DURABLE PREPARATION OF AN INJECTABLE OF MELATONIN EXHIBITING LONG-TERM STABILITY

FIELD OF THE INVENTION

The present invention is comprised in the field of medicine and pharmacy and relates to an injectable composition of melatonin having high stability. The present invention also relates to the use of said composition as a medicinal product and to its use in the treatment of various conditions, such as sepsis for example.

STATE OF THE ART

Melatonin (N-acetyl-5-methoxy-tryptamine) is an endogenous neurohormone physiologically produced by the pineal gland (epiphysis cerebri). Its rate of secretion follows a circadian rhythm linked to the light-dark cycle, and it plays a fundamental role in inducing sleep. Furthermore, it has been found that melatonin plays a fundamental role in inflammatory response regulation, since it acts as a potent scavenger of oxygen free radicals that are generated, for example, during sepsis and the subsequent development of systemic inflammatory response syndrome (SIRS) and the subsequent multiple organ dysfunction syndrome (MODS), which is also known as multiple organ failure; during myocardial infarctions; in mitochondrial damage; in abdominal surgery processes; in pulmonary edema; and in kidney or liver failure.

It is known to activate antioxidant enzyme pathways (superoxide dismutase, glutathione peroxidase, glutathione reductase) (Reiter et al. 1995. J Pineal Res 18:1-11), to regulate mitochondrial homeostasis (Acuña-Castroviejo et al. 2011. Curr Top Med Chem 11:221-240), to reduce the total number of circulating polymorphonuclear leukocytes and serum malondialdehyde levels (Gitto et al., 2004. J Pediatr Surg 39: 184), to modulate monocytes, NK and the production of cytokines, and to inhibit apoptosis (Mundigler et al., 2002. Crit Care Med 30:536-40; Carrillo-Vico et al. 2005. J Pineal Res 39:400-408), and to reduce proinflammatory cytokine levels and oxidative damage in patients with Duchenne muscular dystrophy (Chahbouni et al. 2010, J Pineal Res 48:282-289), among other functions. It has also been found that in critical patients with sepsis there is a disruption in the circadian rhythm of melatonin, while endogenous secretion of melatonin is preserved in patients without sepsis (Mundigler et al., 2002. Crit Care Med 30:536-40).

Its therapeutic usefulness has been demonstrated in different pathologies (Sánchez-Barceló et al. 2010, Curr Med Chem 17:2070-2095), where as a general rule it shows a lack of toxicity after administration (Acuña-Castroviejo et al. 2014. Cell Mol Life Sci DOI: 10.1007/s00018-014-1579-2). It has therefore been used successfully in the treatment of epilepsy (Molina-Carballo et al., 1997. J Pineal Res 23:97-105), as a regulator of the sleep-wake cycle in general (Burke et al. 2013. Sleep 36:1617-1624) and in patients admitted to Intensive Care Units (Mohan and Brunner, 2005. Acta Anaesthesiol Scand 49:1397). Its intravenous use in newborns with sepsis caused a significant drop in mortality without side effects (Gitto et al., 2004. J Pediatr Surg 39: 184). In this case, melatonin was administered intravenously using an ethanol:water composition (1:50). It has also been demonstrated to have a cardioprotective effect after an acute myocardial infarction (Kücükakin et al., 2008. J Pineal Res 44:426-31). Its ability to reduce the inflammatory response and oxidative stress induced by aggressive procedures during surgery, as well as its safety, efficacy and lack of side effects when administered intravenously at different doses have been demonstrated (Kücükakin et al., 2009. J Surg Res 152:338-347; Naguib et al., 2001. British J Anaesth 90:504-507). In this last reference, the carrier used for administering melatonin is a 2:1:1 mixture of water, propylene glycol (PPG) and 1-methyl-2-pyrrolidone (NMP). Nevertheless, 40 NMP, a widely used solvent, seems essential in this case for solubilizing water-insoluble melatonin. However, the use of NMP as a pharmaceutical carrier is now posing problems due to its reproductive toxicity.

Therefore, in view of the results and of the scientific evidence on the effect, efficacy and safety of the administration of melatonin, it is necessary to produce improved melatonin compositions that exhibit long-term stability and therefore allow storage.

In this regard, international patent application WO2012/156565 discloses a “stable” aqueous melatonin composition comprising 10 mg/ml of propylene glycol. This aqueous composition is stable for 3 months after preparation, but 6 months after preparation, the following characteristics are observed:

- The preparation at room temperature, both autoclaved and not autoclaved, has a yellowish appearance, which probably occurs as a result of melatonin oxidation.
- The preparation at 4° C., both autoclaved and not autoclaved, crystallizes and does not completely resuspend when at room temperature.
- The preparation at −20° C., both autoclaved and not autoclaved, is cloudy and this cloudiness is not successfully eliminated when the preparation is taken to room temperature.

Therefore, preparations of this type do not allow storage periods exceeding 3 months given their scarce long-term stability in all storage conditions described in patent application WO2012/156565. For this reason, the production of improved melatonin compositions that exhibit long-term stability and therefore allow storage exceeding 3 months is still required.

BRIEF DESCRIPTION OF THE INVENTION

The authors of the present invention have developed an aqueous melatonin composition exhibiting surprising long-term stability and allowing high concentrations of said water-insoluble active ingredient. The properties of said composition render it useful as an injectable, for example, for the intravenous administration thereof.

Therefore, a first aspect of the invention relates to a composition comprising propylene glycol, polyethylene glycol also referred to as poly(oxy-1,2-ethyndiyl), alpha-hydro-omega-hydroxy, PEG, Carbowax, poly(ethylene oxide), polyoxyethylene, polyethylene oxide or Macrogol; and melatonin or a derivative, salt, prodrug, or solvate thereof. This type of composition is suitable for preparing injectable compositions of melatonin which are useful, for example, for the intravenous administration thereof. In a preferred embodiment of the first aspect of the invention, this type of composition is lyophilized.

Any polyethylene glycol (hereinafter, PEG) suitable for use in an intramuscularly, subcutaneously or intravenously injectable formulation can be used to carry out the present invention. The PEG will preferably have a molecular weight between 200 and 600 atomic mass units (amu), and more preferably of 400 amu (PEG 400).

PEG (Polyethylene glycol) is a polyether that is widely used in industry and is expressed with the following general formula:

HO—(CH2-CH2-O—)n-H.

It is also known by the name “Macrogol,” so PEG400 can also be described as Macrogol 400 and is found as a component in the pharmaceutical industry in drops, injectable solutions, artificial tears, gelatin capsules, etc.

The molecular weight differences between the different types of PEG mean that in addition to giving a “last name” to the type of polyethylene glycol, they have a different presentation and affinity for water. For example, PEG 400 is a colorless viscous liquid with high hygroscopicity close to that of PG, while PEG 6000 is a solid substance with a waxy appearance and low hygroscopicity.

They all have low toxicity, for example the PEG400 LD50 is about 30 g/Kg (oral administration in rats). If the results are extrapolated, for a person weighing 70 kg, the toxic dose would be 2100 g. These characteristics make PEG or macrogol ideal for use as a base material for the solution of the present invention.

A second aspect of the invention relates to a composition comprising water or a saline solution, propylene glycol, polyethylene glycol and melatonin or a derivative, salt, prodrug, or solvate thereof.

A third aspect of the invention relates to the use of the composition described in any of the first or second aspects of the invention in the production of a medicinal product.

A fourth aspect of the invention relates to the composition described in any of the first or second aspects of the invention for use as a medicinal product or for use in therapy.

A fifth aspect of the invention relates to the use of the composition described in any of the first or second aspects of the invention in the production of a medicinal product useful in human subjects for the treatment of circadian rhythm regulation, inflammatory response regulation, the treatment of systemic inflammatory response syndrome (SIRS), the treatment of multiple organ dysfunction syndrome (MODS), the treatment of sepsis in newborns and children; the treatment of sepsis in adults, the treatment of myocardial infarctions, the treatment of mitochondrial damage, the treatment of pulmonary edema, the treatment of kidney or liver failure, or the treatment of an oxidative stress situation generated during surgery, and particularly during abdominal surgery.

An alternative aspect with respect to the fifth aspect of the invention relates to the composition described in any of the first or second aspects of the invention for the treatment in a human subject of circadian rhythm regulation, inflammatory response regulation, the treatment of systemic inflammatory response syndrome (SIRS), the treatment of multiple organ dysfunction syndrome (MODS), the treatment of sepsis in newborns and children; the treatment of sepsis in adults, the treatment of myocardial infarctions, the treatment of mitochondrial damage, the treatment of pulmonary edema, the treatment of kidney or liver failure, or the treatment of an oxidative stress situation generated during surgery, and particularly during abdominal surgery.

In the context of the present invention, an adult human is considered to be patient that is 18 years old or older. A newborn is generally considered to be a patient between 0 and 27 days old, a baby between 28 days and 23 months old, a child from 24 months to 11 years old, and an adolescent from 12 to 17 years old. Although there is a correlation between weight and dose, said correlation is not always linear and must be identified for each group of patients.

The compositions of the invention (see compositions of the first or second aspect of the invention) are prepared using standard methods such as those described or those that are referred to in the Spanish and U.S. Pharmacopoeias and similar reference texts.

A sixth aspect relates to the preparation of the composition of the invention, which comprises mixing water, propylene glycol, polyethylene glycol and melatonin or a derivative, salt, prodrug, or solvate thereof, their salts, prodrugs, derivatives or solvates.

DETAILED DESCRIPTION OF THE INVENTION

The authors have discovered that propylene glycol (PPG) alone without being complemented with the use of co-solvents, many of which are potentially toxic, such as ethanol or NMP, is effective in solubilizing melatonin; however, PPG alone does not allow the production of melatonin compositions exhibiting long-term stability. In that sense, the authors of the present invention have discovered how surprisingly PPG complemented with polyethylene glycol allows not only solubilizing melatonin but also producing compositions exhibiting long-term stability.

Therefore, a first aspect of the invention relates to a composition suitable for being combined with water or a saline solution and for preparing an injectable composition of melatonin comprising propylene glycol, polyethylene glycol and melatonin or a derivative, salt, prodrug, or solvate thereof. In a preferred embodiment of this aspect of the invention, the concentrations of each of the components of the composition of the first aspect of the invention must allow obtaining any of the injectable compositions defined in the second aspect of the invention.

In another preferred embodiment of the first aspect of the invention, the composition is lyophilized and comprises a suitable proportion of each of the following components: propylene glycol, polyethylene glycol and melatonin or a derivative, salt, prodrug, or solvate thereof, in order to be able to obtain, once rehydrated, any of the injectable compositions defined in the second aspect of the invention.

A second aspect of the invention relates to a composition in the form of a pharmaceutically acceptable injectable solution comprising water or a saline solution, propylene glycol, polyethylene glycol and melatonin or a derivative, salt, prodrug, or solvate thereof.

According to a preferred embodiment of the second aspect of the invention, the injectable composition or solution comprises:

- between 5 and 50 grams for every 100 ml of the total solution (w/v) of propylene glycol, preferably between 10 and 30 grams for every 100 ml of the total solution (w/v), more preferably about 20 grams for every 100 ml of the total solution (w/v);
- between 5 and 50 grams for every 100 ml of the total solution (w/v) of polyethylene glycol, preferably between 20 and 40 grams for every 100 ml of the total solution (w/v), more preferably about 30 grams for every 100 ml of the total solution (w/v);
- between 0.1 and 30 grams for every 100 ml of the total solution (w/v) of melatonin, preferably between 0.3 and 30 grams for every 100 ml of the total solution (w/v), more preferably between 0.3 and 20 grams for every 100 ml of the total solution (w/v), more preferably between 0.3 and 10 grams for every 100 ml of the total solution (w/v), more preferably between 0.3 and 2 grams for every 100 ml of the total solution (w/v), more preferably between 0.3 and 1 gram for every 100 ml of the total solution (w/v), more preferably between 0.3 and 0.8 grams for every 100 ml of the total solution (w/v) and even more preferably about 0.6 g/100 ml of the total solution; and
- a sufficient amount of water or saline solution.

Therefore, the composition described in the second aspect of the invention allows surprisingly high loads of melatonin while at the same time being stable as it was found that at relatively low concentrations of propylene glycol (PPG) used in the present invention, melatonin is significantly solubilized, thereby reducing the risk of irritation or pain which can present as a side effect with the administration of PPG at high concentrations. It is therefore possible to administer high doses of melatonin without administering at the same time large amounts of propylene glycol, which can have toxic effects at very high doses, and in any case reducing the risk of side effects.

The composition of any of the first or second aspects of the invention can also comprise other pharmaceutically acceptable excipients. According to the EMEA's definition, an excipient is considered any component in the composition other than an active ingredient. Examples of excipients that can be used in the injectable composition of the composition include antimicrobial preservatives, such as methylparaben, propylparaben; antioxidants, such as sodium metabisulfite, propyl gallate; stabilizing and suspending agents, such as modified soluble or swellable celluloses, for example sodium carboxymethyl cellulose (Aquasorb, Blanose, Nymcel); tonicity agents, such as sodium chloride; or solubilizers, such as propylene glycol or polyethylene glycols. These excipients must be within the bounds of the definition of the invention.

According to the present invention, a “pharmaceutically acceptable” composition or component thereof indicates that they are physiologically tolerable and the administration thereof entails a low risk of allergies, side effects, adverse events or other similar reactions, such as gastric disorders, dizziness and the like, when administered to a human being. Preferably, as it is used herein, the expression “pharmaceutically acceptable” means that it has been approved by a regulatory agency of the state or federal government or that it is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in human beings. The composition of the invention is therefore pyrogen-free.

The composition of the invention includes melatonin, as well as a derivative, a salt, a prodrug or a solvate thereof. For example, pharmaceutically acceptable salts are synthesized from melatonin by means of conventional chemical methods, generally by making it react with a suitable acid in water or in an organic solvent or in a mixture of both. Non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are generally preferred. Examples of acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate.

As it is used in this application, the term “prodrug” is defined herein to mean a chemical compound that has experienced a chemical derivatization, such as a substitution or addition of an additional chemical group to change (for pharmaceutical use) any of its physicochemical properties, such as solubility or bioavailability, for example ester, ether or amide derivatives of an active compound providing the active compound itself after administration to a subject. Those skilled in the art know examples of well-known methods for the production of a prodrug of a given active compound and said methods can be found, for example, in Krogsgaard-Larsen et al., Textbook of Drug Design and Discovery, Taylor & Francis (April 2002).

Particularly preferred prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a patient (for example, allowing an orally administered compound to be more readily absorbed into the blood) or that improve the delivery of the original compound to a biological compartment (for example, the brain or lymphatic system) with respect to the original species.

According to this invention, the term “solvate” must be understood to mean any form of melatonin according to the invention having another molecule (most likely a polar solvent) bound by means of a non-covalent bond. The examples of such solvates include hydrates and alcoholates, for example methanolates. The preparation of salts, solvates and prodrugs can be carried out by means of methods known in the art. It must be noted that non-pharmaceutically acceptable salts, solvates or prodrugs are also within the scope of the invention since they can be useful in the preparation of pharmaceutically acceptable salts, solvates or prodrugs.

Various derivatives of melatonin which are also included in the present invention are known in the state of the art. According to a particular embodiment, the derivative of melatonin is defined according to formula (I), a salt, prodrug or solvate thereof

embedded image

wherein,

n is an integer selected from the group consisting of 1, 2, 3 and 4;

R₁and R₃are selected independently from the group consisting of linear or branched C₁-C₄alkyl; and

R₂is selected from the group consisting of hydrogen, linear or branched C₁-C₄alkyl, —C(═O)O—Ra and —C(═O)—N(H)—Ra, wherein Ra is a linear or branched C₁-C₄alkyl group.

In a particular embodiment, the composition of the invention is intravenously injectable. A particular aspect includes the presence of a second medicinal product in the composition of the invention. Said second medicinal product can be part of the composition or can be provided as a separate composition for the administration at the same time or at different times.

Generally, a “therapeutically effective amount” of the composition of the invention, and therefore of melatonin, will depend on various factors, such as the severity of the disorder being treated, the sex, age or weight of the patient, among many others. For example, the composition of the invention can be administered in the range of 0.1 to 1000 mg/kg/day one or more times a day with standard total daily dosages.

The present invention relates to the use of melatonin, its salts, prodrugs, derivatives or solvates in the preparation of a medicinal product for the treatment in humans or animals of processes such as sepsis, for the treatment of systemic inflammatory response syndrome (SIRS) or for the treatment of multiple organ dysfunction syndrome (MODS), the treatment of myocardial infarctions, the treatment of mitochondrial damage, the treatment of pulmonary edema, the treatment of kidney or liver failure, or the treatment of a surgery-induced oxidative stress situation. In a particular embodiment, said use involves the administration of between 5 and 1,000 mg, between 5 and 700 mg, between 5 and 600 mg or between 5 and 300 mg of melatonin every 24 hours. In another particular embodiment, the amount of melatonin administered to a patient is comprised between 30 and 90 mg every 4 hours, preferably between 40 and 70. In a particular embodiment, between 55 and 75 mg of melatonin are administered to the patient every 24 hours. In general and according to the human equivalent dose calculation (Reagan-Shaw et al. 2007. Faseb J 22:659-661), the minimum doses of melatonin would range between 50 and 500 mg/day (Venegas et al. 2012. J Pineal Res 52:217-227).

In another preferred embodiment of the invention, said use involves the administration of at least 300 mg, preferably at least 400 mg and even more preferably of at least 500 mg of melatonin every 24 hours. In this preferred embodiment of the invention, said use preferably refers to the treatment of sepsis.

In a particular embodiment, the administration is performed by perfusion. In another embodiment, melatonin, its salts, prodrugs, derivatives or solvates, is administered 1, 2, 3, 4, 5 or 6 times or more a day until reaching the required total daily dose. The treatment period can vary according to the patient's progression, and it usually lasts between 1 and 30 days.

In a particular embodiment, said sepsis in adults is severe sepsis. SIRS is a generalized inflammatory response of a range of severe clinical injuries. According to the definition agreed on by the American College of Chest Physicians/Society of Critical Care Medicine, this syndrome is clinically recognized by the presence of two or more of the following symptoms (i) to (iv):

- (i) Temperature >38° C. or <36° C.
- (ii) Heart rate >90 beats/min.
- (iii) Respiratory rate >20 breaths/min or PaCO₂<32 mmHg.
- (iv) White blood cell count >12,000 cells/mm₃, <4,000 cells/mm₃, or >10% of immature (band) forms.

Sepsis corresponds to SIRS due to a clear focus of infection. Diagnosis thereof requires two or more SIRS criteria and the presence of a clear clinical picture of infection or microbiological studies (the presence of pathogenic microorganisms in normally sterile fluids, more than 100,000 CFU/ml in urine or in quantitative cultures of bronchial secretions). In addition, sepsis is considered severe when it is associated with organ dysfunction, hypoperfusion or hypotension (<90 mm Hg of systolic blood pressure). Manifestations of hypoperfusion can be included but are not limited to lactic acidosis (lactic acid >3 mmol/l), oliguria (diuresis 50<30 ml/h for 3 hours or 700 ml in 24 hours), coagulopathy (prolongation of the prothrombin time or thrombocytopenia less than 100,000/ml), or an acute change in mental state (agitation, obnubilation).

The term “treatment” or “treating” in the context of this document refers to the administration of a compound or formulation according to the invention to prevent, improve or eradicate the disease or one or more symptoms associated with said disease. “Treatment” also covers the prevention, improvement or eradication of the physiological sequelae of the disease.

Throughout the description and claims the term “comprises” and variants thereof do not seek to exclude other technical characteristics, supplements, components or steps. For the persons skilled in the art, other objects, advantages and features of the invention will be deduced in part from the description and in part from putting the invention into practice.

The following examples and drawings are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES

The invention will be illustrated below by means of tests conducted by the inventors, clearly showing the stability and effectiveness of the composition of the invention.

Example 1. Preparation of the Composition of the Invention

The melatonin for the injectable solution was prepared at a concentration of 6 mg/ml in about 20% of propylene glycol and about 30% of polyethylene glycol and with pyrogen-free water in a sufficient amount (API).

TABLE 1

Qualitative and quantitative composition of the tested composition

Component
Composition (per ml)
Function

Melatonin
6.0 mg
Active ingredient

Propylene glycol
200.0 mg
Excipient

Polyethylene glycol
300.0 mg
Excipient

(macrogol)

Water for injectables
Sufficient amount 1 ml
Solvent

The material used for packaging the composition described in Table 1 were type I glass ampoules (EP) previously sterilized in an oven.

Example 2. Stability Tests
2.1. General Principles

The present study is a stability study of the product specified in Table 1 after preparation and over a 6-month period. Three industrial-sized baths each comprising 1,000 ampoules of the product specified in Table 1 were used to that end.

2.2 Stability Test Results

TABLE 2

Long-term stability data containing the solution comprising 6 mg/ml

described in Table 1.

Solution comprising 6 mg/ml of melatonin for injection in ampoules.

Batch size: 1,000 ampoules
Type of bath: Scaled-down batch

Manufacturing date: 27/02/2013
Product container once closed:

Test conditions:
Temperature 25° C. +/− 2° C.
Type I glass ampoules containing

Relative Humidity 60% +/− 5%
a colorless, clear and particle-free

Position of the product: right
solution.

Technical
0
3
6

Parameter
specifications
(2013/03)
(2013/06)
(2013/06)

Appearance of the
Glass ampoule
Complies with
Complies with
Complies with

product
containing a
the technical
the technical
the technical

clear, colorless
specification
specification
specification

solution.

Particle-free.

Identification of
Positive
Positive
Positive
Positive

melatonin/UV-

visible)

Determination of
5.5-7.5
6.6
6.6
6.5

pH

Density
1.04-1.08
1.07
1.06
1.06

Extractable
Greater than or
9.5
9.4
9.3

volume
equal to 9 mL

Total

5-
< or =1.5%
n,d
n,d
0.11

methoxytryptamine

individual
< or =0.50%
n,d
n,d
n,d

unknown
< or =0.10%
n,d
n,d

degradation

products

TRR0.7
< or =0.10%

0.01

TRR2.0
< or =0.10%

0.08

Melatonin content
5.7-6.5 mg/ml
5.96
5.84
5.76

Subvisible
>=10 um: < or
115
135
108

particles
=6,000

particles/vial
21
25
18

>=25 um: < or

=600

particles/vial

Tightness
Leak-tight
Complies with
Complies with
Complies with

ampoule
the technical
the technical
the technical

specification
specification
specification

Bacterial
< or =35.1 EU/ml
<35.1
—
—

endotoxins (LAL

test)

Sterility test
Sterile
Sterile
—
—

From the long-term stability test shown, it can be concluded that the described ampoules containing the solution comprising 6 mg/ml of melatonin for injection, after storage over a 6-month period, comply with the technical specifications required for a product having these characteristics.

Example 3. Clinical Study with Septic Patients

The composition of the invention, the solution comprising 6 mg/ml of melatonin for injection in ampoules described in Example 1, hereinafter “injectable of melatonin”, was used in a clinical study with 14 septic patients after abdominal surgery randomly distributed into 2 study groups (A and B). Group A corresponds to patients who, in addition to standard treatment, received the injectable of melatonin at a dose of 60 mg/day for 5 days, blood samples being taken daily to perform successive analytical determinations. Treatment group B received standard treatment and placebo, the latter being the same vial with the same excipients but without the melatonin active ingredient; daily blood samples are also obtained from each patient in this group to perform successive analytical determinations. The blood samples are referred to as T0, T1, T2, T3, T4 and T5.

The following blood parameters were analyzed for each of the participating patients from these samples: number of leukocytes, number of red blood cells, hemoglobin, hematocrit, percentage of neutrophils, percentage of lymphocytes, and number of platelets. The biochemical parameters determined in each patient participating in the study were: transaminases (GOT and GPT), gamma-glutamyl transferase, creatinine, urea, alkaline phosphatase (ALP) and lactic dehydrogenase (LDH).

Justification of the Determinations Performed

The blood parameters comprising the number of leukocytes, neutrophils and lymphocytes, as well as the number of platelets, are parameters that are indicative of a septic state. In this regard, sepsis is known to cause a drop in the percentage of lymphocytes.

The determined biochemical parameters are related to liver function, such as:

- Glutamate oxalacetate transaminase or aspartate amino transferase (GOT/AST): enzyme belonging to the transaminase group which, by transferring amino groups, catalyzes the conversion of amino acids into corresponding α-oxoacids and vice versa. It is found in the cytoplasm and mitochondria. It is highly specific for a liver disease.
- Glutamate pyruvate transaminase or alanine amino transferase (GPT/ALT): enzyme also belonging to the transaminase group and which, by transferring amino groups, catalyzes the conversion of amino acids into corresponding α-oxoacids and vice versa. Its highest level of activity is found in the liver.
- Gamma-glutamyl transferase (GGT): contributes to the diagnosis and control of liver-bile diseases. The enzyme activity of GGT is often the only parameter that increases with respect to diseases of this type, and it is highly sensitive.
- Alkaline phosphatase (ALP/FA): in the human serum, there are four structural genotypes: liver-bone-kidney type, the intestinal type, the placental type and the stem cell variant. This enzyme is found in osteoblasts, hepatocytes, the kidneys, the spleen, the placenta, the prostate, leukocytes and in the small intestine. The liver-bone-kidney type is of particular importance.

In relation to renal function, the following parameters have been determined:

- Creatinine is a waste molecule that is generated from muscle metabolism. Creatinine comes from the creatine, a very important molecule for the production of energy in muscles. About 2% of the body's creatine is converted to creatinine every day. Creatinine is transported from the muscles to the kidney by means of blood. The kidneys filter most of the creatinine and eliminate it in urine. The determination of serum creatinine is a test indicating with fairly accurate reliability the state of renal function.
- Urea, the determination of which is the most widely used test to assess renal function. Urea is the end product of protein and amino acid metabolism. In protein degradation, proteins are broken down into amino acids and deaminated. With the ammonia that is formed, urea is synthesized in the liver. This is the most important pathway in the human body for excess nitrogen degradation.

To detect lesions in tissues such as the liver, the enzyme lactate dehydrogenase (also called “lactic acid dehydrogenase” (LDH)) an enzyme which is found in virtually all tissues in the human body, has been determined. It plays an important role in cellular respiration (the process in which the glucose coming from foods is turned into energy that can be used by cells).

Even if LDH is abundant in tissue cells, blood levels are generally low. However, when tissues become damaged due to a lesion or disease, they release more LDH into the blood stream. The conditions usually causing this increase in the amount of LDH in the blood stream are the following: liver diseases, heart attacks, anemia, muscle trauma, bone fractures, cancer, infections such as meningitis, encephalitis or HIV.

Results and Discussion

The data from the study has been statistically analyzed by an independent statistician and the following results are obtained:

- Leukocytes: The results show a decline in the leukocyte levels both in group A and group B; although leukocyte values decline on average in both groups, the differences are not statistically significant.
- Red blood cells: Although red blood cell values decline on average in both groups, the differences are not statistically significant.
- Hemoglobin: Although hemoglobin values decline on average in both groups, the differences are not statistically significant.
- Hematocrit: Although hematocrit values decline on average in both groups, the differences are not statistically significant.
- Platelets: Although platelet values increase on average in both groups, the differences are not statistically significant.
- Lymphocytes: There are significant differences between the groups (p=0.015), i.e., the lymphocyte average is greater in group A (treated with the injectable of melatonin) than in group B (placebo), regardless of the time (the differences are the same at all measured moments). Furthermore, the effect of time is statistically significant (p=0.005, since sphericity is not complied with), which means that the increase in lymphocyte levels is different at the different measured moments in time. Specifically, the differences are due to times 4 and 5 with respect to the initial time.
- Neutrophils: Like in the case of lymphocytes, there are significant differences between the study groups (p=0.007), i.e., the neutrophil average is greater in group B (placebo) than in group A (melatonin), regardless of the time (the differences are the same at all measured moments). The differences over time are statistically significant (p=0.042, since sphericity is not complied with). It can virtually be said that after moment 3, the decline starts to be significant in group A (melatonin).
- GOT: Even though initial GOT levels are higher in patients subjected to treatment (group A), the differences in average values are not statistically significant (p=0.633). Nor is the change in the different moments in time significant, i.e., GOT levels remain virtually identical.
- GPT: The change in GPT levels is not significant in the different measured moments. Nor are the average values statistically significant.
- GGT: The change in GGT levels is not significant in the different measured moments. Nor are the average values statistically significant.
- ALP (alkaline phosphatase): The change in alkaline phosphatase is not significant in the different measured moments. Nor are the average values statistically significant.
- Urea: There are no significant differences in urea levels over time; they remain virtually identical in both groups (Friedman test, p=0.205 for group A and p=0.959 for group B). If urea levels between the two groups are compared at each measured moment in time, it can be seen that urea levels are higher in group B (placebo) at all the measured moments (except the last one). The differences are statistically significant.
- Creatinine: There are no significant differences in creatinine levels over time; they remain identical in both groups (Friedman test, p=0.122 for group A and p=0.831 for group B). If creatinine levels between the two groups are compared at each measured moment in time, it can be seen that there are no statistically significant differences in creatinine levels between groups A and B.
- LDH: There are no significant differences in LDH levels over time; they remain virtually identical in both groups (Friedman test, p=0.355 for group A and p=0.921 for group B). If LDH levels between the two groups are compared at each measured moment in time, it can be seen that LDH levels are higher in group A (melatonin) at all the measured times, although the differences are not statistically significant.
  
  Analyses with Non-Parametric Tests:

Even though the hypothesis of normality of all the variables is complied with despite the small sample size, non-parametric tests were used as they are more robust and suitable when the samples are so small.

Taking this into account, on one hand the progression over time of the different parameters in each group have been compared separately using the Friedman test for independent samples. For progression over time, statistically significant differences (p<0.10) are obtained in the following parameters:

- The hemoglobin level observed at different times for group A (p=0.069).
- The platelet level observed at different times for group A (p=0.056) and B (p=0.069).
- The lymphocyte level observed at different times for group A (p=0.030) and B (p=0.085).
- The neutrophil level observed at different times for group A (p=0.070).
- The GOT level observed at different times for group A (p=0.013) and B (p=0.077).
- The GPT level observed at different times for group B (p=0.004).
- The GGT level observed at different times for group A (p=0.079).

In addition, the direct differences between groups A and B have been compared at each moment in time independently using the Mann-Whitney test for independent samples, giving the following results:

- At moment T2, there are differences between A and B in red blood cell (p=0.026), hemoglobin (p=0.038) and hematocrit (p=0.053) levels.
- There are differences in lymphocyte levels at all times.
- There are differences in neutrophil levels between A and B at T2 (p=0.007) and T3 (p=0.011).
- There are differences in GPT levels between A and B at T5 (p=0.038).

CONCLUSIONS

Treatment with the injectable of melatonin in septic patients in group A receiving the injectable shows a progressive increase in the percentage of lymphocytes. This increase is statistically significant. These patients also show a statistically significant decline in the percentage of neutrophils. Both the increase in lymphocytes and the decline in neutrophils occur at all times of the study, reaching levels close to normal values in healthy individuals at the end of the study period. This situation entails an immunological recovery in patients receiving treatment with the injectable of melatonin, as the balance between lymphocytes and neutrophils in patients receiving treatment with the injectable is achieved.

In addition, treatment with the injectable of melatonin does not involve any liver or kidney damage in patients receiving treatment with the injectable.

Statistical Analysis Details

The details of the analysis performed on the parameters measured at all the moments (T0, . . . , T5) of the study are provided below.

Leukocytes

Group
Mean
Standard Deviation
N

Leukocytes 10_3 ml
A
14.7329
9.09637
7

T0
B
19.2857
7.82250
7

Total
17.0093
8.48601
14

Leukocytes 10_3 ml
A
14.4200
9.59540
7

T1
B
19.0529
8.86479
7

Total
16.7364
9.19473
14

Leukocytes 10_3 ml
A
11.6614
4.49294
7

T2
B
16.6786
5.94305
7

Total
14.1700
5.69169
14

Leukocytes 10_3 ml
A
10.1457
4.71706
7

T3
B
15.5086
5.02559
7

Total
12.8271
5.44697
14

Leukocytes 10_3 ml
A
11.9529
8.60995
7

T4
B
15.4971
3.56829
7

Total
13.7250
6.59341
14

Leukocytes 10_3 ml
A
12.1114
6.91874
7

T5
B
12.8900
2.85623
7

Total
12.5007
5.10116
14

Although leukocyte values decline on average in both groups, the differences are not statistically significant.

Red Blood Cells

Group
Mean
Standard Deviation
N

red blood cells 10_3 ml
A
4.1243
0.83320
7

T0
B
3.4043
1.06878
7

Total
3.7643
0.99358
14

red blood cells 10_3 ml
A
3.8457
0.62612
7

T1
B
3.7671
0.74904
7

Total
3.8064
0.66449
14

red blood cells 10_3 ml
A
3.8100
0.32655
7

T2
B
3.1486
0.64300
7

Total
3.4793
0.59818
14

red blood cells 10_3 ml
A
3.6071
0.31700
7

T3
B
3.2843
0.49315
7

Total
3.4457
0.43207
14

red blood cells 10_3 ml
A
3.6371
0.52506
7

T4
B
3.4357
0.56151
7

Total
3.5364
0.53262
14

red blood cells 10_3 ml
A
3.6900
0.52173
7

T5
B
3.2643
0.66795
7

Total
3.4771
0.61672
14

Although red blood cell values decline on average in both groups, the differences are not statistically significant.

Hemoglobin

Group
Mean
Standard Deviation
N

hemoglobin 10_3 ml
A
12.0714
2.59597
7

T0
B
10.1143
2.72484
7

Total
11.0929
2.75107
14

hemoglobin 10_3 ml
A
11.0857
1.94202
7

T1
B
11.3143
1.88275
7

Total
11.2000
1.84140
14

hemoglobin 10_3 ml
A
11.0571
0.85021
7

T2
B
9.4143
1.63649
7

Total
10.2357
1.51536
14

hemoglobin 10_3 ml
A
10.6429
1.01301
7

T3
B
9.8143
1.15243
7

Total
10.2286
1.12758
14

hemoglobin 10_3 ml
A
10.6143
1.37408
7

T4
B
10.1429
1.28693
7

Total
10.3786
1.30217
14

hemoglobin 10_3 ml
A
10.7286
1.52065
7

T5
B
9.5143
1.54103
7

Total
10.1214
1.60009
14

Although hemoglobin values decline on average in both groups, the differences are not statistically significant.

Hematocrit

Group
Mean
Standard Deviation
N

hematocrit (%)
A
34.2857
7.21097
7

T0
B
29.4714
8.19445
7

Total
31.8786
7.82503
14

hematocrit (%)
A
32.1143
5.31583
7

T1
B
32.9571
5.82662
7

Total
32.5357
5.37610
14

hematocrit (%)
A
31.9714
2.38447
7

T2
B
27.6143
5.00447
7

Total
29.7929
4.39256
14

hematocrit (%)
A
30.3857
2.65796
7

T3
B
28.7143
3.62971
7

Total
29.5500
3.17702
14

hematocrit (%)
A
30.6571
4.25435
7

T4
B
30.0571
3.99452
7

Total
30.3571
3.97680
14

hematocrit (%)
A
31.1143
4.22588
7

T5
B
28.5714
5.12826
7

Total
29.8429
4.70331
14

Although hematocrit values decline on average in both groups, the differences are not statistically significant.

Platelets

Group
Mean
Standard Deviation
N

platelets 10_3 ml
A
249.0000
165.65526
7

T0
B
387.7143
215.01451
7

Total
318.3571
197.94778
14

platelets 10_3 ml
A
234.4286
183.64355
7

T1
B
374.5714
229.87958
7

Total
304.5000
212.70375
14

platelets 10_3 ml
A
240.2857
196.55594
7

T2
B
316.8571
194.91146
7

Total
278.5714
192.20771
14

platelets 10_3 ml
A
256.1429
187.80879
7

T3
B
301.7143
213.66930
7

Total
278.9286
194.70469
14

platelets 10_3 ml
A
317.7143
250.15576
7

T4
B
320.8571
201.71880
7

Total
319.2857
218.32313
14

platelets 10_3 ml
A
380.1429
309.16847
7

T5
B
277.7143
220.31017
7

Total
328.9286
263.32941
14

Although platelet values increase on average in both groups, the differences are not statistically significant.

Lymphocytes

Group
Mean
Standard Deviation
N

lymphocytes 10_3 ml
A
7.9571
3.79599
7

T0
B
4.8286
2.78132
7

Total
6.3929
3.58554
14

lymphocytes (%) T1
A
8.9571
5.23159
7

B
4.6714
1.12207
7

Total
6.8143
4.26125
14

lymphocytes (%) T2
A
11.9714
5.03545
7

B
5.6143
2.59257
7

Total
8.7929
5.06807
14

lymphocytes (%) T3
A
14.3571
7.43928
7

B
5.5714
2.36130
7

Total
9.9643
6.99270
14

lymphocytes (%) T4
A
16.6143
11.13903
7

B
7.4143
3.56718
7

Total
12.0143
9.26971
14

lymphocytes (%) T5
A
17.1571
9.21590
7

B
8.2571
5.01825
7

Total
12.7071
8.49402
14

Tests of within-Subject Effects
Transformed Variable: Average

Type III Sum of

Source
Squares
df
Mean Square
F
Sig.

Intercept
7497.630
1
7497.630
62.512
0.000

Group
964.252
1
964.252
8.040
0.015

Error
1439.268
12
119.939

There are significant differences between groups (p=0.015), i.e., the lymphocyte average is greater in group A than in group B, regardless of the time (the differences are the same at all measured moments).

Furthermore, the effect of time is statistically significant (p=0.005, since sphericity is not complied with), which means that the increase in lymphocyte levels is different at the different measured moments in time.

Specifically, the differences are due to times 4 and 5 with respect to the initial time:

Tests of within-Subject Effects

Type III Sum

Source
of Squares
df
Mean Square
F
Sig.

time
Sphericity assumed
478.435
5
95.687
6.345
0.000

Greenhouse-Geisser
478.435
2.190
218.472
6.345
0.005

Huynh-Feldt
478.435
2.921
163.772
6.345
0.002

Lower-bound
478.435
1.000
478.435
6.345
0.027

time * Group
Sphericity assumed
119.374
5
23.875
1.583
0.179

Greenhouse-Geisser
119.374
2.190
54.511
1.583
0.223

Huynh-Feldt
119.374
2.921
40.862
1.583
0.212

Lower-bound
119.374
1.000
119.374
1.583
0.232

Error(time)
Sphericity assumed
904.861
60
15.081

Greenhouse-Geisser
904.861
26.279
34.433

Huynh-Feldt
904.861
35.056
25.812

Lower-bound
904.861
12.000
75.405

Tests of within-Subject Contrasts

Type III

Sum of

Source
Time
Squares
df
Mean Square
F
Sig.

time
Level 2 with respect to level 1
2.486
1
2.486
0.119
0.736

Level 3 with respect to level 1
80.640
1
80.640
4.410
0.058

Level 4 with respect to level 1
178.571
1
178.571
4.678
0.051

Level 5 with respect to level 1
442.406
1
442.406
6.974
0.022

Level 6 with respect to level 1
558.183
1
558.183
8.240
0.014

time * Group
Level 2 with respect to level 1
4.686
1
4.686
0.225
0.644

Level 3 with respect to level 1
36.483
1
36.483
1.995
0.183

Level 4 with respect to level 1
112.011
1
112.011
2.934
0.112

Level 5 with respect to level 1
129.018
1
129.018
2.034
0.179

Level 6 with respect to level 1
116.583
1
116.583
1.721
0.214

Error(time)
Level 2 with respect to level 1
249.977
12
20.831

Level 3 with respect to level 1
219.437
12
18.286

Level 4 with respect to level 1
458.117
12
38.176

Level 5 with respect to level 1
761.286
12
63.440

Level 6 with respect to level 1
812.914
12
67.743

Neutrophils

Group
Mean
Standard Deviation
N

neutrophils (%)
A
87.1143
6.82799
7

T0
B
92.4286
5.35435
7

Total
89.7714
6.50792
14

neutrophils (%)
A
86.3143
8.33175
7

T1
B
92.2714
2.84413
7

Total
89.2929
6.73252
14

neutrophils (%)
A
81.2143
6.01953
7

T2
B
90.6429
4.01325
7

Total
85.9286
6.93480
14

neutrophils (%)
A
79.0714
8.00411
7

T3
B
91.7571
3.77618
7

Total
85.4143
8.91497
14

neutrophils (%)
A
77.8143
12.25988
7

T4
B
87.7857
6.93023
7

Total
82.8000
10.87693
14

neutrophils (%)
A
76.7286
12.21757
7

T5
B
87.9571
6.84930
7

Total
82.3429
11.15752
14

Like in the preceding case, there are significant differences between groups (p=0.007), i.e., the neutrophil average is greater in group B than in group A, regardless of the time (the differences are the same at all measured moments).

Tests of within-Subject Effects
Transformed Variable: Average

Type III Sum of

Source
Squares
df
Mean Square
F
Sig.

Intercept
103363.479
1
103363.479
3804.053
0.000

Group
289.683
1
289.683
10.661
0.007

Error
326.063
12
27.172

The differences over time are statistically significant (p=0.042, since sphericity is not complied with). It can virtually be said that after moment 3, the decline starts to be significant.

Tests of within-Subject Effects

Type III Sum of

Source
Squares
df
Mean Square
F
Sig.

time
Sphericity assumed
685.940
5
137.188
3.898
0.004

Greenhouse-Geisser
685.940
1.725
397.654
3.898
0.042

Huynh-Feldt
685.940
2.156
318.204
3.898
0.030

Lower-bound
685.940
1.000
685.940
3.898
0.072

time * Group
Sphericity assumed
148.626
5
29.725
0.845
0.524

Greenhouse-Geisser
148.626
1.725
86.162
0.845
0.428

Huynh-Feldt
148.626
2.156
68.947
0.845
0.449

Lower-bound
148.626
1.000
148.626
0.845
0.376

Error(time)
Sphericity assumed
2111.492
60
35.192

Greenhouse-Geisser
2111.492
20.700
102.007

Huynh-Feldt
2111.492
25.868
81.626

Lower-bound
2111.492
12.000
175.958

Tests of within-Subject Contrast

Type III

Sum of

Mean

Source
Time
Squares
df
Square
F
Sig.

time
Level 2 with respect to level 1
3.206
1
3.206
0.073
0.791

Level 3 with respect to level 1
206.746
1
206.746
14.423
0.003

Level 4 with respect to level 1
265.786
1
265.786
3.255
0.096

Level 5 with respect to level 1
680.411
1
680.411
4.826
0.048

Level 6 with respect to level 1
772.571
1
772.571
4.499
0.055

time * Group
Level 2 with respect to level 1
1.446
1
1.446
0.033
0.859

Level 3 with respect to level 1
59.246
1
59.246
4.133
0.065

Level 4 with respect to level 1
190.183
1
190.183
2.329
0.153

Level 5 with respect to level 1
75.911
1
75.911
0.538
0.477

Level 6 with respect to level 1
122.426
1
122.426
0.713
0.415

Error(time)
Level 2 with respect to level 1
525.577
12
43.798

Level 3 with respect to level 1
172.009
12
14.334

Level 4 with respect to level 1
979.811
12
81.651

Level 5 with respect to level 1
1691.737
12
140.978

Level 6 with respect to level 1
2060.663
12
171.722

GOT

Group
Mean
Standard Deviation
N

GOT
A
65.1667
51.40201
6

T0
B
27.9500
25.27795
6

Total
46.5583
43.23398
12

GOT
A
56.3333
48.38457
6

T1
B
38.8333
22.24785
6

Total
47.5833
37.04901
12

GOT
A
41.0000
41.26984
6

T2
B
45.6667
53.67184
6

Total
43.3333
45.71121
12

GOT
A
38.3333
34.93232
6

T3
B
35.0333
24.83881
6

Total
36.6833
28.94954
12

GOT
A
38.1667
35.92446
6

T4
B
40.7667
33.31556
6

Total
39.4667
33.06020
12

GOT
A
44.8333
48.67614
6

T5
B
35.2500
29.48856
6

Total
40.0417
38.69488
12

Even though initial GOT levels are higher in patients in treatment A, the differences in average values are not statistically significant (p=0.633). Nor is the change at the different moments in time significant, i.e., GOT levels remain virtually identical.

Tests of within-Subject Effects
Transformed Variable: Average

Type III Sum of

Source
Squares
df
Mean Square
F
Sig.

Intercept
21448.926
1
21448.926
17.157
0.002

Group
303.343
1
303.343
0.243
0.633

Error
12501.881
10
1250.188

GPT

Group
Mean
Standard Deviation
N

GPT
A
51.0000
54.22791
7

T0
B
15.7167
9.56147
6

Total
34.7154
42.93710
13

GPT
A
45.0000
46.54747
7

T1
B
25.5000
18.09696
6

Total
36.0000
36.36161
13

GPT
A
40.2857
29.78654
7

T2
B
24.0000
12.69646
6

Total
32.7692
24.12866
13

GPT
A
32.7143
21.06114
7

T3
B
17.7500
7.27839
6

Total
25.8077
17.43982
13

GPT
A
30.5714
19.26012
7

T4
B
17.6000
7.75113
6

Total
24.5846
15.99405
13

GPT
A
30.2857
21.47645
7

T5
B
13.6667
6.59293
6

Total
22.6154
17.97470
13

The change in GPT levels is not significant in the different measured moments. Nor are the average values statistically significant.

GGT

Group
Mean
Standard Deviation
N

GGT
A
150.2857
158.16206
7

T0
B
94.9333
50.15283
6

Total
124.7385
119.91896
13

GGT
A
131.7143
144.03786
7

T1
B
94.6667
53.64202
6

Total
114.6154
109.27911
13

GGT
A
130.2857
140.32904
7

T2
B
82.3333
35.61835
6

Total
108.1538
104.85136
13

GGT
A
142.4286
160.26526
7

T3
B
128.0000
94.47963
6

Total
135.7692
128.91027
13

GGT
A
147.7143
134.40575
7

T4
B
211.5000
186.63735
6

Total
177.1538
156.97709
13

GGT
A
240.8571
204.12858
7

T5
B
194.5000
194.53611
6

Total
219.4615
192.82445
13

The change in GGT levels is not significant in the different measured moments. Nor are the average values statistically significant.

Alkaline Phosphatase

Group
Mean
Standard Deviation
N

Phosphatase
A
116.6667
87.96969
6

T0
B
95.0000
22.47665
6

Total
105.8333
62.25145
12

Phosphatase
A
100.5000
87.36761
6

T1
B
84.0000
30.02665
6

Total
92.2500
62.87813
12

Phosphatase
A
97.0000
54.85618
6

T2
B
74.8333
16.40020
6

Total
85.9167
40.29992
12

Phosphatase
A
106.3333
65.98081
6

T3
B
109.1667
70.30339
6

Total
107.7500
65.02045
12

Phosphatase
A
108.8333
54.57258
6

T4
B
159.3333
118.81021
6

Total
134.0833
92.00836
12

Phosphatase
A
112.1667
43.56336
6

T5
B
143.8333
102.89104
6

Total
128.0000
77.12446
12

The change in alkaline phosphatase is not significant in the different measured moments. Nor are the average values statistically significant.

Analyses with Non-Parametric Tests:

On one hand, the progression over time of the different parameters in each group is compared separately (using the Friedman test for independent samples), statistically significant differences (p<0.10) are obtained in:

- The hemoglobin level observed at different times for group A (p=0.069):

hemoglobin
hemoglobin
hemoglobin
hemoglobin
hemoglobin
hemoglobin

Group
10_3 ml T0
10_3 ml T1
10_3 ml T2
10_3 ml T3
10_3 ml T4
10_3 ml T5

A
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
12.0714
11.0857
11.0571
10.6429
10.6143
10.7286

Median
12.3000
11.4000
11.0000
10.7000
10.3000
10.9000

SD
2.59597
1.94202
.85021
1.01301
1.37408
1.52065

Minimum
7.40
6.90
9.50
8.80
9.20
8.40

Maximum
16.10
12.90
12.30
11.50
13.30
13.00

B
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
10.1143
11.3143
9.4143
9.8143
10.1429
9.5143

Median
9.1000
11.1000
9.0000
9.9000
10.0000
9.6000

SD
2.72484
1.88275
1.63649
1.15243
1.28693
1.54103

Minimum
8.40
8.60
7.60
8.70
8.80
7.40

Maximum
16.10
14.10
12.50
11.90
12.30
11.40

- The platelet level observed at different times for groups A (p=0.056) and B (p=0.069):

platelets
platelets
platelets
platelets
platelets
platelets

Group
10_3 ml T0
10_3 ml T1
10_3 ml T2
10_3 ml T3
10_3 ml T4
10_3 ml T5

A
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
249.0000
234.4286
240.2857
256.1429
317.7143
380.1429

Median
205.0000
147.0000
152.0000
185.0000
220.0000
269.0000

SD
165.65526
183.64355
196.55594
187.80879
250.15576
309.16847

Minimum
87.00
44.00
33.00
33.00
59.00
97.00

Maximum
521.00
558.00
578.00
549.00
784.00
963.00

B
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
387.7143
374.5714
316.8571
301.7143
320.8571
277.7143

Median
350.0000
243.0000
185.0000
195.0000
267.0000
196.0000

SD
215.01451
229.87958
194.91146
213.66930
201.71880
220.31017

Minimum
163.00
155.00
149.00
67.00
61.00
37.00

Maximum
635.00
738.00
595.00
638.00
611.00
607.00

- The lymphocyte level observed at different times for groups A (p=0.030) and B (p=0.085):

lymphocytes
lymphocytes
lymphocytes
lymphocytes
lymphocytes
lymphocytes

Group
10_3 ml T0
(%) T1
(%) T2
(%) T3
(%) T4
(%) T5

A
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
7.9571
8.9571
11.9714
14.3571
16.6143
17.1571

Median
7.2000
8.2000
11.0000
11.9000
12.5000
15.3000

SD
3.79599
5.23159
5.03545
7.43928
11.13903
9.21590

Minimum
3.00
2.70
4.50
2.80
2.60
5.40

Maximum
13.40
18.90
18.20
24.60
36.60
29.70

Percentiles
25
4.3000
5.2000
8.0000
10.6000
11.5000
9.7000

50
7.2000
8.2000
11.0000
11.9000
12.5000
15.3000

75
12.1000
10.8000
17.2000
20.6000
25.6000
27.1000

B
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
4.8286
4.6714
5.6143
5.5714
7.4143
8.2571

Median
3.9000
5.2000
4.9000
5.4000
5.8000
7.7000

SD
2.78132
1.12207
2.59257
2.36130
3.56718
5.01825

Minimum
3.30
2.60
3.10
2.20
4.30
2.00

Maximum
11.10
5.70
10.30
9.40
14.10
17.90

Percentiles
25
3.5000
3.9000
3.8000
4.3000
4.6000
4.5000

50
3.9000
5.2000
4.9000
5.4000
5.8000
7.7000

A
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
7.9571
8.9571
11.9714
14.3571
16.6143
17.1571

Median
7.2000
8.2000
11.0000
11.9000
12.5000
15.3000

SD
3.79599
5.23159
5.03545
7.43928
11.13903
9.21590

Minimum
3.00
2.70
4.50
2.80
2.60
5.40

Maximum
13.40
18.90
18.20
24.60
36.60
29.70

Percentiles
25
4.3000
5.2000
8.0000
10.6000
11.5000
9.7000

50
7.2000
8.2000
11.0000
11.9000
12.5000
15.3000

75
12.1000
10.8000
17.2000
20.6000
25.6000
27.1000

B
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
4.8286
4.6714
5.6143
5.5714
7.4143
8.2571

Median
3.9000
5.2000
4.9000
5.4000
5.8000
7.7000

SD
2.78132
1.12207
2.59257
2.36130
3.56718
5.01825

Minimum
3.30
2.60
3.10
2.20
4.30
2.00

Maximum
11.10
5.70
10.30
9.40
14.10
17.90

Percentiles
25
3.5000
3.9000
3.8000
4.3000
4.6000
4.5000

50
3.9000
5.2000
4.9000
5.4000
5.8000
7.7000

75
4.1000
5.6000
8.0000
7.6000
9.9000
10.1000

- The neutrophil level observed at different times for group A (p=0.070):

neutrophils
neutrophils
neutrophils
neutrophils
neutrophils
neutrophils

Group
(%) T0
(%) T1
(%) T2
(%) T3
(%) T4
(%) T5

A
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
87.1143
86.3143
81.2143
79.0714
77.8143
76.7286

Median
88.6000
86.9000
81.1000
75.8000
79.5000
79.7000

SD
6.82799
8.33175
6.01953
8.00411
12.25988
12.21757

Minimum
74.00
73.30
72.90
71.30
56.80
61.90

Maximum
95.20
95.40
91.80
92.90
94.10
91.90

Percentiles
25
83.1000
78.7000
77.5000
74.1000
67.3000
65.7000

50
88.6000
86.9000
81.1000
75.8000
79.5000
79.7000

75
91.0000
94.8000
84.5000
87.6000
85.0000
89.6000

B
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
92.4286
92.2714
90.6429
91.7571
87.7857
87.9571

Median
94.3000
92.5000
92.2000
92.4000
88.4000
89.9000

SD
5.35435
2.84413
4.01325
3.77618
6.93023
6.84930

Minimum
80.50
87.30
83.80
84.90
77.30
76.30

Maximum
96.20
96.10
95.60
96.10
94.50
97.30

Percentiles
25
92.9000
90.4000
87.0000
89.5000
80.5000
81.7000

50
94.3000
92.5000
92.2000
92.4000
88.4000
89.9000

75
94.9000
94.1000
93.3000
95.3000
94.2000
90.8000

- The GOT level observed at different times for groups A (p=0.013) and B (p=0.077):

Group
GOT T0
GOT T1
GOT T2
GOT T3
GOT T4
GOT T5

A
N
Valid
7
7
7
6
7
7

Lost
0
0
0
1
0
0

Mean
60.4286
54.0000
41.7143
38.3333
36.2857
42.0000

Median
36.0000
40.0000
32.0000
22.5000
22.0000
25.0000

SD
48.56905
44.59821
37.72141
34.93232
33.16984
45.06292

Minimum
16.00
13.00
8.00
15.00
18.00
21.00

Maximum
147.00
145.00
122.00
106.00
110.00
144.00

Percentiles
25
29.0000
26.0000
18.0000
17.2500
20.0000
23.0000

50
36.0000
40.0000
32.0000
22.5000
22.0000
25.0000

75
108.0000
74.0000
46.0000
61.0000
38.0000
30.0000

B
N
Valid
6
6
7
7
7
7

Lost
1
1
0
0
0
0

Mean
27.9500
38.8333
43.4286
33.8857
38.2286
33.2143

Median
19.2500
28.5000
25.0000
23.0000
23.0000
24.5000

SD
25.27795
22.24785
49.35199
22.87702
31.14534
27.45277

Minimum
11.00
20.00
13.00
15.00
13.00
15.00

Maximum
78.00
74.00
151.00
72.20
99.60
94.00

Percentiles
25
11.9000
22.2500
16.0000
19.0000
18.0000
19.0000

50
19.2500
28.5000
25.0000
23.0000
23.0000
24.5000

75
40.5000
62.7500
53.0000
61.0000
60.0000
34.0000

- The GPT level observed at different times for group B (p=0.004):

Group
GPT T0
GPT T1
GPT T2
GPT T3
GPT T4
GPT T5

A
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
51.0000
45.0000
40.2857
32.7143
30.5714
30.2857

Median
36.0000
32.0000
27.0000
31.0000
31.0000
27.0000

SD
54.22791
46.54747
29.78654
21.06114
19.26012
21.47645

Minimum
7.00
7.00
8.00
5.00
9.00
10.00

Maximum
167.00
143.00
79.00
56.00
59.00
73.00

Percentiles
25
15.0000
16.0000
15.0000
15.0000
12.0000
13.0000

50
36.0000
32.0000
27.0000
31.0000
31.0000
27.0000

75
60.0000
55.0000
76.0000
54.0000
49.0000
40.0000

B
N
Valid
6
6
7
7
7
7

Lost
1
1
0
0
0
0

Mean
15.7167
25.5000
22.2857
16.2143
16.2286
12.2857

Median
14.0000
19.5000
20.0000
15.0000
17.0000
12.0000

SD
9.56147
18.09696
12.44607
7.78812
7.95188
7.04070

Minimum
5.50
12.00
11.00
7.00
8.00
4.00

Maximum
33.50
60.00
43.00
28.00
31.00
23.00

Percentiles
25
9.6250
12.7500
12.0000
10.0000
9.0000
4.0000

50
14.0000
19.5000
20.0000
15.0000
17.0000
12.0000

75
20.6000
36.7500
36.0000
22.0000
19.0000
18.0000

- The GGT level observed at different times for group A (p=0.079):

Group
GGT T0
GGT T1
GGT T2
GGT T3
GGT T4
GGT T5

A
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
150.2857
131.7143
130.2857
142.4286
147.7143
240.8571

Median
68.0000
56.0000
69.0000
74.0000
98.0000
163.0000

SD
158.16206
144.03786
140.32904
160.26526
134.40575
204.12858

Minimum
33.00
28.00
29.00
33.00
34.00
39.00

Maximum
418.00
377.00
398.00
481.00
432.00
610.00

Percentiles
25
37.0000
30.0000
34.0000
46.0000
76.0000
112.0000

50
68.0000
56.0000
69.0000
74.0000
98.0000
163.0000

75
335.0000
296.0000
246.0000
210.0000
190.0000
436.0000

B
N
Valid
6
6
7
7
7
7

Lost
1
1
0
0
0
0

Mean
94.9333
94.6667
81.7143
119.1429
190.0000
176.5714

Median
108.0000
79.0000
83.0000
87.0000
110.0000
120.0000

SD
50.15283
53.64202
32.55618
89.37455
179.62090
183.81227

Minimum
31.60
32.00
39.00
40.00
37.00
37.00

Maximum
144.00
191.00
140.00
281.00
476.00
554.00

Percentiles
25
35.6500
64.2500
51.0000
41.0000
40.0000
54.0000

50
108.0000
79.0000
83.0000
87.0000
110.0000
120.0000

75
141.7500
131.7500
94.0000
193.0000
396.0000
271.0000

- If direct differences between groups A and B are compared at each moment in time independently (Mann-Whitney test for independent samples), at moment T2, there are differences between A and B in red blood cell (p=0.026), hemoglobin (p=0.038) and hematocrit (p=0.053) levels.
- In lymphocyte levels, there are differences at all times:

lymphocytes
lymphocytes
lymphocytes
lymphocytes
lymphocytes
lymphocytes

10_3 ml T0
(%) T1
(%) T2
(%) T3
(%) T4
(%) T5

Mann-Whitney U
11.000
9.500
6.500
6.000
10.000
9.000

Wilcoxon W
39.000
37.500
34.500
34.000
38.000
37.000

Z
−1.727
−1.919
−2.302
−2.366
−1.853
−1.981

Asymp. sig. (2 −
0.084
0.055
0.021
0.018
0.064
0.048

tailed)

Exact sig. [2 * (1 −
0.097a
0.053a
0.017a
0.017a
0.073a
0.053a

tailed sig.)]

- There are differences in neutrophil levels between A and B at T2 (p=0.007) and T3 (p=0.011)
- There are differences in GPT levels between A and B at T5 (p=0.038)
- There are no significant differences in urea levels over time; they remain virtually identical in both groups (Friedman test, p=0.205 for group A and p=0.959 for group B):

UREA

Group
urea T0
urea T1
urea T2
urea T3
urea T4
urea T5

A
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
51.4286
47.7143
45.0000
39.4286
40.2857
43.5714

Median
57.0000
61.0000
60.0000
57.0000
54.0000
55.0000

SD
26.13973
27.13985
28.56571
25.81897
30.05946
31.57455

Minimum
9.00
5.00
10.00
7.00
2.00
4.00

Maximum
83.00
74.00
77.00
67.00
72.00
79.00

Percentiles
25
23.0000
19.0000
14.0000
11.0000
8.0000
10.0000

50
57.0000
61.0000
60.0000
57.0000
54.0000
55.0000

75
69.0000
73.0000
71.0000
58.0000
66.0000
78.0000

B
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
111.2857
115.0000
109.0000
114.2857
117.5714
115.7143

Median
117.0000
120.0000
92.0000
100.0000
90.0000
99.0000

SD
37.94168
33.78856
46.38965
54.75921
73.16160
85.85397

Minimum
62.00
68.00
66.00
69.00
44.00
30.00

Maximum
169.00
172.00
191.00
213.00
250.00
260.00

Percentiles
25
64.0000
84.0000
71.0000
71.0000
61.0000
55.0000

50
117.0000
120.0000
92.0000
100.0000
90.0000
99.0000

75
133.0000
130.0000
150.0000
166.0000
182.0000
208.0000

- However, if urea levels between the two groups are compared at each measured moment in time, urea levels are higher in group B at all the measured moments except the last one. The differences are statistically significant:

Group
N
Mean
SD
SEM
p

urea T0
A
7
51.4286
26.13973
9.87989
0.011

B
7
111.2857
37.94168
14.34061

urea T1
A
7
47.7143
27.13985
10.25790
0.002

B
7
115.0000
33.78856
12.77087

urea T2
A
7
45.0000
28.56571
10.79682
0.004

B
7
109.0000
46.38965
17.53364

urea T3
A
7
39.4286
25.81897
9.75865
0.001

B
7
114.2857
54.75921
20.69704

urea T4
A
7
40.2857
30.05946
11.36141
0.026

B
7
117.5714
73.16160
27.65248

urea T5
A
7
43.5714
31.57455
11.93406
0.073

B
7
115.7143
85.85397
32.44975

In addition, the progression over time of the following parameters in each group separately has not shown significant differences:

CREATININE

- There are no significant differences in creatinine levels over time; they remain identical in both groups (Friedman test, p=0.122 for group A and p=0.831 for group B):

creatinine
creatinine
creatinine
creatinine
creatinine
creatinine

Group
T0
T1
T2
T3
T4
T5

A
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
1.7614
1.4957
1.3343
1.1929
1.1300
1.0571

Median
1.4400
1.1000
.9700
1.1200
1.2200
0.7600

SD
1.15338
0.99052
0.90526
0.79229
0.61709
0.61302

Minimum
0.49
0.48
0.46
0.39
0.46
0.40

Maximum
3.70
2.80
2.47
2.18
2.05
1.94

Percentiles
25
0.7500
0.6800
0.5000
0.4100
0.5200
0.5900

50
1.4400
1.1000
0.9700
1.1200
1.2200
0.7600

75
2.9100
2.7400
2.2200
1.9500
1.6700
1.7100

B
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
2.1157
2.1000
1.8843
1.8357
1.7600
1.5829

Median
2.0400
1.9700
1.9600
1.4100
1.6900
1.7200

SD
1.11884
0.84766
0.79521
0.93259
0.99698
1.05880

Minimum
0.54
0.80
0.64
0.67
0.54
0.39

Maximum
4.18
3.47
2.85
3.10
2.77
2.89

Percentiles
25
1.6000
1.5800
1.1900
1.2300
00.7600
0.6100

50
2.0400
1.9700
1.9600
1.4100
1.6900
1.7200

75
2.6300
2.7000
2.7400
2.8900
2.7600
2.8200

- If creatinine levels between the two groups are compared at each measured moment in time, it can be seen that there are no statistically significant differences in creatinine levels between groups A and B:

Group
N
Mean
SD
SEM
p

creatinine T0
A
7
1.7614
1.15338
0.43594
0.456

B
7
2.1157
1.11884
0.42288

creatinine T1
A
7
1.4957
0.99052
0.37438
0.318

B
7
2.1000
0.84766
0.32039

creatinine T2
A
7
1.3343
0.90526
0.34216
0.318

B
7
1.8843
0.79521
0.30056

creatinine T3
A
7
1.1929
0.79229
0.29946
0.165

B
7
1.8357
0.93259
0.35249

creatinine T4
A
7
1.1300
0.61709
0.23324
0.209

B
7
1.7600
0.99698
0.37682

creatinine T5
A
7
1.0571
0.61302
0.23170
0.318

B
7
1.5829
1.05880
0.40019

LDH

- There are no significant differences in LDH levels over time; they remain virtually identical in both groups (Friedman test, p=0.355 for group A and p=0.921 for group B):

Group
LDH T0
LDH T1
LDH T2
LDH T3
LDH T4
LDH T5

A
N
Valid
7
7
7
7
7
7

Lost
0
0
0
0
0
0

Mean
708.5714
685.8571
638.0000
658.7143
602.4286
707.8571

Median
561.0000
631.0000
418.0000
499.0000
380.0000
552.0000

SD
345.48992
345.16538
474.81435
445.02723
458.08255
354.42462

Minimum
504.00
320.00
327.00
377.00
364.00
397.00

Maximum
1474.00
1369.00
1677.00
1659.00
1623.00
1421.00

Percentiles
25
524.0000
421.0000
405.0000
480.0000
367.0000
491.0000

50
561.0000
631.0000
418.0000
499.0000
380.0000
552.0000

75
713.0000
836.0000
714.0000
570.0000
556.0000
912.0000

B
N
Valid
6
7
7
7
7
7

Lost
1
0
0
0
0
0

Mean
430.6667
591.4286
580.2857
570.5714
544.4286
609.5714

Median
420.0000
565.0000
545.0000
532.0000
590.0000
537.0000

SD
156.18024
142.31989
189.49557
159.89982
187.55964
257.69805

Minimum
197.00
359.00
391.00
329.00
305.00
351.00

Maximum
617.00
781.00
979.00
811.00
789.00
1066.00

Percentiles
25
306.5000
540.0000
468.0000
465.0000
343.0000
408.0000

50
420.0000
565.0000
545.0000
532.0000
590.0000
537.0000

75
594.5000
756.0000
611.0000
697.0000
683.0000
819.0000

- If LDH levels between the two groups are compared at each measured moment in time, LDH levels are higher in group A at all the measured times, although the differences are not statistically significant:

Group
N
Mean
SD
SEM
p

LDH T0
A
7
708.5714
345.48992
130.58292
0.073

B
6
430.6667
156.18024
63.76032

LDH T1
A
7
685.8571
345.16538
130.46025
0.805

B
7
591.4286
142.31989
53.79186

LDH T2
A
7
638.0000
474.81435
179.46296
0.535

B
7
580.2857
189.49557
71.62259

LDH T3
A
7
658.7143
445.02723
168.20448
0.710

B
7
570.5714
159.89982
60.43645

LDH T4
A
7
602.4286
458.08255
173.13893
0.710

B
7
544.4286
187.55964
70.89088

LDH T5
A
7
707.8571
354.42462
133.95991
0.620

B
7
609.5714
257.69805
97.40071

DURABLE PREPARATION OF AN INJECTABLE OF MELATONIN EXHIBITING LONG-TERM STABILITY

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

Priority Claims (1)

PCT Information