UDC 615.21: 615.076.9

Leningrad Region
2021

It is carried out in accordance with the GLP OECD Principles

STUDY REPORT

Study of the accumulation of substance Lithium Ascorbate following repeated intragastric administration to sexually mature rats

(final)

Study Leader  [Signature] A.V. Popova

ABSTRACT

Key results

Keywords: Lithium ascorbate, cumulation, rats, intragastric administration

The report is presented on 129 pages, including 6 tables and 19 figures.

STUDY DATES

LIST OF EXECUTORS

STUDY DECLARATION OF GLP COMPLIANCE 
FROM THE STUDY LEADER

This study was carried out in accordance with the standard operating procedures of the institution, and the Study plan № 4 mutually agreed with the Sponsor (Normopharm LLC, Russia).

The study complies with the GLP requirements, except for the fact that no identification, purity, and stability studies of the test object have not been carried out by the research institution.

There were no deviations from the Study Plan that would affect data interpretability or the scientific integrity and results of the study.

I, the undersigned, hereby confirm that I take overall responsibility for the technical conduct of the study; analysis, interpretation, documentation and presentation of results, as well as archiving of the study-related materials.

The objectives set out in the study plan have been achieved. There were no unforeseen circumstances that could affect the quality or integrity of the study.

This report presents the results reliably. I am fully responsible for the accuracy of the data obtained, as well as the confidentiality of the preclinical study.

I guarantee that after the study completion, the study plan, the final report, source data and all relevant documentation will be transferred to the archives of the research institution.

Study leader A.V. Popova
/name/

STATEMENT BY THE MANAGEMENT OF THE RESEARCH INSTITUTION ON THE PROVISION OF RESOURCES TO CONDUCT THE STUDY IN ACCORDANCE WITH THE GLP PRINCIPLES, REGULATORY REQUIREMENTS AND STANDARDS FOR THE ETHICAL HANDLING OF ANIMALS

The management of the research institution shall ensure that the following has been provided for the proper conduct of the study:

• availability of a sufficient number of qualified and experienced personnel with a clear understanding of their responsibilities, as confirmed by training data;

• equipping the research institution with the necessary equipment, facilities and materials;

• availability of a quality service responsible for the quality assurance system;

• availability of approved standard operating procedures, as well as access to them by all personnel involved in the conduct of the study;

• appointment of a study leader in accordance with the established procedure, who has qualifications appropriate to the study objectives;

• interaction of the study leader, employees of quality service and personnel involved in the study.

Compliance with the Principles of Good Laboratory Practice

This study was carried out in accordance with the principles of GLP OECD (GOST 33044-2014 “Principles of Good Laboratory Practice”; Decree of the EEC Council № 81 “On Approval of the Rules of Good Laboratory Practice of the Eurasian Economic Union in Circulation of Medicines” dated 03.11.2016). The manipulations were performed in accordance with the standard operating procedures of the institution and Study plan № 4.

Regulatory Compliance

The design of this study was based on the selection of the study goal, in accordance with the regulatory legal acts and guidelines laid down in “Regulatory Standards” section of this Report.

Compliance with the Standards of Ethical Handling of Animals

This study was reviewed at a meeting of the Bioethics Commission for compliance of the draft study with the “Three R’s” principles and Directive 2010/63/EU. The study was approved for conduct (№ BEC 3.42/21 dated 23.08, 2021, 9 persons voted).

General Director of CJSC “Saint Petersburg Institute of Pharmacy”

V.G. Makarov                 [Signature]         24.11.2021

/Name/             /Signature/ /Date/

STATEMENT OF THE QUALITY SERVICE ON CONDUCTING AND DOCUMENTING THE INSPECTION OF THE KEY STAGES OF THE STUDY

The Quality Service conducted and documented all stages of the study inspection. The results were reported to the study leader and the management of the research institution.

The study was inspected to ensure that the procedures and manipulations performed were in accordance with the standard operating procedures of the institution, the study plan, and the regulatory requirements of the Good Laboratory Practice standards.

The final study report was reviewed by a quality officer and found to be an accurate statement of the data obtained and the procedures applied. The results presented in the final report accurately and fully reflect the data obtained during the study.

The conclusion of the quality service on this study is an Annex to the final report.

S.S. Sapynov                 [Signature]         24.11.2021

/Name/             /Signature/ /Date/

TABLE OF CONTENTS

LIST OF ABBREVIATIONS AND ACRONYMS 13

INTRODUCTION 14

STUDY GOAL AND OBJECTIVES 15

1. MATERIALS AND METHODS 16

1. Study objects 16

1. Test object 16

2. Animals 16

3. Method of administration and selection of doses 19

1. Method and duration of administration 19

2. Selection and calculation of doses 19

3. Dosing procedure 20

4. Methodology 20

1. Study design  20

2. Determination of cumulation factor 21

3. Feed deprivation 21

4. Body weight recording 22

5. Recording of timing of intoxication development and clinical examinationn of aninals ..22

6. Euthanasia 23

7. Pathomorphological examination 23

8. Data analysis 24

9. Study quality assurance and control  24

2. STUDY RESULTS 25

1. Тoxicometry 25

1. Lethality 25

2. Picture of intoxication. Clinical examination 25

2. Determination of cumulative properties of the test object 26

3. Pathomorphological examination data 26

FINDINGS 28

CONCLUSION 29

TABLES AND FIGURES 30

DATA ARCHIVING 38

NORMATIVE DOCUMENTS 39

REFERENCES  40

ANNEX A 42

ANNEX B 49

ANNEX C 71

ANNEX D 74

ANNEX E 78

ANNEX F 82

ANNEX J 107

ANNEX G 127

In this R&D report, the following abbreviations and acronyms are used:

INTRODUCTION

The test object is Lithium Ascorbate, substance (Normopharm LLC).

Lithium ascorbate is a highly absorbable and low-toxic organic lithium salt [1]. Lithium salts are widely used as normothymics in various affective disorders [2]. Lithium ions have a significant effect on the homeostasis of acetylcholine, enkephalins, catecholamines, serotonin, and other neurotransmitters [3]. Lithium (primarily as lithium carbonate) for the treatment of bipolar disorder or depression [4, 5] is administered in doses in the hundreds of milligrams. Such doses may lead to severe undesirable effects during therapy (renal pathology, teratogenesis). Compared to lithium carbonate therapy, lithium ascorbate has also shown efficacy in ultra-low doses [1].

This study aimed at investigating the accumulation of the substance Lithium Ascorbate following repeated intragastric administration into sexually mature rats, is part of the complex of preclinical studies required for the recording of the drug in the Russian Federation (RF) [6, 7].

The study was carried out with the engagement of employees of the necessary departments [Appendix A], the approved study plan [Appendix B] and approved by the bioethics commission [Appendix C].

The information obtained during the study did not duplicate the results of previous studies.

STUDY GOAL AND OBJECTIVES

Study goal:

Study of the accumulation of lithium ascorbate substance (Normopharm LLC) following repeated intragastric administration to sexually mature rats.

Study objectives:

1. analysis of the clinical picture of intoxication;

2. recording of death and time to death of animals;

3. study of the cumulative effect of the test object, determination of the cumulation coefficient.
   

1. Materials and methods

1. Study objects

Information on the test object and vehicle is given in Tables 1.1.1.1 and 1.1.2.1.

1. Test object

Table 1.1.1.1 - Test object

2. Vehicle

Table 1.1.2.1 - Vehicle

The documents of the pharmacist service are given in Appendix D. The research institution did not carry out identification, purity and stability studies of the test object. These values were determined by the Sponsor according to standard methods. The Sponsor of the study is responsible for the reliability of the submitted data on the identification, purity and stability of the test object.

2. Animals

Species: Wistar rats

Sex: Males

Source: JSC “NPO “HOUSE OF PHARMACY”

Animal Birth Certificate: № 2.1-31.08.2021 dated 31.08.2021 [Annex E]

Rationale for selection of animal species/line Wistar Rats are successfully used in animal toxicological experiments [6].

The animals were kept under standard conditions in accordance with Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes [8].

1. Method of administration and selection of doses

1. Method and duration of administration

The test object was administered into experimental animals repeatedly, intragastrically, since this method is an analogue of the oral one, which is planned to be used in clinical practice.

2. Dose Selection and Calculation

To determine the cumulation coefficient, the test object is administered in doses corresponding to 0.1 LD50, 0.15 LD50, 0.22 LD50, 0.34 LD50, 0.5 LD50, 0.75 LD50 and 1.12 LD50, according to the Lim R.K.’s method [9, 10]. According to the results of the “Study of the toxic properties of substance Lithium Ascorbate following a single intragastric administration to sexually mature rats” carried out under agreement № 0109-SPIF/2021 dated June 1, 2021 under the study code (BEC №) 2.28/21,  LD50 for males is 4810±386 mg/kg. Cumulation doses were calculated based on the data obtained and were 481 mg/kg, 721.5 mg/kg, 1058.2 mg/kg, 1635.4 mg/kg, 2405 mg/kg, 3607.5 mg/kg, and 5387.2 mg/kg.

Each dose was administered for 4 days, then the investigators switched to the next dose. The experiment was started with the administration of the object in a dose of 0.1 LD50 (481 mg/kg). The death of all animals was recorded by the 21st day. Doses of 3607.5 mg/kg and 5387.2 mg/kg were not tested.

Thus, the test object was repeatedly administered to the animals intragastrically in doses of 481 mg/kg, 721.5 mg/kg, 1058.2 mg/kg, 1635.4 mg/kg and 2405 mg/kg.

3. Dosing procedure

The test object (as solution) was repeatedly administered into animals intragastrically, if necessary, in fractions, using syringes and special probes.

The volumes of administration and concentration of the test object in the group are presented in Table 1.3.3.1.

Table 1.3.3.1 – Dosing volumes of administration and concentration of the test subject

The test object in a dose of 2405 mg/kg was administered in fractions, in two equal parts, with an interval between injections of at least 30 minutes.

3. Methodology

1. Study design

The total number of animals involved in the experiment is 10 male Wistar rats. The characteristics of the experimental group and the design of the experiment are presented in Tables 1.4.1.1 and 1.4.1.2.

Remarks

1 - a - due to the death of all animals by day 21 of the experiment, the dose was not used;

2 - b - due to the death of all animals by day 21 of the experiment, the manipulation was not performed.

Table 1.4.1.2 - Manipulation schedule.

Remark - a - due to the death of all animals by day 21 of the experiment, the manipulation was not performed.

1.4.2 Determination of the cumulation factor

The accumulation was calculated using the Lim R.K.’s method, as this method also allows evaluating the habituation [9, 10]. The cumulation factor was calculated as the ratio of LD50 in n-fold administration to LD50 in single administration: Кcum = LD50n/LD501. LD50  in n-fold administration was defined as the cumulative dose received by the animals on the day of the experiment, in which the lethality rate was 50%.

2. Feed deprivation

The animals were deprived of feed 16 hours before dosing, each body weight record and euthanasia. Water was given ad libitum throughout the experiment.

3. Body weight recording

Body weight was recorded in the morning hours immediately before the first administration, then on days 5, 9, 13 and 17 of the experiment. Source data are presented in raw charts [Annex Е].

The procedure of weighing rats was carried out on an electronic balance Vibra AJ-1200CE (Shinko Denshi, Japan). The maximum weighing limit is 1200 g, the minimum weighing limit is 0.5 g. Calibration mark is 0.1 g. Accuracy class is 2 [Appendix B].

4. Recording of the timing of intoxication development and clinical examination of animals

1. Recording of the timing of intoxication development

The animals were observed daily, at least once in the first 30 minutes after administration. In the case of fractional administration, observation was carried out after the administration of the last dose of the test object.

The following was recorded:

• Behavior: distress/agitation;

• Response to stimuli: decreased/increased;

• Skin: redness / paleness / cyanosis / jaundice;

• Mucous membranes: redness/pallor/cyanosis/jaundice;

• Discharge: from the eyes / from the nose / from the anus / from the urethra;

• Muscle tone: decrease/increase;

• Motor coordination disorders: ataxia/hyperkinesis;

• Dyspnoea;

• Death.

2. Clinical examination

The animals were clinically examined prior to administration, then on days 2, 8 and 14 of the experiment. A detailed examination of the animal was carried out in the cage, in the hands and in the open area. The manifestation and severity, where acceptable, of signs of intoxication were noted.

1. Examination in the cage:

• Behavior: normal/distress/agitation;

• Attitude towards other animals: normal/ aggression;

2. Examination when picking up an animal:

• Response to stimuli: normal/decreased/increased;

• Nutritional status: normal /dystrophic / obese;

• Muscle tone: normal/decreased/increased;

• Fur: normal (smooth, shiny) / ruffled / hair loss / dull / dirty / discoloration.

• Skin:

• Turgor: normal/reduced;

• Color: normal / redness / paleness / cyanosis / jaundice / hemorrhage;

• Integrity: normal (not impaired)/abrasions/cracks/wounds;

• Palpable masses.

• Mucous membranes:

• Color: normal / redness / paleness / cyanosis / jaundice;

Impaired integrity.

• Eyes: normal/exophthalmos (bulging eyes)/impaired integrity/discharge;

• Nasal cavity: normal / serous discharge / purulent discharge / bloody discharge;

• Oral cavity: normal/drooling.

3. Outdoor inspection:

• Body position in space: normal / forced lying down / forced wandering in a circle / forced movement forward and backward / forced desire to lie on one side; 

• Impaired motor coordination: normal/ataxia/hyperkinesis;

• Breathing type: normal / toracic /abdominal /dyspnea;

• Bowel movements: normal / diarrhea / presence of blood in the stool / change in stool color;

• Urination: normal/discoloration;

6. Euthanasia

By day 21 of the experiment, the lethality rate was 100%.

7. Pathomorphological examination

Pathomorphological examination included necropsy with the completion of necropsy charts [Appendix G].

Histology

Histological examination was performed if macroscopic changes were found in the internal organs during necropsy (only the tissue of the altered organ was examined to clarify the diagnosis). The adrenal glands (in one animal), intestinal fragments (in several animals), the stomach (in several animals) and the pancreas (in several animals) were taken for histological examination.

For histological examination, the material was fixed in a 10% solution of neutral formalin for 24 hours [11], after which it was embedded into paraffin according to the generally accepted method [12]. Then sections with a thickness of 5-7 μm were made, which were stained with hematoxylin and eosin. Histological specimens were analyzed using an Accu-Scope 3000 SERIES light-optical microscope (USA) at a magnification of 40, 100, and 400. Microphotography was carried out using a digital camera TOUPCAM UCMOS05100KP (China) and ToupView software 3.7.7892.

8. Data analysis

Descriptive statistics was applied to the body mass data: the data were checked against the normal distribution law using the Shapiro-Wilk’s W test. The mean value and standard error of the mean were calculated, which are presented in the summary tables.

The cumulation factor was calculated using the Lim R.K’s method [9, 10].

9. Study quality assurance and control

The quality service of the research institution carried out [Appendix I]: 

-   verification of the study plan

• checking the study plan - schedule

• incoming audit of the preclinical study

• audit of the experimental part of the study

• checking the chronology of the study and the completeness of the study report

• verification of the final study report. 
  

2 Study results

The cumulative properties of lithium ascorbate, substance (Normopharm LLC, Russia), were evaluated following repeated intragastric administration to male Wistar rats.

The range of animal body weight values at the beginning of the experiment was 187.0±3.21, body weight data are presented in Table 2.1.

The data obtained during the experiment are presented in full in the source charts [Appendix E].

1. Toxicometry

1. Lethality

During the experiment, the death of 10 male rats was recorded. Data on the timing of animal mortality are presented in tables 2.1.1.1 and 2.1.1.2. The first lethal effects were recorded on day 15 of administration (day 14 of the experiment). Dead animals were found in the morning, before the daily administration of the test object, so the cumulative dose received at the time of death was 12313.6 mg/kg. On day 17 (day 16 of the experiment) the death of animals was 40%, the cumulative dose was 15,584.4 mg/kg, on day 18 (day 17), the death was 60%, the cumulative dose was 17,989.4 mg/kg. Taking into account the incidence of lethal outcomes, LD50 following repeated administration in males was 17989.4 mg/kg.

By day 21 of the experiment, the mortality rate reached 100%.

2. Picture of intoxication. Clinical examination

The data are presented in Table 2.1.2.1. The first signs of intoxication in animals were recorded on day 10 of administration (the total dose of the test object was 6926.4 mg/kg) and were manifested as ruffled hair, in a single case nasal discharge was observed. In the following days, the pattern of intoxication gradually increased. On day 11 of observation (the total dose of the test object was 7984.6 mg/kg), in addition to ruffled hair and nasal discharge, inhibition of behavior and a decrease in skin turgor were observed in animals. A similar pattern of intoxication persisted until day 15 of administration of the test object. On day 15 of administration, the first lethal effects were recorded (the total dose of the test object was 12313.6 mg/kg). Further, the severity of toxic effects continued to increase, on day 19 of administration (the total dose of the object was 22799.4 mg/kg) the survived animals had ruffled hair, inhibition of the condition, bloody discharge from the nose, ataxia, tremor, and a decrease in response to stimuli. By day 21 of the experiment, the mortality rate was 100%.

2. Determination of the cumulative properties of the test object

The cumulation coefficient was calculated as the ratio of LD50 following repeated intragastric administration to LD50 following a single administration. The obtained data suggest at least 50% of animals (60%) died on day 18 of administration (day 17). The total dose of the test object at this time was 17989.4 mg/kg (Table 2.2.1). The cumulation factor was determined by the formula: 

Kcum = LD50(p)/ LD50 (1); Kcum = 17989.4/4810 = 3.74. Since Kcum>1, we may suggest the occurrence of habituation in animals to lithium ascorbate [9, 10]. In accordance with the accepted classification, lithium ascorbate belongs to the group of substances with moderate cumulative properties (3 < Kcum < 5) [13].

3.  Pathomorphological examination data

The incidence of pathological signs found in unplanned necropsy is given in Table 2.3.1.

All the animals of this group died. In all animals, venous congestion of internal organs, cerebral edema and congestion of its membranes were found (Figure 2.3.1), and in 50% of animals at necropsy mild pulmonary edema was found (Figure 2.3.2).

In 40% of animals, single punctate hemorrhages were found on the glandular part of the gastric mucosa (Figure 2.3.3). Male № 1.6 had erosive-ulcerative lesions of the glandular part of the gastric mucosa (Figure 2.3.4), and male № 1.9 had a loose gastric mucosa (Figure 2.3.5). In both animals, blood was found in the stomach contents (Figure 2.3.4). Histological examination shown erosive lesions of the gastric mucosa in animals № 1.3 and 1.6 (Figure 2.3.6). In rats № 1.3 and 1.9, focal impairment of integrity and exfoliation of the epithelium of the glandular part of the gastric mucosa, mild edema and mononuclear infiltration were shown (Figure 2.3.7).

Hemorrhages in the mucosa of the small intestine were found in 40% of animals (Figure 2.3.8), in the mucosa of the large intestine in 50% (Figure 2.3.9). Half of the animals were found to have blood in the contents of the large and small intestines (Figures 2.3.10 and 2.3.11). Male № 1.9 had erosive-ulcerative lesions in the mucous membrane of the large intestine (Figure 2.3.12). As a result of histological examination, ulcerative defects of the mucous membrane were shown in rat № 1.3 in the small intestine (Figure 2.3.13) and in rat № 1.9 in the large intestine (Figure 2.3.14).

In the pancreas of males № 1.3 and 1.9, multiple white miliary inclusions were found (Figure 2.3.15), as a result of histological examination, in both animals focal hypertrophy of cells of the exocrine part of the pancreas was found, in male № 1.9 the changes were accompanied by focal autolysis (Figure 2.3.16).

Male rat № 1.7 was found to have multiple hemorrhages in the adrenal cortex (Figure 2.3.17). Histological examination showed congestion as well as hemorrhages in the adrenal tissue (Figure 2.3.18).

In rats 1.3 and 1.7, full-blooded mesenteric lymph nodes were found (Figure 2.3.19).

The immediate cause of death of all the animals was acute heart failure.

FINDINGS

The study on the accumulation of lithium ascorbate substance (Normopharm LLC, Russia) following repeated intragastric administration to sexually mature rats concluded the following:

1. The pattern of intoxication (inhibition of the general condition of animals, ruffled hair, nasal discharge) was shown in animals from day 10 of the observation, when the cumulative dose of the test object reached 6926.4 mg/ml.

2. Lethal effects were recorded from day 15 of the administration, by day 21 the mortality rate reached 100%;

3. The cumulation factor of lithium ascorbate was 3.74. In accordance with the accepted classification, lithium ascorbate is assigned to the group of substances with moderate cumulative properties (3 < Kcum < 5).
   

CONCLUSION

All study activity to investigate Lithium Ascorbate substance (Normopharm LLC, Russia) was planned and implemented in strict accordance with the requirements of the Ministry of Health of the Russian Federation and international standards in preclinical studies of the safety of pharmacological agents - the GLP system (Good Laboratory Practice) [14]. According to the results of the study on the cumulative properties of the test object, Lithium ascorbate in sexually mature rats following repeated intragastric administration, the cumulative factor of lithium ascorbate was 3.74. It has been established that the test object has moderate cumulative properties (3 < Kcum< 5).