Technical field

The invention relates to solid,liquid,enteral and parenteral stable combinations of pharmaceutical forms comprising N-[4-[[(2,4-Diamino-6-pteridinyl)methyl] methylamino] benzoyl]-L- glutamic acid (or N-[4-((2,4-Diamino pteridinyl -6-ylmethyl)

methylamino)benzoyl]-L(+)-glutamic acid ) (hereafter refer to as API I) and N-[4-[[(2- Amino-3,4- dihydro -4-oxo-6- pteridinyl) methyl] methyl amino] benzoyl]-L-glutamic acid (or N-(4-(2-Amino-4- hydroxypteridine-6- ylmethyl amino)benzoil)-L(+)-glutamic acid) (hereafter refer to as API II) or pharmaceutically acceptable salts,particulary

monohydrate,sodium and disodium, as active ingredients.

API I :

State of the Art

The formulation of the invention relates to a pharmaceutical dosage form and its combination which is not exist in Turkey and the world.

It is known that the formulation of 50 mg/ml methotrexate and protein binding methotrexate derivatives is used for the treatment of inflammatory autoimmune diseases,

EP2046332 describes using 50 mg/ml methotrexate for the treatment of inflammatory autoimmune diseases.

EP2046813 describes protein binding methotrexate derivatives and pharmaceutical compositions comprising the methotrexate derivatives. The formulation of the protein binding methotrexate derivative is intended to be used for the treatment of cancer and rheumatic disease.The structural formula which mentioned in the independent claim will be explained in detail in the following;

Technical problems that the invention aims to solve

The object of the invention is to provide the use of the most stable pharmaceutical dosage forms that containing API I and API II combination,indicating a long shelf life, used for preventive and therapeutic purpose,increasing patient compliance.

Description of the invention

The invention relates to solid,liquid,enteral and parenteral stable combinations of pharmaceutical forms comprising N-[4-[[(2,4-Diamino-6-pteridinyl)methyl] methylamino] benzoyl]-L- glutamic acid (or N-[4-((2,4-Diamino pteridinyl -6-ylmethyl)

methylamino)benzoyl]-L(+)-glutamic acid ) and N-[4-[[(2-Amino-3,4- dihydro -4-oxo-6- pteridinyl) methyl] methyl amino] benzoyl]-L-glutamic acid (or N-(4-(2-Amino-4- hydroxypteridine-6- ylmethyl amino)benzoil)-L(+)-glutamic acid) (hereafter refer to as API II) or pharmaceutically acceptable salt as active ingredient.

API I is an antineoplastic drug. However, it is used to treat many other diseases unrelated with cancer in lower doses. It is being used via intravenous and higher doses are being used to treat cancer.

API I is a cycle specific drug that is showing both antineoplastic and immunosuppressant activity .As an antimetabolite it inhibits dihydrofolate reductase enzyme which is responsible for reduction of API II to tetrahydro structure . Reduction of tetrahydro API II decreases the required level of one-carbon groups and accordingly, purines' ,DNA's and cellular protein synthesis is inhibited.

API I is being administered by orally and intravenously. It is well absorbed when given as a low single dose (below 50mg / ml). The absorption is irregular when given above this dosage and it is suitable administering in divided doses. It is completely absorbed when administered by intramuscular injection.

Regarding to API I, the British Pharmacopoeia (BP) is reported that the content is not less than %97 on dry basis and the American Pharmacopoeia (USP) reported that the content is not less than %98 on dry basis.lt is yellow to brown crystallized powder. Practically insoluble in water,chloroform and ether. It dissolves in dilute mineral acids and in dilute solutions of alkali hydroxides and carbonates. It must be protected from light.

API II is a water soluble vitamin B-complex used for oral and parenteral administration. Deficiency can cause various hematological complications including megaloblastic anemia.

API II is a biochemically inactive compound that it is precursor of tetrahydrofolic acid and methyltetrahydrofolate. Tetrahydrofolic acid, methyltetrahydrofolate and other folic acid derivatives are necessary substances to sustain erythropoiesis normally. These substances are needed as cofactors for the synthesis of purine and thymidylate nucleic acid. API II and its derivatives are necessary for the conversion of amino acids to each other. API II is given by oral and parenteral administration. Peak levels have been seen within 1 hour.

API II is yellow to orange-brown coloured, odorless or very lightly scented crystalline powder.According to British Pharmacopoeia (BP), practically it is insoluble in water and other organic solvents. According to US Pharmacopeia (USP), it is very slightly soluble in water; insoluble in alcohol, acetone, chloroform and ether; rapidly soluble in dilute solutions of alkali hydroxides and carbonates; soluble in hydrochloric acid and sulfuric acid. According to USP pH range of solution for injection is 8-1 1. It must be protected from light and avoided freezing.

Parenteral drugs are given to circulatory system directly or indirectly excepting gastrointestinal system with a syringe as a sterile single dose or multi-dose form of ready to use syringe or vial.The pharmaceutical form is containing N- [4 - [[(2,4-Diamino-6- pteridinyl) methyl] methylamino] benzoyl] -L-glutamic acid (or N- [4 - ((2,4-Diaminopteri pyrimidin-6- ylmethyl) methylamino) benzoyl] -L (+) - glutamic acid) and N- [4 - [[(2- Amino-3,4-dihydro-4-oxo,-6-pteridinyl) methyl] methylamino] benzoyl] -L -glutamic acid (or N- (4- (2-Amino-4-hidroksipteridin-6-ylmethylamino) benzoyl) -L (+) - glutamic acid) or their pharmaceutically acceptable salts, individually and / or in combination.

Production of parenteral solution is comprising the production of small volume parenteral of different volumes of liquid dosage form. Production of parenteral drug can be carried out in the fully automated production line named ready to use syringe filling line.

Parenteral solutions are filled into sterile pre-filled syringe, vial or PVC containers.They are administered by injection . Thus,parenteral solutions may also be called injectable solutions. Injectable solutions must be sterile.The pH of the solution must be equal or close to the pH of body fluids (7.4). Osmolality of the solution must be isosmotic with body fluids or close to them.lt must not contain pyrogen which has bacterial lipopolysaccharide structure and can cause fever (should be apyrogenic ). In addition, the solution must not contain particulate matter (micro or macroparticle).

Parenteral treatment is administering therapeutic agents by other than gastrointestinal system. In this way, fluid and electrolytes, vitamins, sugars, protein, blood or components, vaccines, several drugs for therapeutic or diagnostic purposes can be administered. The pharmaceutical effect starts faster than oral route. Drugs which can not be absorbed easily and degraded in the digestive tract can be given in this way.

There are many advantages of drug administered parenterally. Parenteral administration is preferred when it is desired to act quickly, the patient can not take the drug orally

(unconscious patients),the drug is destroyed by gastro-intestinal fluids and providing local effect and complete bioavailability.

The way of administration is determined by the suitability of the vessel, the patient's choices and preferences of the physician.For instance; Although the irritant drugs administered intramuscularly, easily absorbable drugs such as insulin are given by subcutaneously. Effect starts quickly and continues during the desired time. Sterile preparations are produced in clean room conditions for helping to minimize the risk of pyrogenic and particulate matter. Quality assurance of these requirements in sterile manufacturing has great importance and this manufacturing process must be carried out according to the validated production methods and procedures.

The solutions sterilized by filtration during the process are prepared in class C

environment. If sterile filtration is not possible,the solution is prepared in class A environment which has a background of class B environment.

Including small and large volume parenteral preparations, filling and other operations of aseptically prepared products are done in class A environment which has a background of class B environment.

Because of parenteral drugs are manufactured generally in the form of ready to use syringes or vials in a single dose or rarely multidose, it can be mentioned two types of production process: Mostly the issue depends on the education and behavior of the staff.

It is loaded to the production vessel according to process flow chart. Usually 85% of the volume of WFI (water for injection) is loaded into the reactor firstly. Then, active substance and excipients are loaded.

80-90% of water for injection is loaded into the reactor. Before loading into the production reactor, WFI is cooled gradually 50 ° C then 40 ° C. Cooling is performed by flowing WFI which is passing through opposite direction in the coil .

The solution is made isotonic or isohydric.Osmotic pressure of the solution is adjusted up to the pressure of body fluids.

The volume of solution (final volume in the recipe) is completed with water for injection.

Control and adjustment of pH is made.

Solution is stirred in accordance with the processes for production program. Dissolved oxygen in the solution is removed by sending nitrogen continuously during stirring.

The solution preparation process ends when dissolved oxygen completely removed by passing through nitrogen.

Solution is passed through firstly 0,45 μπι then 0.22 μπι membrane filter and samples are taken for analysis.lt is filtered in this way and samples are taken for the following in- process controls.

In-process controls;

a) pH

b) Viscosity

c) Particulate matter

d) Osmolality e) Clarity and homogeneity

Product is transferred to the reactor when in-process control results are in the

specifications.

Sending the inert gas such as nitrogen continuously during the preparation process in the production tank is performed to provide removing oxygen and also mechanical stirring of the solution and mixing gas flow .Thus, a complete dissolution and homogeneity is obtained.

In-process controls are performed after preparation of the solution. When in-process controls correspond to specifications,the solution is filtered 0.45 μιη and 0.22 μπι membrane filter with nitrogen respectively.

Filtered product is sent to filling line storage reactor. Preparation for filling and filling are performed after appropriate analysis results.

The present invention is formed based on these features of the formulation more detailed below.

Detailed description of the invention;

Stable formulation for cancer and supportive care is established by using production methods mentioned above with API I and API II as active ingredients and the following excipients.

Table 1

The development of the product had been done with using different base or salt of API I and API II. As a result, optimal form of API I and API II is found and it has been decided to use API I and API II forms because of their high solubility.

Also in the present invention the ratio and the order of addition of buffering agents in the manufacturing process is determined to provide dissolving of the combination of the active ingredients. Solubilizing and buffering agent in the pharmaceutical form; sodium hydroxide is selected from sodium carbonate and derivatives. Strong acid or alkali solutions cause damage in the area where they are injected. Adjusting the pH is needed to increase the stability of the drug, prevent tissue pain, damage and irritation at the injection site, prevent the growth of microorganisms and increase physiological activity. The reason of the pH changing is degradation of drug and substances that contaminated from glass or plastics containers and stoppers. Buffer solutions are used if the drug is degraded out of stable pH range. It should be considered that buffering agent must be compatible with the drug.Injectable dosage form is desirable to adjust the pH using a suitable buffering agent. pH of the blood is between 7.3-7.5. The injected solution is at the same pH with plasma to prevent pain and irritation. Blood has a buffer capacity. pH adjusment may not be needed because of rapid dilution when intravenous solutions are injected very slowly

Priority is the stability of the preparation. This is why the pH of the solution is adjusted to 7.5 to 9.0 which the active agent is stable.

Chelating agent is added to protect patients from the toxicity of metal ions and make active ingredients stable. These stable combined pharmaceutical form can be applied parenterally, when we prefer to use a chelating agent; as edetic acid or edetate disodium hydrate, sodium, calcium disodium, dipotassium, cobalt, disodium, trisodium, disodium and derivatives .For any reason very low concentrations of heavy metals in the environment can accelerate oxidation. Therefore, chelating agents are being used to hold these substances. They generate stable water-soluble complexes.

In the present invention,solution of the active ingredients combination was prepared.

Isotonicity agent is added to the formula not to cause any damage to the patients' cells. Isotonicity agent can be selected as sodium chloride,mannitol and- derivatives in this stable parenteral combined pharmaceutical form.Isotonic solutions have equivalent pressure to the osmotic pressure of blood plasma. Hypotonic solution has lower osmotic pressure than the osmotic pressure of the blood plasma and the solution which has more osmotic pressure is called hypertonic solutions.

Sterile parenteral pharmaceutical preparation is equal to body pH (7.4) or close to it, preferably should be isotonic and pyrogen free. The solution is desired to be isotonic to reduce the damage of the parenteral drug to the tissue and maintain the blood electrolyte balance. Osmotic pressure of the solution (osmolality) is expressed in units of osmole / kg.

The solution which has lower osmotic pressure from body fluids or 0.9% NaCl is hypotonic. It causes hemolysis of erythrocytes. The solution which has more osmotic pressure from body fluid is hypertonic and it causes constriction of erythrocytes. Any cosolvent or surfactan are not required because of the active substance is dissolved.

There is no need to use any microbial preservative such as benzyl alcohol because aqueous solution which is containing API I and API II is stable during shelf life.

The solvent used during production in unit formula is water for injection and it is suitable for preparing parenteral solutions. The raw material of the injection water is deionized water known as WPU.WPU is obtained by distillation by passing through the water heat exchanger at 96 ° C to 102 ° C. Pyrogenic substances substantially purified in this water. The Water for Injection (WFI) obtained from the autoclave sterilization with contacting with water vapor.This sterilization is identified SIP (sterilization in place) and the process is carried out in situ sterilization.

Combined stable aqueous solution comprising API I and API II is filled into glass vials and use pre-filled syringe for parenteral use. Both filtration and terminal sterilization were administered. Parenteral solutions is maintained at 121 ⁰ C for 15-20 minutes in autoclave sterilization.

Parenteral solutions that sensitive to heat is sterilized by filtration with defined pore size of membrane filter made of cellulose ester. 0.22 um pore size filters are efficient for removing very small organisms. But the solutions that filled in the ready for use syringe filling line is subjected to sterilization at 121 ° C for 20 minutes like ampoules. After the sterilization process samples are sent to the microbiology.

Parenteral solutions must be sterile because of directly injected to people. Sterility is absence of living organisms such as bacteria and fungi which are reproduced in standard culture media.Sterilization is the destruction of these organisms.

Parenteral solutions are kept at 121 ° C during 15 to 20 minutes in autoclave sterilization. Parenteral solution that unstable to the heat sterilization is filtered through membrane filters which is made of cellulose esters and derivatives and has undefined pore size . 0.22 μπι pore size filters are efficient for the removal of very small organisms.

Ready to use syringes are small primary packaging containers that made of glass, plastic, polymer or copolymer materials which drug or drug solvent is filled for parenteral purpose.Pre-filled syringe and vial containers should not contain any extractable substances that may change stability and increase toxicity of the product.

Stability is related to shelf life of the product. Time dependent changing of bacterial or chemical compound in the solution is undesired . Therefore stability enhancers and preservatives are added to the parenteral dosage forms.

We can provide stable formulation comprising API I and API II together.Also it is possible to market the product separately.