**3. Preclinical toxicological studies**

160 Rheumatoid Arthritis – Treatment

allograph (Janković, 1991), arthritis (Janković, 1991) and the experimentally induced allergic

In studies conducted at the Charité University in Berlin on the effect of intraarticular application of opioids in comparison to the intraarticular application of glucocorticoids in patients with the chronic inflammatory arthritis accompanied by pain and functional impairment, according to the established hypothesis, local application of opioids led to a significant reduction of pain and inflammation, which can be explained by the activation of peripheral opioid receptors (that have been identified on peripheral sensory nerve endings), and subsequent decreases in neural excitability, transmission of nociceptive impulses, and reduced release of proinflammatory neurotransmitters (Stein et al, 2001; Mousa et al, 2001). In summary, preclinical studies indicate that the activation of melanocortin receptors could be a new strategy to control inflammation (Catania et al, 2004). Similarly to any new therapeutic approaches, this strategy may have both advantages and disadvantages in comparison to the currently available medications. The main advantage of melanocortin in the treatment of inflammation is that its anti-inflammatory activity is not restricted to a specific mediator or a chemical process. In fact, as a consequence of reduced activation of the nuclear factor NF-B, the collective reduction of all important molecules involved in the

Another positive aspect is the fact that the treatment with melanocortin peptides never reverses the inflammatory response, but modulates it. It is well known that the inflammatory response is a crucial reaction of the host that contributes to the elimination of pathogens and harmful molecules. Cytokines, which are a major component of the inflammatory process, also have a significant function in regulating the recovery of tissues, haematopoiesis and immune responses. Any agent that completely inhibits their production or action would have a detrimental impact on the host defence mechanism. Melanocortin peptides modulate the increased production of cytokines during infection or inflammation, but do not prevent their release. Besides, they do not affect the production of mediators of inflammation when being on complete rest. So, for example, alpha-1-13 corticotrophin modulates the febrile response caused by pyrogens, while on the other side, it does not

The main advantage of the melanocortin in relation to the currently used anti-inflammatory drugs, particularly corticosteroids, is that neuropeptides do not reduce the microbicidal activity of neutrophils, but instead increase it. This feature could be very important in the

The lack of selectivity could be a potential problem when it comes to the use of natural melanocortin peptides. Today, it is clear that the melanocortins affect many body functions, including the regulation of food consumption, sexual behaviour and pigmentation. Systemic injection of non-selective peptide could, therefore, cause adverse effects by stimulating all subtypes of receptors. However, the design and synthesis of new melanocortin analogues with a selective affinity for specific receptors could greatly facilitate the achievement of target effects. Knowledge of amino acid substitution that reduces binding to all receptors can also help prevent unwanted receptor activation. (Catania et al, 2004). Another potential problem lies in the rapid splitting, or degradation of peptide molecules in the circulation

The relatively short half-life could be a problem if it is necessary to maintain the concentration in the blood. On the other hand, the fact that the peptides are not accumulated

encephalomyelitis (Veljić et al, 1991).

inflammatory process is evident.

cause any changes to the non-febrile temperature of the body.

treatment of inflammation in immunecompromised persons.

and other body fluids, and natural melanocortins are no exception.

Enkorten toxicity testing was conducted according to the Instructions and Rules of Good Laboratory Practice (GLP) and Harmonized Tripartite Guideline issued in 1998.

Objectives of preclinical toxicological studies were to determine the degree of toxicity of the investigated substance; to identify the target organs of toxicity and to determine the reversibility of the toxic effects; to detect the toxic effects that may help in the identification of the parameters for subsequent clinical trials and to determine the maximum tolerated dose.

Preclinical toxicological studies were carried out in rats and rabbits. The experimental animals were Wistar albino rats bred in the breeding quarters in Harlan, Italy, and the rabbit strain was HY plus giant PS 19 gene, from France. Environmental conditions for animals in the vivarium were maintained constant with daily temperature and humidity monitoring. Semisynthetic food for laboratory animals *ad libitum* and the tap water that meets the Sarajevo city water-supply criteria for drinking water were used during study. Animals were randomly divided in groups according to dose levels. Three dose levels were determined using the method of multiplication of the anticipated maximum human therapeutic dose of 10 mg of met-enkephalin (substance a) and 2 mg of alpha-MSH (substance b): the dose equivalent to the anticipated human therapeutic dose (0.071 mg /kg **a** + 0.014 mg/kg **b**); 5 times higher dose (0.355 mg/kg **a** + 0.07 mg/kg **b**); 10 times higher dose (0.71 mg/kg **a** + 0.14 mg/kg **b**). Enkorten was applied as a constant volume dose of 0.0005 ml/g according to the following dosage regime: during the first month - three times a week; during the second month - twice a week, and during the third month - once a week. The control group was treated by 0.9% of physiological NaCl solution.

Treatment was followed by a 10-day observation period. The appearance of toxic signs was monitored on a daily basis. Weighing of individual animals was performed before dosing, once a week, and before the animals were sacrificed. Daily food and water consumption per cage, as well as the stool mass were recorded on a weekly basis.

In subchronic and chronic studies, haematological and biochemical analyses were performed of the fasting blood samples taken from 12 animals from each of the experimental groups and from 24 animals from the control group, before the animals were sacrificed. The following haematological analyses were performed: hematocrit, haemoglobin concentration, red blood cell count, red blood cell morphology, total and differential white blood cell count, platelets. The following biochemical analyses were performed: Calcium (Ca+2), Potassium (K+), Sodium (Na+), Chloride (Cl - ), Phosphorus (PO24-), glucose, Aspartal aspartate aminotransferase (AST) , Alanine aminotransferase (ALT), alkaline phosphatase (AP), Gamma-glutamyl transpeptidase (GGT), blood urea nitrogen (BUN), total proteins, albumin, globulin, total bilirubin, creatinine, lipids, and cholinesterase.

Necropsy was performed only on the sacrificed animals (no lethality observed). Upon completion of the experiment, animals were sacrificed and both external and internal examinations of the sacrificed animals were performed. The following organs: brain, heart, lungs, liver, spleen, thymus, kidneys and adrenal glands, as well as samples of other macroscopically changed tissues were subjected to histopathological analysis.

Enkorten – A Potential Drug for the Treatment of Rheumatoid Arthritis 163

combination to the rats did not result in a permanent adverse toxicological effect on the examined organs. Reversible signs of toxicity disappeared with substance elimination and

mood changes, miosis, respiratory depression and gastrointestinal disturbances (Plotnikoff et al., 1997; Brunton and Parker, 2008). The evidence of ptosis, motor activity changes, horizontal positioning of the tail or increased phonation could suggest opioid pathway engagement. Increased intensity of phonation was clearly noted for all i.v. dosed treatment

The subacute toxicity study with the subcutaneous (s.c.) administration of Enkorten was conducted with three experimental groups, each of 10 Wistar albino rats (both males and females) and the control group of 20 Wistar albino rats (both males and females) during four weeks. Neither lethal outcomes nor toxic signs were observed during the study. The absence of pronounced toxicity of the substances was confirmed by the body weight gain in both experimental and control groups of animals during the study period. The ability of gaining or maintaining the body weight is considered to be a non-specific indicator of the health status of the animal (Gad and Chengelis, 1998), especially in toxicity studies with multiple applications. Furthermore, the average food and water consumption as well as the percentage ratio of stool mass were balanced between male and female groups. Neither external nor internal examination of animals sacrificed at the end of the study revealed any macroscopic pathological changes. Histopathological analysis revealed no microscopic pathological changes in tissue samples of liver, kidneys, lungs, heart, brain, spleen and thymus. All examined tissues showed a normal structure. No statistically significant differences in post-mortem organ weights were noted between male and female groups. The results of subacute toxicity study indicated that the tested combination of substances was not toxic when administered s.c. and repeatedly during

The subchronic toxicity study with intramuscular administration of Enkorten was conducted with three experimental groups, each of 10 Wistar albino rats (both males and females) and the control group of 15 Wistar albino rats (both males and females) during

As a part of this study, the electrocardiogram (ECG) was recorded in 6 animals from each of the experimental groups (3 males + 3 females) and from the control group (3 males + 3 females), which in total amounts to 24 animals. The ECG was recorded on the day before the study commenced, as well as during the third, fifth, seventh, ninth and the eleventh week of the study and on the day before the planned sacrificing of animals. The electrocardiograph Schiller Resting ECG, connected to a personal computer was used for ECG registration. The computer program SEMA - 200 Vet (a program for ECG analysis in veterinary medicine) was used for the analysis of the registered curves. This program calculated the average values of the heart rate (FSR bits/min) and the duration of the RR interval (ms), P wave

(ms), PQ interval (ms), QRS complex (ms) and QT interval (ms).

*)* receptors in the range of sedation,

usually were not followed by a permanent tissue damage. High doses of met-enkephalins produce effects on *OP3 (*

**3.2 Subacute toxicity study** 

four weeks (Todić et al, 2007).

**3.3 Subchronic toxicity study** 

three month.

groups, but not at all in control animals (Rakanović-Todić et al, 2011).

Collected data were statistically analyzed using the Microsoft® Excel 2002 and SigmaStat programs for statistical analysis.

#### **3.1 Acute toxicity study**

The acute toxicity study was performed as the initial step of the prospective drug toxicological evaluation. Acute toxicity provides the important safety parameters for the prospective human overdose scenario and its expected clinical presentation. Traditionally, international authorities and governmental agencies had adopted the mean lethal dose as the sole measurement of acute toxicity. Testing of the rationally high doses, without detected lethality, is also acceptable and complies with the Limit Test concept (ICH, 2009; EMA, 2010).

Acute toxicity was assessed on Wistar albino rats. The study was performed as the single dose testing following intravenous (i.v.), subcutaneous (s.c.) and intraperitoneal (i.p.) application, in accordance with the Limit Test methodology. The administered multiplications were: via i.v. route 50, 100 and 200 times; via s.c. route 100, 250 and 500 times; and via i.p. route 100, 250, 500 times plus 1000 times in and additional group of males. Neither lethality, nor significant macroscopic and microscopic changes were observed at the necropsy following planned sacrificing. The necropsy and histopathological evaluation revealed no significant changes. No statistically significant differences in post-mortem organ weights were noted.

The most prominent clinical signs were noted on day 1 of the observation period. Following all three application routes, slightly reduced motor activity and the horizontal positioning of the tail was observed in all animals treated with the highest i.v. dose. There was a slight ptosis (eyelids down ¼) in a few animals in all i.v. treatment groups. Slight cyanosis and general vasoconstriction was noted in one male treated with the highest i.v. dose, and one male treated with the medium i.v. dose. Slightly slower motor activity of all treated and control animals was observed following the s.c. application. Also, slight muscular hypotonia was noted on day 3 in three males treated with the highest dose and one male treated with the lowest dose. Irregular breathing, decrease in motor activity, somnolence, ataxia, catalepsy and muscular hypotonia were observed in all males from the additional group administered i.p. with 1000 dose multiplication. A proportion of the effects noted could be attributed to the application of the large amount of fluid, since the constant volume was not respected for this group. Afterwards, the frequency and character of breathing were observed once a week and were found to remain within the physiological limits.

Intensive phonation during the manipulation with animals was registered for i.v. dosed groups. The frequency of intensive phonation intensified between days 9 and 14 of the observation period, which was not noted in control animals. Statistically significant difference was detected for increased phonation in all treatment groups of males, IVF3 and IVF2, compared to controls (p 0.000001). Following the s.c. application, each phonation was registered, and there was no prominent difference in phonation in males, while a statistically significant difference in phonation was noted for group SCF3 (p = 0.000001), compared to the control.

The tested combination induced no lethality and demonstrated a low level of toxicity in high doses. The fact that neither organ or tissue damage, nor organ mass ratio differences in control and experimental groups were detected indicates that the application of the tested combination to the rats did not result in a permanent adverse toxicological effect on the examined organs. Reversible signs of toxicity disappeared with substance elimination and usually were not followed by a permanent tissue damage.

High doses of met-enkephalins produce effects on *OP3 ()* receptors in the range of sedation, mood changes, miosis, respiratory depression and gastrointestinal disturbances (Plotnikoff et al., 1997; Brunton and Parker, 2008). The evidence of ptosis, motor activity changes, horizontal positioning of the tail or increased phonation could suggest opioid pathway engagement. Increased intensity of phonation was clearly noted for all i.v. dosed treatment groups, but not at all in control animals (Rakanović-Todić et al, 2011).
