Technical field:

This invention relates to a stable and ease of oral application liquid formulation comprising liposomal iron comprising iron (III) source and in particular to the removal of preservatives from this preparation. It is used thereof for human beings for treating iron deficiency.

Prior Art:

Iron is an essential mineral that is naturally present in many foods, added to some food products, and available as a dietary supplement. It is the most abundant trace element in humans; also it is structurally essential to the formation of haemoglobin and myoglobin, and imperative in oxygen transport, energy production, and DNA synthesis. Iron is also necessary for growth, development, normal cellular functioning, and synthesis of some hormones and connective tissue.

The average iron content in healthy human body is around 40 mg/kg body weight for women and 50 mg/kg for men. Approximately 30 mg/kg of iron is present within red cells as haemoglobin, with 4 mg/kg as myoglobin in muscle tissue and 2 mg/kg as iron containing enzymes in cells. The remainder is held in reserve as storage iron in the form of ferritin or haemosiderin predominantly in the liver, spleen and bone marrow. In absolute terms, the quantity of storage iron is usually 0.2 g, the actual amount present at any one time depending on the balance between dietary intake and physiological requirements. Under normal circumstances, ferritin predominates as the principal form of tissue in the liver and spleen.

Iron deficiency is the most common micronutrient deficiency in the world, affecting 1.3 billion people. Data from 187 countries from 2010 revealed that anemia affected up to one-third of the global population, though prevalence varied widely across regions, and iron deficiency was responsible for about 50% of anemia cases. It is known to occur at all stages of life; children aged 0 to 5 years, women of childbearing age, and pregnant

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SUBSTITUTE SHEETS (RULE 26) women are particularly at risk. In a systematic analysis in the Global Burden of Disease Study 2016, iron-deficiency anemia was the fourth leading cause of years lived with disability, especially in women. Thus, prophylaxis and management of iron deficiency is a first order public issue. The main causes of iron deficiency are increased demands, reduced absorption and/or increased loss of iron. (Susana Gomez-Ramirez et al., 2018)

Main causes of iron deficiency are grouped as:

1) Increased demands

• Body growth (infancy and childhood)

• Pregnancy and lactation

• Recovery from blood loss

• T reatment with erythropoiesis stimulating agents

2) Limited external supply or absorption

• Poor intake

• Inappropriate diet with deficit in bioavailable iron and/or ascorbic acid (including excess of dietary fiber, phenolic compounds from tea or coffee, and soya products)

• Malabsorption (autoimmune atrophic gastritis, gastric resection, bariatric surgery, inflammatory bowel disease, celiac disease, non-celiaz gluten sensitivity, Heliobacter pylori infection)

• Medications (AntiH ₂ , PPI, antacids, etc.)

• Increased hepcidin levels (e.g., IRIDA or ACI)

• Molecular defects in iron transport proteins (e.g., heme oxygenase or DMT1 deficiencies)

3) Increased iro loses

• Bleeding trauma

• Gastrointestinal bleeding (peptic ulceration, neoplasia, inflammatory bowel disease, vascular malformations, medications [anti-inflammatory, anti-platelet or anticoagulant agents])

• Genitourinary bleeding

• Menses and multi-parity

• Multiple diagnostic phlebotomies (medical ‘vampirism’)

• Blood donation

• Dialysis (particularly hemodialysis) Iron deficiency can lead to the development of a form of anemia, resulting in lower than normal levels of hemoglobin, the molecule in red blood cells responsible for transporting oxygen. Symptoms of iron deficiency include fatigue; shortness of breath; palpitations or rapid heartbeat; dizziness or lightheadedness; pallor; weakness; frequent infections; headache; cold extremities; restless leg syndrome; poor appetite in children or altered appetite for nonedible substances such as ice or dirt; and in women of reproductive age, altered menstrual cycles (very light or very heavy). Not surprisingly, therefore, iron deficiency anemia independently increases morbidity and mortality.

Iron containing supplements are most often used for treating iron deficiency. Iron supplements can be delivered intramuscularly, intravenously, or orally. Intravenous (IV) iron formulations are increasingly used, but there is still a risk of infusion, hypersensitivity reactions and the need for venous access and infusion monitoring. Nevertheless, though increasingly safer, IV iron formulations are more expensive than oral iron and still carry the need for venous access (side effects at the injection site may occur) and infusion monitoring (there is still a risk of infusion and hypersensitivity reactions). In this regard, the European Medicines Agency states that “IV iron products should be administered only when staff is trained to evaluate and manage anaphylactic reactions, and resuscitation facilities are immediately available”. According to studies oral administration is preferred due to its convenience.

It is well known to treat an iron deficiency with orally administered iron supplements. Conventional oral iron fortificants can be divided into 4 groups: (1) freely water soluble (e.g., ferrous sulphate, ferrous gluconate, ferrous lactate and ferric ammonium citrate); (2) poorly water soluble (e.g., ferrous fumarate, ferrous succinate, and ferrous saccharate); (3) water insoluble (e.g., ferric pyrophosphate, ferric orthophosphate, and elemental iron); and (4) experimental [e.g., sodium-iron EDTA and iron-porphyrin (heme) complexes isolated from bovine hemoglobin].

Oral iron supplements, provided as ferrous or ferric salts, are usually the first line of treatment for iron deficiency, because of their availability, ease of administration, and relatively low cost. Oral iron has usually been prescribed at a high dose (100-200 mg elemental iron), to be taken 1-3 times a day. However, the bioavailability is 10% to 15% for ferrous iron preparations (sulfate, gluconate, fumarate, etc.), and it is even lower for ferric iron salts or ferric iron complexes (amino acids, polysaccharide, ovo- albumin, etc.). In addition, up to 50% of patients on oral iron (depending on the iron formulation) report gastrointestinal side effects due to the direct toxicity of ionic iron, which may lead to reduced tolerance and adherence to iron supplementation. Therefore, a low single daily dose (40-60 mg) and/or single alternate day dose (80- 100 mg) are preferred in order to reduce the side effects and maximize fractional absorption.

Commonly used oral iron salts are poorly absorbed, with unabsorbed iron leading to gastrointestinal side effects. Newer oral iron supplements have been formulated to increase their tolerability. However, there was still a need for new carriers that not only protect the iron but also enhance its intestinal absorption, thus reducing dosage and side effects.

The choice of the type of iron salt, the iron (II) salt or the iron (III) salt, the choice of the compounds or substances used to formulate said salt and prepare the composition, and the choice of the type of process used to prepare the composition play an important role in the composition to be obtained. It would be optimal to have an iron (II)- or iron (lll)-based composition in which iron is highly bioavailable and, at the same time, devoid of any limits or drawbacks from the organoleptic point of view (taste, smell, color, long-term stability) and the composition is devoid of limits and disadvantages related to, for example, its hygroscopicity, particle agglomeration, color changing and its solubility. However, water-soluble and bioavailable iron (II) salts, such as for example ferrous sulphate, often cause unacceptably color, taste, flavor and smell changes, in particular when said salts are mixed with other components or ingredients to form a final composition. On the other hand, iron (III) salts are less water soluble and bioavailable than iron (II) salts, such as for example ferric pyrophosphate. The reduced bioavailability of, for example ferric pyrophosphate, is related to its moderate solubility in diluted acid, such as that present in gastric juice. Nevertheless, iron (III) salts, such as for example ferric pyrophosphate, have the advantage to be more stable and, thus, they change much less their smell, flavor and taste or their color, when said iron (III) salts are mixed with other components or ingredients to form a final composition. In the present invention ferric pyrophosphate is used for iron source.

In addition to these, encapsulation of iron in a micronized form into liposomes is the recent approach to improve iron tolerance and absorption. This new, promising strategy for delivering iron orally is associated with greater gastrointestinal (Gl) absorption, higher bioavailability with reduced incidence of adverse effects. It is believed that because of no direct contact of iron with intestinal mucosa, it is better absorbed and tolerated. Therefore, supplementing liposomal iron in pregnancy can be helpful to improve tolerability, compliance, and outcomes of the therapy.

Ferric pyrophosphate is the usual form of iron used for liposomal iron delivery. According to clinical studies conducted by Blanco-Rojo et al. (2011), Plesea- Condratovici et al. (2012) and Parisi et al. (2012), it is suggested that, ferric pyrophosphate in liposomal iron delivery is safe for treatment of iron deficiency.

There are some advantages with liposomal Iron (Biniwale P. et al. 2018);

- Quicker absorption and restoration of the iron content: Experimental evidence suggests liposomal iron recovers iron levels in liver quickly than conventional oral iron. Multiple studies suggest that liposomal encapsulation of iron is associated with enhanced iron absorption compared to non-capsulated conventional oral iron.

- No induction of oxidative damage: Evidence suggests that liposomal iron is associated with decreased malondialdehyde levels and increase in super-oxide dismutase levels. This may help in minimizing the oxidative damage that is possibly induced by conventional iron.

- Absorption with improved capacity: It has good absorption and has reduced incidence of adverse effects probably due to lower oxidative damage.

- Physical stability and gradual release property: Liposomes are unilamellar vesicles and are nano-sized particles. Lipid bilayer provides stability and may release the contents gradually. Gradual release may help in better absorption of liposomal contents.

While non-liposomal oral iron is an effective strategy to increase haemoglobin levels in iron-deficiency anaemia, its efficacy in replenishing iron stores may be limited by its ineffective absorption, potential gastrointestinal events, non-compliance and inflammation, a common condition in patients, often associated with increased hepcidin levels, which lead to impaired absorption of iron from the gastrointestinal tract and retention of iron in the reticuloendothelial system. However, liposomal iron, a preparation of ferric pyrophosphate carried within a phospholipid and sucrose esters of fatty acid membrane, is a new generation of oral iron, which shows a high gastrointestinal absorption and high bioavailability with a low incidence of side effects. The absorption or bioavailability of liposomal pyrophosphate iron is 3.5 times greater than the free pyrophosphate iron, 2.7 times higher than iron sulfate, and 4.1 times higher compared with iron gluconate.

US 5534268 discloses to propose an additive to supplement the iron intake, which comprises bioavailable iron (II) in an aqueous medium, encapsulated in liposomes; said medium comprises reducing agents, such as ascorbic acid or its salts, as stabilizers of the reduced state of the iron.

Preservatives are frequently used in oral pharmaceutical compositions to increase the shelf life of drugs by reducing/inhibiting the oxidation of active substances and excipients or microbial growth in the product. However, there are problems encountered in the use of preservatives. The use of preservatives in high concentration can create toxic effects and cause undesirable potential side effects. Within the scope of general safety measures, it would be advantageous to reduce the concentration of preservatives in the compositions to the appropriate level or to introduce products that do not contain preservatives completely. Therefore, the present invention is specifically aimed at developing a stable oral liquid liposomal iron composition without using preservatives.

Therefore, there is a need for a composition that offers a solution for the stability and bioavailability problems encountered in compositions comprising liposomal iron with preservative free form. In the present invention, the composition which is intended to be developed for the treatment of iron deficiency, comprising liposomes containing iron (III) source provides a solution to the stability and bioavailability problems of the oral liquid drug product, particularly with the dosage form without comprising preservatives.

Description of the Invention:

The present invention relates to a pharmaceutical liquid preservative free composition for use in the treatment of disorders or diseases related to or derived from iron deficiency.

In the present invention, it is aimed to produce a pharmaceutical composition that can provide stability without using preservatives (antimicrobial and antioxidant) to produce oral liquid liposomal iron composition in preservative-free form.

The present invention relates to a pharmaceutical liquid composition comprising liposomal iron, methods for the preparation thereof, and also an oral drop formulation comprising liposomal iron analogue, which is a preparation of ferric pyrophosphate carried within a phospholipidic membrane, wherein the said formulation do not comprise any preservative and providing simple application procedure with high stability. The present invention relates to a stable liquid oral pharmaceutical composition in a form of a drop treating iron deficiency anemia in a patient comprising liposomal iron, which is a preparation of ferric pyrophosphate carried within a phospholipidic membrane; wherein the composition is free of preservative.

The present invention relates to a stable liquid oral preservative free pharmaceutical composition in a form of a drop comprising liposomal iron, wherein the composition comprises pharmaceutical sugar.

The present invention relates to a stable liquid oral preservative free pharmaceutical composition in a form of a drop comprising liposomal iron, wherein the composition comprises sucrose as a pharmaceutical sugar.

The present invention relates to a stable liquid oral preservative free pharmaceutical composition in a form of a drop comprising liposomal iron, wherein the composition comprises sucrose lower than 20 g per 30 mL product.

The present invention relates to a stable liquid oral preservative free pharmaceutical composition in a form of a drop comprising liposomal iron, wherein the composition comprises sucrose lower than 15 g per 30 mL product.

The present invention relates to a stable liquid oral preservative free pharmaceutical composition in a form of a drop comprising liposomal iron, wherein the concentration of iron (III) is 8.5 mg/mL.

The invention relates to a composition of administering an oral drop to the human being; the liposomal iron liquid preservative free composition can be simply dropped into the mouth, it finds the way into the proper body processes by absorption through the mucous membranes and/or simple swallowing according to normal salivary mechanisms.

The present invention relates to a process for preparing said pharmaceutical liquid liposomal iron preservative free composition.

The composition of the present invention is suitable for pediatric subjects, adolescents, athletes, men, women, pregnant women and elderly.

The poor absorption of iron salts is a challenge in formulating iron compounds.

The term ‘liposomal iron’ is a preparation of ferric pyrophosphate carried within a phospholipidic membrane. The term ‘preservative’ is intended to encompass antioxidants and antimicrobials. Preservatives are chemicals used to increase the shelf life of drugs by reducing/inhibiting the oxidation of active substances and excipients or microbial growth in the product. The use of preservatives, like alcohols, benzoates, sorbates, and parabens is common in liquid formulations. Preservatives are effective in controlling mold, inhibiting yeast growth and protecting against bacterial proliferation, thus, finally, to allow compliance with the European Pharmacopoeia microbiological specifications (Ph. Eur. 6.7, S5.1.4) for "aqueous preparations for oral use" or "aqueous preparations for oromucosal use". Consequently most of the supplementary products contain preservatives. Microbial preservatives may include but not limited to sodium benzoate, benzoic acid, boric acid, sorbic acid and their salts thereof, benzyl alcohol, benzalkonium chloride, parahydroxybenzoic acids and their alkyl esters, methyl and propyl parabens or their mixtures thereof.

However, preservatives are generally toxic and usage of these may cause potential undesirable side effects. For general safety reasons, it would therefore be advantageous to reduce the concentration of preservatives to an appropriate low level or remove of preservatives from such liquid compositions. In the present invention, ‘preservative free or free of preservative’ indicates that there is no antioxidant or antimicrobial preservative present in the specified pharmaceutical dosage form.

The term 'stable' as used herein refers to an oral liquid pharmaceutical liposomal iron composition provided results within the accepted limits in microbial growth tests without reduction in iron content over short term and long term stability conditions.

As used herein, an "iron deficiency anemia" includes a disorder or disease related to iron deficiency, iron uptake, and/or iron metabolism. Examples of iron deficiency disorders include iron deficiency anemia, such as iron deficiency anemia caused by insufficient dietary intake or absorption of iron. Iron deficiency anemia may be related to, for example, malnutrition, pregnancy (including the postpartum period), heavy uterine bleeding, chronic disease (including chronic kidney disease), cancer, renal dialysis, gastric by-pass, multiple sclerosis, diabetes (e.g. Type I and Type II diabetes), insulin resistance, and attention deficit disorders.

The term "therapeutically effective amount" is an amount of the composition indicated for treatment while not exceeding an amount which may cause significant adverse effects.

A "patient’ refers to any patient or subject who could benefit from the inventive composition. Compositions of this invention are detailed below showing examples. However, pharmaceutical compounds of this invention are not restricted to the following examples.

Example 1. Compositions comprising ascorbic acid

F-01 F-02 F-03 F-04 F-05 F-06

Component _{per 30 mL}

Liposomal iron 3 g 3 g 3 g 3 g 3 g 3 g

Ascorbic acid , „ , „ „„ „„

10 mg 10 mg 20 mg 20 mg 30 mg 30 mg

T utti frutti flavor T , “ T , — T —

Raspberry flavor + - + - +

Stevia 5.5 mg 5.5 mg 5.5 mg 5.5 mg 5.5 mg 5.5 mg

Water q.s. q.s. q.s. q.s. q.s. q.s.

Total Volume 30 mL 30 mL 30 mL 30 mL 30 mL 30 mL

Samples F-01 to F-06 were prepared as follows: Pharmaceutical ascorbic acid (vitamin C) is mixed in about 1/3 of the total batch volume in water until complete dissolution is observed. Stevia extract is added to the resulting mixture and mixing is continued. Liposomes comprising 8% iron (III) are used for the iron source. Liposomal iron active ingredient is added to this mixture and mixing is continued until it becomes homogeneous. Samples F-01 to F-06 were studied both with and without flavoring agents. Flavorings (tutti frutti and raspberry flavors) are added to the homogeneous mixture (F-01, F-03 and F-05) and mixing is continued. The resulting mixture is completed to the total volume of the batch size studied with deionized water. Samples, F-02, F-04 and F-06, which did not add flavorings, are also completed to the total volume of the batch size studied with deionized water. Liquid food supplements prepared in appropriate specifications are packaged under nitrogen gas. (Packaging: 30 ml amber glass bottle)

Prepared samples were tested for taste, iron content and accelerated stability tests for microbial content.

When ascorbic acid (vitamin C) is used at 30mg or more, the finished product cannot be sweetened even if with using additional flavoring agents. In addition, vitamin C, which is used for preservative purposes, interacted with active substance in the finished product, causing a decrease in the amount of ascorbic acid (as preservative) in the stability conditions. The decrease in the amount of Vitamin C preservative negatively affected the results of the product in terms of microbial content.

Example 2. Compositions comprising sugar and free of preservatives 4 F-07 F-08 F-09

Component __ . r per 30 mL

Liposomal iron 3 g 3 g 3 g

Pharmaceutical sugar (Sucrose) 10 g 15 g 20 g

T utti frutti flavor

Raspberry flavor + + +

Stevia 5.5 mg 5.5 mg 5.5 mg

Water q.s. q.s. q.s.

Total Volume 30 mL 30 mL 30 mL

Samples F-07 to F-09 were prepared as follows: Pharmaceutical sugar is mixed in about 1/3 of the total batch volume in water until complete dissolution is observed. Stevia extract is added to the resulting mixture and mixing is continued. Liposomes comprising 8% iron (III) are used for the iron source. Liposomal iron active ingredient is added to this mixture and mixing is continued until it becomes homogeneous. Flavorings (tutti frutti and raspberry flavors) are added to the homogeneous mixture and mixing is continued. The resulting mixture is completed to the total volume of the batch size studied with deionized water. Liquid food supplements prepared in appropriate specifications are packaged under nitrogen gas. (Packaging: 30 ml amber glass bottle)

Prepared samples were tested for taste, iron content, pH, density and accelerated stability tests for microbial content.

Density changes were observed according to the amount of sugar used. The density and pH were measured as 1.16 (pH 3.10), 1.23 (pH 3.03) and 1.32 (pH 2.98) for F-07, F-08 and F-09, respectively. Sample F-09, which was prepared using 20 grams of sugar, left very sweety and mouth-burning feeling in taste. Microorganism growth was observed in F-09 product at stability conditions.

The samples F-07 and F-08, containing 10 grams and 15 grams of sugar, were found to be suitable in the taste tests. Also, no microorganism growth was observed during the short term and long term stability conditions.

Stability Studies

The stability of a drug substance is an important factor in the manufacture of safe and effective pharmaceutical products. Stability studies are required to be submitted by any applicant seeking approval for a new pharmaceutical product. Stability study requirements are covered, for example in the United States Pharmacopeia, in the Good Manufacturing Practices (GMP) as well as in FDA and ICH Guidelines. It is known that many drugs exhibit poor or modest shelf stability. The diminution of the concentration of a drug as a result of its degradation is inherently undesirable, as it makes therapy with the drug less certain. Stability issues can be caused by environmental factors such as humidity, temperature and the like.

In the development of liposomal iron oral drop dosage form, stability was assessed under three different isothermal conditions (25°C ± 2°C, 30°C ± 2°C and 40°C ± 2°C) in temperature-programmable control cabinets. A temperature of 25°C ± 2°C represents ambient temperature, 30°C ± 2°C represents intermediate temperature and 40°C ± 2°C is a temperature that can be reached under extreme conditions in homes without air conditioning in the summer. The developed liposomal iron oral drop dosage forms (F- 07 and F-08) were subjected to accelerated stability condition having temperature of 40°C ± 2°C and 75 % ± 5°C RH for up to 6 months; intermediate stability condition having temperature 30°C ± 2°C and 65 % ± 5°C RH for up to 12 months; ambient stability condition having temperature of 25°C±2°C and 60% ± 5% RH for up to 24 months. No unexpected results of iron content and microbial growth were observed in the liposomal iron in the stability tests examined for short term (6 months) and long term (12 months and 24 months) stability conditions.

It is concluded that the product developed in the present invention is biologically natural in the content and it is almost safe for human beings consumption. Also, the product intrinsically is not subject to rancidity, oxidation or degradation over the shelf life of the composition according to results obtained in the period of 3 different stability conditions. There is little change in content of liposomal iron within the short term and long terms stability periods.

Despite the product contains no preservative, the stability of product is provided with sugar. Therefore this formulation achieves the improvement of stability for the liposomal iron oral drop dosage form.