	0.02	4.73	1.22	12.46
0	N	10	10	10	10	10	10	10
G4 &	Mean	12.36	8.33	136.50*	0.42*	49.84	16.4	328.7
	SD	2.59	0.31	4.99	0.02	1.68	0.8	6.86
2500	N	10	10	10	10	10	10	10
Group & Dose		Platelets	MPV	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Leukocyte	Basophils	Retic	PT	APTT	CT
(mg/kg b.w/Day)		(109/L)	(fL)	(%)	(%)	(%)	(%)	(%)	(%)	109/µL	(s)	(s)	(s)
G1 &	Mean	853.2	7.73	22.55	68.91	2.67	4.44	1.44	0	0.18	17.05	20.55	150
	SD	103.72	0.44	10	9.45	0.65	1.52	0.14	0	0.08	2.05	0.85	20
0	N	10	10	10	10	10	10	10	10	10	10	10	10
G4 &	Mean	1049.80*	7.61	25.16	65.87	2.81	4.87	1.26	0	0.13	17.3	20.09	150
	SD	239.27	0.31	8.86	8.96	1.51	1.86	0.36	0	0.03	0.58	0.9	24.49
2500	N	10	10	10	10	10	10	10	10	10	10	10	10

Notes:

Values are expressed as Mean (X̄) ± SD, N = Number of animals/group/sex.

Note.: WBC - White Blood Corpuscles, RBC - Red blood cell count, Hgb –Hemoglobin, HCT - Hematocrit, MCV - Mean corpuscular volume,

MCH - Mean corpuscular hemoglobin. MCHC - Mean corpuscular hemoglobin concentration, MPV- Mean platelet volume; time (APTT),

CT – Clotting Time in seconds (s)

*Statistically significant from G1 (P value<0.05)

Table 5.

Summary of haematology values at the end of treatment – rodent females (high dose group).

Group & Dose			WBC	RBC	HGB	HCT	MCV	MCH		MCHC
(mg/kgb.w/Day)			109/L	1012/L	g/L	L/L	fL	Pg		g/L
G1 &		Mean	10.05	7.94	148.1	0.43	54.56	18.74		343.3
		SD	3.33	0.67	5.61	0.03	1.56	1.5		24.71
0		N	10	10	10	10	10	10		10
G4 &		Mean	9.19	8.22	143.8	0.44	53.12	17.50*		329.3
		SD	2.92	0.35	4.18	0.01	1.65	0.63		4.24
2500		N	10	10	10	10	10	10		10
Group & Dose			Platelets	MPV	Neutrophils	Lymphocytes	Monocytes		Eosinophil	Leukocytes	Basophils	Retic	PT	APTT	CT
(mg/kgb.w/Day)			(109/L)	(fL)	(%)	(%)	(%)		(%)	(%)	(%)	109/µL	(s)	(s)	(s)
G1 &	Mean		923.9	7.43	21.63	69.15	2.61		4.9	1.65	0.05	0.14	16.37	20.24	159
	SD		130.37	0.29	6	7.27	0.62		2.54	0.67	0.05	0.03	1.36	1.68	20.25
0	N		10	10	10	10	10		10	10	10	10	10	10	10
G4 &	Mean		1110.70*	7.31	23.58	66	2.69		5.75	1.95	0.04	0.15	16.65	20.43	159
	SD		154.38	0.22	5.65	7.02	1.14		4.28	1.65	0.05	0.06	1.08	1.7	20.25
2500	N		10	10	10	10	10		10	10	10	10	10	10	10

Notes:

Values are expressed as Mean (X̄) ± SD, N = Number of animals/group/sex.

Note.: WBC - White Blood Corpuscles, RBC - Red blood cell count, Hgb –Hemoglobin, HCT - Hematocrit, MCV - Mean corpuscular volume,

MCH - Mean corpuscular hemoglobin. MCHC - Mean corpuscular hemoglobin concentration, MPV- Mean platelet volume; time (APTT),

CT – Clotting Time in seconds (s)

*Statistically significant from G1 (P value<0.05)

Compared with the control animals, female, and male rats in DOPS-F02 groups showed no remarkable abnormalities in hemoglobin, hematocrit WBC, WBC differentials, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, RET, RET%, APTT or platelets at the end of study. All results were within the normal range of Wistar rats [10].

3.2.1.4. Hematology

Results in clinical chemistry (Table 6, Table 7) showed no remarkable abnormalities in the DOPS-F02 dosed female and male rats in the comparison with the control animals in ALB, T PRO, AST, ALT, TBIL, CK, GGT, T.CHOL, TRIG, Creat, GLU, Urea, ALP, LDH, K⁺, Na⁺, Cl^-, A/G, GLB, Ca, Pi, AMYP or LIPC at end of the study (on Day 181) and at the end of the recovery phase [11].

Table 6.

Summary of clinical biochemistry values at the end of study- rodent males (high dose group).

Dose		ALB	ALP	ALT	AST	Ca	T.Chol	CK	Creat	GGT	Glucose	HDL-C	Pi
(mg/kg b.w/Day)		g/L	U/L	U/L	U/L	mmol/L	mmol/L	U/L	µmol/L	U/L	mmol/L	mmol/L	mmol/L
0	Mean	28.44	215.2	62	133	2.47	1.4	192.8	27.6	1	6.18	0.85	1.45
	SD	0.53	91.66	6.82	11.42	0.04	0.1	132.38	2.3	0.71	0.56	0.04	0.11
	N	5	5	5	5	5	5	5	5	5	5	5	5
2500	Mean	28.4	176	57.2	149.6	2.47	1.59	191.4	28.6	3.20*	5.89	0.86	1.46
	SD	1.55	60.15	12.99	33.72	0.06	0.28	101.81	4.56	1.92	0.56	0.18	0.17
	N	5	5	5	5	5	5	5	5	5	5	5	5
Dose		LDH	LDL-C	T.Bil	T Pro	Trig	BUN	Na	K	T3	T4	TSH
(mg/kg b.w/Day)		U/L	mmol/dL	mmol/L	g/L	mmol/L	mmol/L	mmol/L	mmol/L	ng/mL	ng/mL	ng/mL
0	Mean	86.8	0.3	3.24	80.2	0.46	4.49	143.12	3.37	10	342.7	15.7
	SD	22.8	0.03	0.26	4.01	0.1	1.11	0.99	0.12	1.6	60.9	4
	N	5	5	5	5	5	5	5	5	5	5	5
2500	Mean	213.00*	0.38*	2.99	81.98	0.5	5.27	144.38	3.46	10.2	347.4	15.5
	SD	73.3	0.07	0.25	4.28	0.06	1.75	1.27	0.15	2.2	52.9	2
	N	5	5	5	5	5	5	5	5	5	5	5

Notesmg/kg – Milligram/kilogram, b.w – Body weight, Contd- Continued.

Values are expressed as Mean (X̄) ± SD, N = Number of animals/group/sex.

ALB- Albumin, ALP – Alkaline Phosphatase, ALT - Alanine aminotransferase, AST - Aspartate aminotransferase, Ca-Calcium, T.Chol – Total Cholesterol.

CK – Creatine Kinase, Creat - Creatinine, GGT - Gamma-glutamyltransferase, HDL – High density Lipoprotein, Pi – Inorganic Phosphorous.

LDH – Lactate dehydrogenase, LDL – Low density lipoprotein, T. Bil – Total Bilirubin, T Pro – Total protein, Trig – Triglyceride,

BUN-Blood Urea Nitrogen, Na – Sodium, K – Potassium

*Statistically significant (P value<0.05)

Table 7.

Summary of clinical biochemistry values at the end of study- rodent females (high dose group).

Dose		ALB	ALP	ALT	AST	Ca	T.Chol	CK	Creat	GGT	Glucose	HDL-C	Pi
(mg/kg b.w/Day)		g/L	U/L	U/L	U/L	mmol/L	mmol/L	U/L	µmol/L	U/L	mmol/L	mmol/L	mmol/L
0	Mean	29.92	326	75.2	162.8	2.48	1.51	138.8	28.6	0.6	6.12	0.98	1.2
	SD	1.23	120.66	17.41	54	0.03	0.13	35.14	4.39	0.55	0.03	0.09	0.21
	N	5	5	5	5	5	5	5	5	5	5	5	5
2500	Mean	30.78	208.6	72.2	158	2.5	1.67	153.6	27.6	3.80*	6.16	1	1.14
	SD	1.89	57.71	20.62	29.65	0.06	0.27	39.53	1.52	2.59	0.69	0.16	0.24
	N	5	5	5	5	5	5	5	5	5	5	5	5
Dose		LDH	LDL-C	T.Bil	T.Pro	Trig	BUN	Na	K	T3	T4	TSH
(mg/kg b.w/Day)		U/L	mmol/dL	mmol/L	g/L	mmol/L	mmol/L	mmol/L	mmol/L	ng/mL	ng/mL	ng/mL
0	Mean	123.2	0.28	3.42	78.34	0.4	4.2	141.96	3.51	8.1	287	12.5
	SD	81.42	0.02	0.13	0.92	0.09	0.51	1.7	0.13	1.7	78.9	4.2
	N	5	5	5	5	5	5	5	5	5	5	5
2500	Mean	197.8	0.34	3.31	80.24*	0.64	5.27*	143.04	3.45	11.5*	392.2	17.1*
	SD	73.37	0.11	0.58	0.65	0.39	0.88	0.38	0.14	1.6	65.5	1.5
	N	5	5	5	5	5	5	5	5	5	5	5

Notesmg/kg – Milligram/kilogram, b.w – Body weight, Contd- Continued.

Values are expressed as Mean (X̄) ± SD, N = Number of animals/group/sex.

ALB- Albumin, ALP – Alkaline Phosphatase, ALT - Alanine aminotransferase, AST - Aspartate aminotransferase, Ca-Calcium,

T.Chol – Total Cholesterol.

CK – Creatine Kinase, Creat - Creatinine, GGT - Gamma-glutamyltransferase, HDL – High density Lipoprotein,

Pi – Inorganic Phosphorous. LDH – Lactate dehydrogenase, LDL – Low density lipoprotein, T. Bil – Total Bilirubin,

T Pro – Total protein, Trig – Triglyceride, BUN-Blood Urea Nitrogen, Na – Sodium, K – Potassium

*Statistically significant (P value<0.05)

There were no treatment effects of toxicological significance and apparent fluctuations in the results were not corroborative to dosing levels and considered as biological variance and not considered treatment related.

No abnormalities in urinalysis parameters were noted in treatment and recovery groups with their results being comparable to the corresponding vehicle contr

3.2.1.5. Urinalysis

No abnormalities in urinalysis parameter were noted in treatment and recovery groups when comparable with the corresponding vehicle control group.ol group.

3.2.1.6. Pathology

3.2.1.6.1. Bone marrow parameters

The bone marrow parameters did not reveal any treatment-related effects in different dose groups compared to the respective vehicle control groups (Table 8, Table 9)

Table 8.

Summary of bone marrow parameters –rodent males-main study.

(mg/kg b.w/Day)	%	Myeloid	Erythroid	Lymphocytes	megakaryocytes	M:E Ratio	TOTAL
G1 &	Mean	95.7	89.4	12.4	2.5	1.08	200
0.0	SD	6.17	5.54	3.14	2.29	0.12	0
	N	10	10	10	10	10	10
G2 &	Mean	97.9	88.2	11.1	2.8	1.11	200
	SD	3.24	4.69	4.91	2.52	0.06	0
750	N	10	10	10	10	10	10
G3 &	Mean	97.2	91	9.9	1.9	1.08	200
	SD	4.77	6.39	2.3	2.77	0.13	0
1250	N	10	10	10	10	10	10
G4 &	Mean	96.7	89.4	12.1	1.8	1.08	200
	SD	2.65	3.98	2.3	2.36	0.06	0
2500	N	10	10	10	10	10	10

Table 9.

Summary of bone marrow parameters – rodent females-main study.

(mg/kg b.w/Day)	%	Myeloid	Erythroid	Lymphocytes	megakaryocytes	M:E Ratio	TOTAL
G1 &	Mean	96.6	89.4	11.5	2.5	1.09	200
0.0	SD	4.2	6.05	2.97	2.2	0.11	0
	N	10	10	10	10	10	10
G2 &	Mean	94.2	90.4	12.4	3	1.05	200
	SD	5.4	6.26	2.8	2.28	0.12	0
750	N	10	10	10	10	10	10
G3 &	Mean	95.8	90.9	11.1	2.2	1.06	200
	SD	6.48	6.69	4.7	2.4	0.14	0
1250	N	10	10	10	10	10	10
G4 &	Mean	97.8	89.2	11.4	1.6	1.1	200
	SD	4.56	6.21	3.41	1.62	0.12	0
2500	N	10	10	10	10	10	10

Key: mg/kg – Milligram/kilogram, b.w – Body weight. Values are expressed as Mean (X̄) ± SD, N - Number of animals/group/sex

3.2.1.6.2. Absolute organ weights & Relative organ weights

No treatment related statistical changes in organ weights were noted in male and female animals when compared to the corresponding vehicle control groups. A few of the parameters showed statistically significant changes such as; increase in relative organ weight of the thyroid and parathyroid in male animals treated at 750 mg/kg, decrease in relative organ weights of ovaries in 1250 mg/kg dosed female animals, and the increase in liver of recovery group female animals.

Since there were no dose dependent trending effects as compared to vehicle group (Table 10, Table 11; Fig. 14, Fig. 15) these changes were considered as unrelated to treatment. Fluctuations in test values were within normal ranges of the particular species or no trending effects of treatment related, toxicologically significant or dose related effects were apparent and these changes were considered biological variance and not considered as treatment related. Any of the observed statistically significant parameters from the study were considered not related to treatment. Since there were no dose dependent increase or decrease as compared to vehicle group.

Table 10.

Summary of absolute organ weights (g) at the end of study – rodent males.

Dose		Adrenals	Testes	Epididymitis	Liver	Kidneys	Heart	Brain	Spleen	Thymus
(mg/kgb.w/Day)		(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)
0	Mean	0.059	3.361	1.362	10.328	2.369	1.177	1.964	1.497	0.223
	SD	0.009	0.249	0.105	1.536	0.296	0.118	0.109	0.251	0.072
	N	5	5	5	5	5	5	5	5	5
2500	Mean	0.062	3.444	1.426	10.008	2.345	1.043	1.976	1.178	0.24
	SD	0.01	0.272	0.173	2.061	0.275	0.123	0.127	0.379	0.096
	N	5	5	5	5	5	5	5	5	5
Dose		PSVCG	Pituitary	Thyroid para thyroid	“PSVCG” = Prostate & Seminal Vesicle & Coagulation Gland
(mg/kgb.w/Day)		(g)	(g)	(g)
0	Mean	2.03	0.01	0.025
	SD	0.436	0.003	0.006
	N	5	5	5
2500	Mean	1.841	0.011	0.027
	SD	0.262	0.002	0.007
	N	5	5	5

Table 11.

Summary of absolute organ weights (g) at the end of study – rodent females.

Dose		Adrenals	Ovaries	Uterus+Cervix	Liver	Kidney	Heart	Brain	Spleen	Thymus	Thyroid para thyroid	Pituitary
mg/kgb.w/Day		(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)
0	Mean	0.058	0.147	0.791	6.26	1.572	0.752	1.817	0.64	0.238	0.021	0.011
	SD	0.011	0.027	0.35	0.578	0.139	0.047	0.098	0.218	0.049	0.009	0.003
	N	5	5	5	5	5	5	5	5	5	5	5
2500	Mean	0.069	0.122	0.812	6.713	1.636	0.748	1.828	0.722	0.243	0.027	0.012
	SD	0.016	0.018	0.323	0.424	0.118	0.053	0.096	0.135	0.081	0.005	0.002
	N	5	5	5	5	5	5	5	5	5	5	5

Key.: mg/kg – Milligram/kilogram, b.w – Body weight.

Values are expressed as Mean (X̄) ± SD, N - Number of animals/group/sex

Fig. 14.

Mean (X̄ ± SD) organ-to-body weight ratio (%) at the end of treatment – male. Calculated based on mean fasted body weight, e.g., the mean (n = 10) body weight of 2500 mg/kg dosed animals = 419.22 ± 33.87 g or the mean (n = 10) body weight of controlled animals = 413.8 ± 33.21 g.

Fig. 15.

Mean organ-to-body weight ratio (%) at the end of treatment – female. Calculated based on mean fasted body weight, e.g., the mean (n = 10) body weight of 2500 mg/kg dosed animals = 219.59 ± 25.04 g or the mean (n = 10) body weight of controlled animals = 215.14 ± 27.83 g.

The analyzed coagulation parameters of males and females treated at all dose levels (750, 1250 and 2500 mg/kg body weight) were observed to be comparable with vehicle control throughout the experimental period.

3.2.1.7. Histopathology

No treatment related statistical changes in organ weights were noted in male and female animals when compared with the corresponding vehicle control group. A few observed statistically significant parameters such as increase in relative organ weight of thyroid and parathyroid in male animals treated at 750 mg/kg, the decrease in relative organ weights of ovaries in 1250 mg/kg dosed female animals, and the increase in liver of recovery group female animals were considered unrelated to treatment supported by the fact that there were no dose dependent trending effects as compared to vehicle group.

In comparison with the control group, no abnormalities regarding size, morphology, color, and texture were noted in main organs, such as brain, heart, liver, spleen, lung, kidney, adrenal gland, thymus, gastrointestinal tract and reproductive organ, of all necropsied rats in DOPS-F02 groups at the end of the dosing phase and recovery phase. Findings indicated that there were no treatment related microscopic changes in male and female animals since the few minimal changes observed were observed crossing all groups of the control or treated animals.

The rate of occurrence of pathological lesions in different organs of male and female animals of the treatment group (G4) compared well with control (G1). However, there were minimal to slight hyperkeratosis, hyperplasia and inflammation (reactive inflammation) in the non-glandular part of stomach in intermediate (G3, 5 out of 20 animals) and high (G4, 8 out of 20 animals) dose groups. These findings were attributed to local irritation caused by the gavage during administration of a test article and therefore the irritation is considered as non-adverse in nature [12]. There were no lesions in the stomach of G2, G5 and G6 group animals. Test items produced treatment related changes in the non-glandular part of stomach at intermediate (1250 mg/kg b.w.) and high dose level (2500 mg/kg b.w.) as compared to control under the test conditions in the present study. The lesions in non-glandular part of stomach of rats does not have human relevance and these lesions were recovered in the recovery animals (G6). Lesions of the non-glandular stomach are the frequent findings in nonclinical toxicity studies and are attributed to local irritation caused by gavage administration of a test article. Therefore, the lesions in non-glandular stomach were also considered non-adverse.

3.2.1.8. Toxicokinetics

Following once daily oral gavage administration of DOPS-F02 to Wistar rats, the toxicokinetic profiling showed that, for a 3.33-fold increase in dose of DOPS-F02 from 750 to 2500 mg/kg/day, the increase in peak plasma concentration (C_max) was dose proportional in both genders across the tested dose levels, except males on Day 180 which showed more than a dose proportional increase. In addition, the increase in plasma exposure (AUC_last) was more than dose proportional in both genders, on both study days, across the tested dose levels. No gender related differences in exposure were observed on both study days. On repeated oral administration, the exposure of DOPS-F02 showed no tendency for accumulation at the tested dose levels, except in males at 2500 mg/kg/day on Day 180 which showed ∼2-fold increase in exposure (Table 12 and Fig. 16, Fig. 17, Fig. 18).

Table 12.

Summary of toxicokinetic parameters: mean (X̄ ± SD) toxicokinetic parameters of DOPS-F02 following oral gavage administration to Wistar rats on Days 1 and 180 [Mean (n = 3) ± SD).

Day	Gender	Dose(mg/kg/day)	@Tmax(h)	Cmax(ng/mL)	AUClast(h*ng/mL)	Clast(ng/mL)	#Tlast(h)	AUC0-24h(h*ng/mL)
1	Male	750	1.00	60.9 ± 3.58	34.4 ± 1.86	60.9 ± 3.58	1.00 ± 0.00	126 ± 6.73
		1250	1.00	96.0 ± 15.9	262 ± 36.0	41.8 ± 5.18	4.00 ± 0.00	303 ± 40.6
		2500	1.00	229 ± 15.5	806 ± 36.9	59.7 ± 3.91	6.00 ± 0.00	985 ± 47.9
	Female	750	1.00	65.1 ± 5.20	36.8 ± 2.72	65.1 ± 5.20	1.00 ± 0.00	134 ± 7.76
		1250	1.00	141 ± 7.66	399 ± 13.5	68.4 ± 5.29	4.00 ± 0.00	468 ± 17.6
		2500	1.00	257 ± 32.5	859 ± 53.2	56.0 ± 4.49	6.00 ± 0.00	1030 ± 52.8
180	Male	750	1.00	67.5 ± 12.3	36.3 ± 15.8	67.5 ± 12.3	1.00 ± 0.00	138 ± 34.1
		1250	1.00	129 ± 13.1	334 ± 31.2	45.1 ± 2.14	4.00 ± 0.00	380 ± 31.1
		2500	1.00–6.00	705 ± 751	1460 ± 901	570 ± 867	6.00 ± 0.00	3170 ± 3500
	Female	750	1.00	78.9 ± 3.79	38.6 ± 2.98	78.9 ± 3.79	1.00 ± 0.00	157 ± 8.32
		1250	1.00	169 ± 20.3	490 ± 37.4	87.6 ± 5.75	4.00 ± 0.00	578 ± 40.1
		2500	1.00	310 ± 22.4	1000 ± 116	71.3 ± 15.9	6.00 ± 0.00	1220 ± 161

Data represented as mean (X̄) ± SD; @: T_max represented as range; #: T_last represented as median

Fig. 16.

1 Mean (X̄ ± SD) Plasma concentration-time profiles of DOPS-F02 following oral gavage administration to male Wistar rats on Day 1. 2 Mean Plasma concentration-time profiles of DOPS-F02 following oral gavage administration to female Wistar rats on Day 1.

Fig. 17.

1 Mean Plasma concentration-time profiles of DOPS-F02 following oral gavage administration to male Wistar rats on Day 180. 2 Mean Plasma concentration-time profiles of DOPS-F02 following oral gavage administration to female Wistar rats on Day 180.

Fig. 18.

1 Dose-exposure of DOPS-F02 following oral gavage administration to male Wistar rats on Day 1 and Day 180. 2 Dose-exposure of DOPS-F02 following oral gavage administration to female Wistar rats on Day 1 and Day 180.

There was no other abnormality found in the entire studying period.

4. Discussion

Novel excipients are critical enabling technologies for modern drug formulation, offering enhanced bioavailability, enabling new delivery technologies, and improving stability for complex molecules. DOPS-F02 is a novel excipient (as the first application accepted into the Novel Excipient Review Pilot Program by the FDA), originally developed for parenteral applications and this toxicology program was designed to underwrite extending its use for oral drug delivery applications. Due to its very low bioavailability, the majority of pharmacology safety evaluations, reproductive toxicity and immunogenicity of DOPS-F02 were previously conducted via intravenous injections [7]. DOPS-F02 is a synthetic PEG-carbohydrate-lipid polymer with a specific composition and its three main components (oleic acid, lactobionic acid, monomethoxypolyethylene glycol ether have been in clinical use for decades. The intention of this work was to complete 90 day dog and 180 day oral toxicology studies with this novel excipient. The results from such studies in dogs and rats are well accepted for determining any toxicity on long-term administration. In this work, DOPS-F02 at various dose levels did not appear to retard animal growth or affect food consumption and utilization. There were no significant changes in the hematological parameters between the control and the treatment groups indicating that DOPS-F02 was non-toxic as it did not affect circulating red cells, nor the hematopoiesis and leucopoiesis that could otherwise trigger a non-megaloblastic anemia. There were no changes in packed cell volume (PCV) and eosinophils. Furthermore, plasma levels of blood urea, nitrogen, glucose, total proteins, albumin, sodium, and potassium ions were not affected by feeding DOPS-F02 in rats and dogs. These findings also indicated that normal metabolism of the animals was not affected by the polymer. In addition, oral administration of DOPS-F02 did not alter the urinary levels of glucose, protein, hemoglobin, bilirubin, or creatinine indicating normal hepatocellular and nephrotic function.

There is no prior clinical experience with DOPS-F02 at the present time, the two leading regulatory authorized PEG-lipid based polymers; polyoxyl 35 castor oil or Cremophor EL (“Cr-EL”) and polysorbate 80 (PS80) were considered as relevant benchmarks for comparison purposes. For instance, polysorbate 80 (PS80), one of the most studied excipients in pharmaceutical formulations, is used for the same purposes that DOPS-F02 is intended for and is therefore an appropriate reference polymer/benchmark. In addition, PS80 and DOPS-F02 share similarities with respect to their chemistry (Table 13).

Table 13.

Surfactant properties and compositions of Polysorbate 80 (PS80) and DOPS-F02.

Polymer	Lipid moiety	Polymer moiety	Sugar portion	Molecular weightd	CMC (mg/L)	HLBe	Synthesis
PS 80a	Oleic acid (≥ 58 %)b	Branched PEG (20)	Sorbitol	1310	13 – 15 (∼ 0.012 mM)	15.5	Ethoxylation (radical Reaction)
DOPS-F02	Oleic acid (≥ 90 %)c	mPEG (12)	Lactobionic acid	1221	∼ 13 (∼ 0.011 mM)	14.2	Total synthesis

^ahttps://en.wikipedia.org/wiki/Polysorbate_80

^bUSP 44/NF 39; no purity specification for PS80

^cPurity of DOPS-F02

^daveraged value

^eby Griffin method https://en.wikipedia.org/wiki/Hydrophilic-lipophilic_balance

Some of the existing safety information for PS80 provides a useful reference. A Panel of the European Food Safety Authority (EFSA) concluded that “based on the NOAEL of 2500 mg/kg bw/day, identified from an oral carcinogenicity study with polysorbate 80 (E433) in rats, and applying an uncertainty factor of 100, a group ADI of 25 mg/kg bw/day for polysorbates 20, 80, 40, 60 and 65 (E432, E433, E434, E435 and E436, respectively) can be established” [13]. It is important to note however that information on sub-chronic oral toxicity of PS80 in dogs is limited or absent. Other interesting information includes a published study reported evidence of mild and transient clinical signs of hypersensitivity reactions including erythema, edema, and scratching, in a third of animals approximately 20–60 min post oral gavage dosing at 10 mg/kg of polysorbate 80 [14]. These mild clinical signs were largely attributed to the purity profile of PS80 which is a mixture of free PEG, and ethoxylated glycerol, PEG-fatty acid esters. Amounts of free acids or other starting materials in the final PS80 product are due to the radical synthesis which produces unesterified chains in polysorbates [15]. Note that the fatty acid composition of PS80 may not affect a drug product stability [16]. It has also been reported that purer fatty acids do not improve the stability or performance of polysorbates. For instance, oxidative degradation (due to ester hydrolysis) is thought to be the major stability, as well as safety, issue for polysorbates [17]. Much effort in the recent years has been spent on developing and commercializing purer or less heterogeneous polysorbates. In these studies, high purified oleic acid comprised PS 80 s were used, the purity of oleic acid was claimed to be > 98 %. However polysorbate 80 made with so called “ultra-pure” oleic acid showed a fast degradation and poorer performance than the polymer made with less pure of oleic acid [18], [19].

Unlike PS80 or other commercial PEG-lipid based polymers, DOPS-F02 is made using a step-by- step synthesis so that the purity issues caused by common polymerization, e.g., radical reactions, are avoided. The purity of the final product of DOPS-F02 is constantly above 90 % of the current purity level of oleic acid used due to the unique synthesis [7]. The high purity is demonstrated by chromatography, and this is thought to account for its superior safety profile compared to PS80 or other PEG-lipid based polymers, e.g., Cremophor EL (“Cr-EL”). Currently there is no defined purity specification for finished polymer-lipid products found in any up-to-date pharmacopeia, e.g., US Pharmacopoeia (USP) or European Pharmacopoeia (Ph. Eur.).

Current data indicate that the current data do not suggest a potential for pseudo-allergy [7] . Furthermore, the use of this excipient can be supported because the accumulation of DOPS-F02 is insignificant from a required daily amount perspective and is typically less than other PEG-lipid based excipients, e.g., PS80 or Cr-EL. Specifically, there was no accumulation after 180-day daily doses of 1250 mg/kg bw DOPS-F02 in rats.

DOPS-F02 has a poor permeation in gastrointestinal tract (likely due to its relatively larger molecular size, e.g., Mw1221), and therefore the oral bioavailability is extremely low ∼approximately 0.1 % or less, in dog or rats. This translates to extremely small amounts of the polymer being systemically available. For example, only a few mg could be absorbed, e.g., 30 mg/kg dosing level for a 60 kg body weight, The poor gastrointestinal absorption in human of DOPS-F02 can also be predicted by using in silico PK modeling which predicted to be significantly lower than PS80.

Referring to PS80 or Cr-EL, an oral ADI (acceptable daily intake) of 50 mg/kg bw/day of DOPS-F02 could be proposed for human consumption, which aligns with the FDA guidance [20]. The estimated value is based on the NOAEL value of 1000 mg/kg bw/day determined for this polymer in the 90-day oral toxicity study in juvenile beagle dogs and the 180-day oral toxicity study in rats which showed no adverse signs up to 2500 mg/kg∙bw. In addition, no evidence of hypersensitivity or other acute toxicity was observed from oral administration of DOPS-F02 in any of the tested animals, and the dosing level was up to 2000 mg/kg.bw in dogs.

Since dogs are more sensitive to this test article, the repeated dose study in dogs is regarded as the more relevant NOAEL. The ADI value is derived from the NOAEL value to a HED (human equivalent dose) as below [21]:

$$\mathit{HED}\left(\frac{\mathit{mg}}{\mathit{kg}}\right)=\frac{{\mathit{animal}\mathit{dose}\left(\frac{\mathit{mg}}{\mathit{kg}}\right)x\mathit{animal}K}_m}{\mathit{Human}{K}_m}$$

Where the K_m value is 20 for dog and 37 for human [21], a 1000 mg/kg is translated into a HED of approximately 540 mg/kg. Furthermore, by applying a safety factor of 10 [21], an ADI of 50 mg/kg can be established. In addition, the K_m value is 6 for rats, [21], a 2500 mg/kg translates to a HED of approximately 405 mg/kg. After applying a safety factor of 10, an ADI of 40 mg/kg can be established.

Even though there is a lack of systemic exposure in non-rodent toxicity studies with the benchmark polymers Cremophor EL (“Cr-EL”) and polysorbate 80 (“PS80”), comparisons can be made with their accessible clinical data. The “maximum daily exposure” in marketed oral products is 3635 mg for Cr-EL and 907 mg for PS80 accroding to the FDA’s Inactive Ingredient Database [22]. These values correspond to ∼61 mg/kg of Cr-EL and ∼ 15 mg/kg of PS80 for a standard human of 60 kg body weight. Furthermore, significantly higher limits are allowed for their use in intravenous injections, i.e., 27,668 mg for Cr-EL and 4739 mg for PS80 [22].

The safety profile of DOPS-F02 has been established by substantial evidence from the reported nonclinical studies. Based on literature data, in silico predictions, as well as the regulatory authorized limits for polysorbate 80, the results from the DOPS-F02 studies demonstrated that this novel excipient is safer than polysorbate. 80, especially by mitigating specific adverse reactions associated with high doses or impurities and degradation products of PS80. Overall, this information supports justification of the proposed clinically relevant dose range for DOPS-F02. A provisional maximum daily amount of DOPS-F02 at 25–35 mg/kg should be considered as a reasonable use level value for adult oral products, a pediatric value of 50 % or less, e.g., 10 mg/kg has been proposed for children 2 years or above in the DMF (Master Drug File) of DOPS-F02 submitted to the FDA,

5. Conclusion

Overall, no adverse event or severe adverse event was reported to the oral intake of DOPS-F02 at the highest dose levels in both dogs or rats. The excipient also did not affect laboratory parameters in the dogs or rats. These findings show in the studies that DOPS-F02 is non-toxic and well tolerated for 90 days in dogs or 180 days in rats, with no significant changes in hematology, organ weights, or histopathology at all tested doses in both species. In addition, DOPS-F02 did not affect clinical or laboratory parameters in the animal species from other completed GLP studies, e.g., genotoxicity or animal reproductive toxicology studies: Segment I and Segment II, The solubilizing properties, potential stability advantages, of DOPS-F02 have been demonstrated in work that will be published at a later date. It is a novel excipient and safety data reported in this study highlight its suitability for safe use at 25–35 mg/kg/day in adult oral formulations. Further work is planned for conducting first-in-human trials with alternative formulations of various drugs using DOPS-F02 to evaluate its value as a novel oral drug delivery system to overcome drug solubility issues.

CRediT authorship contribution statement

Nian Wu: Writing – review & editing, Writing – original draft, Supervision, Project administration, Investigation, Data curation, Conceptualization. Fang R. Teng: Resources, Project administration, Data curation. Lacarya Scott: Writing – review & editing, Resources, Funding acquisition, Data curation. Siva Rama Krishna Nutalapati: Writing – review & editing, Validation, Formal analysis, Data curation.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nian WU reports financial support was provided by LipoSeuticals Inc. Nian Wu reports a relationship with LipoSeiticals Inc that includes: equity or stocks. Siva Rama Krishna Nutalapati reports a relationship with Aptapharma that includes: equity or stocks. Lacarya Scott reports a relationship with LipoSeuticals Inc that includes: equity or stocks. Fang R. Teng reports a relationship with LipoSeuticals Inc that includes: employment. Nian Wu has patent #Polyethylene Glycol-Saccharide-Lipid Conjugates. PCT: WO2025029758 pending to LipoSeuticals. The authors declare no competing interests. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Handling Editor: Prof. L.H. Lash

Appendix A. Supplementary material

Supplementary material

Data availability

Data will be made available on request.