**3.2 Subacute toxicity study**

162 Rheumatoid Arthritis – Treatment

Collected data were statistically analyzed using the Microsoft® Excel 2002 and SigmaStat

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;

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

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),

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

programs for statistical analysis.

**3.1 Acute toxicity study** 

EMA, 2010).

weights were noted.

compared to the control.

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 four weeks (Todić et al, 2007).

#### **3.3 Subchronic toxicity study**

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 three month.

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).

Enkorten – A Potential Drug for the Treatment of Rheumatoid Arthritis 165

control group in the values of phosphorous, alkaline phosphates, and urea. The difference in triglycerides between the first and the control group, the AST values between the second and third group and the first and the third group, the ALT values between the second and the control group were also noticed. The sifnificant difference in the values of these parameters for males may be coincidental, because the difference between the groups treated by the highest dose level and the control group was not registered. In females a statistically significant difference in calcium and potassium was registered between the treated and the control groups. The values of phosphorus and alkaline phosphatase significantly differed between the first and the control group, and urea value between the first and the control group. Triglyceride statisticaly levels of all three treated groups significantly differed from the control group values. Also, there was a significantly difference in the values of ALT between the second and the control group. These data suggest that tested substance affects the value of calcium, potassium and triglycerides in females. The value difference of other parameters can be random, because the difference between the groups treated with the highest dose level and the control group was not registered. The data on the volume and biochemical parameters of urine showed statistically non significant difference between control and other groups of animals. Statistically significant difference between the control and first group of animals was found only in glucose during the second measurement. Registered difference is probably

Statistical analyses of the results obtained after biochemical analyses were not statistically significant (including both male and female groups) for the following parameters: Ca+2, K+,

The tested substance has no effect on the parameters of erythrocytes, leukocytes, granulocytes, thrombocytes and lymphocytes. However, the tested substance has effects on the value of monocytes when compared to the control group, and, as far as males are concerned, also in the values of lymphocytes. The tested substance did not have effects on the volume and biochemical parameters of urea, except for the glucose report. The noted

Testing was conducted at the Institute following the NTP, 2002 (NTP - National Toxicology Program - Good Science and Good Decisions - NIEHS, USA, 2002), in five months old, adult

In the first phase, Enkorten in a doubled human therapeutic dose (2HD; α-MSH 0.028 mg/kg + met-enkephalin 0.143 mg/kg) was administered once intraperitoneally (i.p.). Afterwards, animals were administered a bilateral intranasal (i.n.) dose of Enkorten eight times higher than the human therapeutic dose (8HD) per day, within two consecutive days. Administration of the substance in the above doses was carried out using the methods of crossing and gender matching. In the second phase, the same dosing protocol but with the i.n. dose increased to twenty times human therapeutic dose (20HD) was administered to the F1 generation of male and female rats, after they reached sexual maturity. By applying the same protocol, the control group received a physiological solution. During the first and the second phase, the typical parameters of the reproductive capacity of the animals from the F0

male and female laboratory Wistar albino rats (the Institute's own breeding colony).

difference is, probably, the result of stress (Bečić et al, 2007).

**3.5 A pilot study of the reproductive toxicity in rats** 

, glucose, total proteins, albumins, globulins, total bilirubin, creatinine, cholesterol,

a consequence of stress.

cholinesterase, AST, GGT.

Na+, Cl-

In the subchronic toxicity study, not a single lethal outcome was registered. No statistically significant differences between experimental and control groups in body weight of animals, in food and water consumption were documented. No statistically significant differences between experimental and control groups in the sensitivity to pain, frequency of breathing, were detected, nor differences in post-mortem organ weights during macroscopic examination and histopathological analysis of the tissue samples. No statistically significant differences were found between experimental and control groups in the average duration of ECG parameters and biochemical parameters in males. The results of the investigation of the Enkorten influence on the biochemical parameters in females showed the following statistically significant differences: in the values of calcium (p = 0.046) between the third and the first group (FG3 *vs.* FG1); in the values of sodium (p = 0.044) although no statistically significant differences were documented in the sodium values between the individual experimental groups and the control group; in the values of urea (p = 0.049) although no statistically significant differences in the urea values between the individual experimental groups and the control group were documented; in the values of ASAT (p = 0.020) between the first and the control group; in the values of cholinesterase (p = 0.036) between the first and the control group (FG1 *vs.* FGK). It may be concluded that Enkorten probably affected the values of ASAT and cholinesterase in females, whereas the differences in the values of calcium, sodium and urea were probably due to chance. The results of the haematological parameters showed no statistically significant differences. It can be concluded that the tested substance had no impact on haematological parameters. Test results of the toxic effect investigation suggested that the maximum tolerated dose for male and female rats approximately corresponds to the dose 10 times higher than the anticipated human therapeutic dose. Based on the estimates obtained, we can conclude that in the study conducted in male and female Wistar albino rats, the investigated product showed no subchronic toxicity when the multiple i.m. application of animal doses equivalent to the either anticipated human therapeutic dose, or 5 or 10 times higher dose was performed.

During the implementation of the subchronic Enkorten toxicity study in rabbits, individual cases of mortality, that cannot be linked with the activity of the test substance, have been documented in all experimental groups.

#### **3.4 Chronic toxicity study**

Based upon results of the chronic toxicity study performed on rats after the subcutaneous administration of Enkorten (three experimental groups, each of 40 Wistar albino rats and a control group) during six months, it was concluded that test substance did not show any toxic effects. In the course of the chronic toxicity study, not one lethal outcome was registered. In the course of pain sensitivity testing, the tested substance did not demonstrate a significant analgesic effect. Moreover, it was found that the tested substance increases the irritability of animals in the sense that it increases the frequency of phonation. The histopathology laboratory reports recorded no changes with respect to the mass of single organs or with respect to the macroscopic and microscopic structure of organs and tissues of experimental animals. The difference is noted in the mass of the left adrenal in the control and the first group in relation to administering the combination to male rats. Impact of tested substances on biochemical parameters in males showed statistically significant difference between the first and the control groups, as well as between the second and the

In the subchronic toxicity study, not a single lethal outcome was registered. No statistically significant differences between experimental and control groups in body weight of animals, in food and water consumption were documented. No statistically significant differences between experimental and control groups in the sensitivity to pain, frequency of breathing, were detected, nor differences in post-mortem organ weights during macroscopic examination and histopathological analysis of the tissue samples. No statistically significant differences were found between experimental and control groups in the average duration of ECG parameters and biochemical parameters in males. The results of the investigation of the Enkorten influence on the biochemical parameters in females showed the following statistically significant differences: in the values of calcium (p = 0.046) between the third and the first group (FG3 *vs.* FG1); in the values of sodium (p = 0.044) although no statistically significant differences were documented in the sodium values between the individual experimental groups and the control group; in the values of urea (p = 0.049) although no statistically significant differences in the urea values between the individual experimental groups and the control group were documented; in the values of ASAT (p = 0.020) between the first and the control group; in the values of cholinesterase (p = 0.036) between the first and the control group (FG1 *vs.* FGK). It may be concluded that Enkorten probably affected the values of ASAT and cholinesterase in females, whereas the differences in the values of calcium, sodium and urea were probably due to chance. The results of the haematological parameters showed no statistically significant differences. It can be concluded that the tested substance had no impact on haematological parameters. Test results of the toxic effect investigation suggested that the maximum tolerated dose for male and female rats approximately corresponds to the dose 10 times higher than the anticipated human therapeutic dose. Based on the estimates obtained, we can conclude that in the study conducted in male and female Wistar albino rats, the investigated product showed no subchronic toxicity when the multiple i.m. application of animal doses equivalent to the either anticipated human therapeutic dose, or 5 or 10 times higher dose was performed. During the implementation of the subchronic Enkorten toxicity study in rabbits, individual cases of mortality, that cannot be linked with the activity of the test substance, have been

Based upon results of the chronic toxicity study performed on rats after the subcutaneous administration of Enkorten (three experimental groups, each of 40 Wistar albino rats and a control group) during six months, it was concluded that test substance did not show any toxic effects. In the course of the chronic toxicity study, not one lethal outcome was registered. In the course of pain sensitivity testing, the tested substance did not demonstrate a significant analgesic effect. Moreover, it was found that the tested substance increases the irritability of animals in the sense that it increases the frequency of phonation. The histopathology laboratory reports recorded no changes with respect to the mass of single organs or with respect to the macroscopic and microscopic structure of organs and tissues of experimental animals. The difference is noted in the mass of the left adrenal in the control and the first group in relation to administering the combination to male rats. Impact of tested substances on biochemical parameters in males showed statistically significant difference between the first and the control groups, as well as between the second and the

documented in all experimental groups.

**3.4 Chronic toxicity study** 

control group in the values of phosphorous, alkaline phosphates, and urea. The difference in triglycerides between the first and the control group, the AST values between the second and third group and the first and the third group, the ALT values between the second and the control group were also noticed. The sifnificant difference in the values of these parameters for males may be coincidental, because the difference between the groups treated by the highest dose level and the control group was not registered. In females a statistically significant difference in calcium and potassium was registered between the treated and the control groups. The values of phosphorus and alkaline phosphatase significantly differed between the first and the control group, and urea value between the first and the control group. Triglyceride statisticaly levels of all three treated groups significantly differed from the control group values. Also, there was a significantly difference in the values of ALT between the second and the control group. These data suggest that tested substance affects the value of calcium, potassium and triglycerides in females. The value difference of other parameters can be random, because the difference between the groups treated with the highest dose level and the control group was not registered. The data on the volume and biochemical parameters of urine showed statistically non significant difference between control and other groups of animals. Statistically significant difference between the control and first group of animals was found only in glucose during the second measurement. Registered difference is probably a consequence of stress.

Statistical analyses of the results obtained after biochemical analyses were not statistically significant (including both male and female groups) for the following parameters: Ca+2, K+, Na+, Cl-, glucose, total proteins, albumins, globulins, total bilirubin, creatinine, cholesterol, cholinesterase, AST, GGT.

The tested substance has no effect on the parameters of erythrocytes, leukocytes, granulocytes, thrombocytes and lymphocytes. However, the tested substance has effects on the value of monocytes when compared to the control group, and, as far as males are concerned, also in the values of lymphocytes. The tested substance did not have effects on the volume and biochemical parameters of urea, except for the glucose report. The noted difference is, probably, the result of stress (Bečić et al, 2007).

#### **3.5 A pilot study of the reproductive toxicity in rats**

Testing was conducted at the Institute following the NTP, 2002 (NTP - National Toxicology Program - Good Science and Good Decisions - NIEHS, USA, 2002), in five months old, adult male and female laboratory Wistar albino rats (the Institute's own breeding colony).

In the first phase, Enkorten in a doubled human therapeutic dose (2HD; α-MSH 0.028 mg/kg + met-enkephalin 0.143 mg/kg) was administered once intraperitoneally (i.p.). Afterwards, animals were administered a bilateral intranasal (i.n.) dose of Enkorten eight times higher than the human therapeutic dose (8HD) per day, within two consecutive days. Administration of the substance in the above doses was carried out using the methods of crossing and gender matching. In the second phase, the same dosing protocol but with the i.n. dose increased to twenty times human therapeutic dose (20HD) was administered to the F1 generation of male and female rats, after they reached sexual maturity. By applying the same protocol, the control group received a physiological solution. During the first and the second phase, the typical parameters of the reproductive capacity of the animals from the F0

Enkorten – A Potential Drug for the Treatment of Rheumatoid Arthritis 167

Plasma Concentration) is the highest concentration of drug in the blood that is measured after a dose; Tmax (Time to Reach Cmax) is the time at which the highest plasma drug concentration occurs Cmax; AUC (Area Under the Curve) is the measure of total plasma exposure of drug over a given time period; T½ (Half-life) is the time required for a given

The study was carried out on a group of 14 healthy volunteers, ages from 18 to 30, males, of

All volunteers received subcutaneously one dose of Enkorten preparation in the presumed therapeutic dose for human application. No serious side-effects were observed during the study. The available kit is not specific for the determination of α-MSH of the test preparation; thus, the cross-reactivity of physiologically present α-MSH and α-MSH component of the preparation was determined. Significant interindividual differences of α-MSH plasma concentration were found. Mean values were 21.31 - 47.56 pg/ml. Mean plasma concentration of endogenous met-enkephalin was 50.40 pg/ml. Maximum plasma concentration after Enkorten application, measured 5 minutes after the application, was 1551.86 pg/ml. The plasma half-life was 15 minutes. The concentration/time curve of met-

Enkorten is an immunomodulatory preparation containing two components, α-melanocyte stimulating hormone (α-MSH) and methionine-enkefalin (met-enkephalin). α-MSH and met-enkephalin are endogenous substances of the neuropeptide group. They participate in the homeostatic processes, maintaining the biochemical link between the brain, neuroendocrine system and the immune system. The mechanism of action of both peptides directly reclines on both mechanisms of immune response development. The effect also

Pharmacodynamics of the combination shown in the studies imply great therapeutic possibilities. The presumed indication range of Enkorten encompasses diseases with inflammatory processes as the primary pathophysiological aspect (asthma, inflammatory

Preclinical findings indicate that the tested combination was nontoxic and well tolerated in doses applied by i.v., s.c., i.m., and i.p. routes, and therefore have the potential for safe

Adachi, S., Nakano, T., Vliagoftis, H., Metcalfe, D.D. (1999). Receptor-mediated modulation

Andersen, G.N., Hagglund, M., Nagaeva, O., Frangsmyr, L., Petrovska, R., Mincheva-

of murine mast cell function by -melanocyte stimulating hormone. *J Immunol* 163:

Nilsson, L., Wikberg, J.E. (2005). Quantitative measurement of the levels of melanocortin receptor subtype 1, 2, 3 and 5 and pro-opiomelanocortin peptide gene expression in subsets of human peripheral blood leucocytes. *Scand J Immunol* 61:

drug concentration to decrease by 50%.

**5. Conclusion** 

future use as a medicine.

3363–3368.

279–284.

**6. References** 

body weight and height within the standard values.

enkephalin implies first-order kinetics (Kusturica et al, 2009).

includes analgesia, antipyretic and anti-inflammatory activities.

bowel disease, rheumatoid arthritis, multiple sclerosis).

and F1 generations (gestation, litter and lactation) as well as the litter of the F1 and F2 generations (number of offspring per litter, size, weight, growth, development and advancement until the age of 8 weeks) were followed. At the end of the study, *post mortem* analysis was performed on the tissue and reproductive organs of young male and female rats from the F2 generation.

Comparing to controls, Enkorten did not cause any changes in the first phase of testing. In the second phase of the study, no gestation appeared in the experimental group from the F1 generation, whereas in the remaining four groups the reproduction process was completely in accordance with the control. The number of offspring per litter ranged from 8 to 12. The size and weight of youth as well as gender contribution were fully in compliance with the control group. The development, growth, advancement of youth and lactation were progressing normally. Development was monitored until the age of 8 weeks of life, after which histopathological analysis of tissues and reproductive organs was performed. *Post mortem* analyses did not reveal significant changes in any of the young animals. No signs of teratogenicity were documented in the two generations of young rats.

Based on the observed results, it can be concluded that Enkorten did not significantly affect the process of reproduction in rats. Enkorten did not cause any teratogenic changes. Histopathological reports were completely normal.
