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The basic purpose of this work was to develop accelerated stability study of Cholecalciferol (Vitamin D3) tablets and injections and to understand the drug stability at different intervals at accelerated climatic conditions.
The other objectives were, to determine the storage conditions, identification of degradation pathways , shock and vibration , to determine which type of stress affect the molecule(high or low temperature, oxidation, pH extremes) and the in-use stability.
The stability studies played a major role in drug development; allow understanding of the molecule, necessary for developing analytical methods and selecting packaging material.
For tablet dosage form, tests like disintegration, hardness, friability, moisture content and assay of the active ingredients of three commercially available brands that is, Osam-D Tablets, Calgo Tablets and Qalsan D Tablets were performed and for Injection dosage form, tests like Color, Clarity, Particulate contamination, Sterility, Bacterial Endotoxins test, and assay of active ingredients of three commercial available brands that is, Indrop D, ED-3 and Cara-In-D injections were performed.
The assay for the active ingredient(s) in the tablet and injection formulations were performed by using U.V spectroscopic method (newly developed or non-pharmacopoeia method). Similarly the assays of active ingredients were studied by analysis through Double beam U.V Spectrophotometer.
Climatic chamber was used to maintain temperature and relative humidity for accelerated climate.
In Accelerated stability studies, minimum of 3 points including the initial and final time points (e.g. 0, 3, 6 months) from a 6-month study were analyzed. Actually, the assay results data was analyzed by comparison between different commercial brands and were found to be sensitive to high temperatures as well as to high relative humidity while physical tests as mentioned above are not focused due to degradation assay content and our purpose of study.
In ambient climate, the active ingredient should have best result while in accelerated climate; there were little bit degradation of active drug material.
Aims and Objectives:
Purpose Of Study:
Cholecalciferol (Vitamin D3) undergoes degradation reactions at elevated temperatures and high humidity conditions .Our goal in this work was to expand investigation of degradation of various Cholecalciferol samples of tablets and injections to a wider range of conditions than reported previously. Degradation of Cholecalciferol (Vitamin D3) tablets and injections was compared to pure Cholecalciferol.
The evaluation of the quality of Cholecalciferol (Vitamin D3) tablets and injections available in different brands in market of Pakistan
Specific objectives are given below;
To determine the storage conditions.
Key to dealing with temperature and relative humidity.
To take information about Shock and Vibrations.
Permit and allow understanding of molecule.
Necessary for developing analytical methods.
Determination of the drug potency of product.
Identification of degradation pathways during experiment.
Identification of degradants of products.
To evaluate degree of variability of individual batches.
An approach to determine "Significant Change" during practical processes.
Play role in drug development.
To select packaging material for drug product.
Introduction and Structure of Cholecalciferol :
The vitamin that mediates intestinal calcium absorption, bone calcium metabolism and very likely, muscle activity, usually acts as a hormone precursor, is called as Cholecalciferol.
Cholecalciferol is also called as:
. Calciferol because of its function in calcium metabolism and
. Antirachitic factor as it prevents rickets.
Cholecalciferol could be thought of as a hormone instead of a vitamin:
. Because it can be synthesized in the body
. It is released in the blood circulation and
. Has a clearly different target organ.
But it is still included in the list of vitamins, as it becomes an essential dietary factor when endogenous synthesis is too low to meet the physiological requirements.
Structure of Cholecalciferol:
Figure1: Cholecalciferol-activated 7-dehydrocholesterol.
Introduction and Definition of Vitamin:
The name 'Vitamine' was proposed for the nutrient compound required to stop the nutritional deficiency disease beriberi, because of its vital need and because chemically it was found to be an amine. Later after a number of other necessary and important organic nutrients were discovered, the 'e' was dropped, when it was found that not all of them are amines
The term 'Vitamin' has now been adopted universally and applied to a group biologically important and necessary compounds that includes 14 compounds which cannot be synthesized by human beings. They, must therefore, be provided through food.
History and Classification of vitamin:
Vitamin D is the name given to certain sterols occurring in cod-and fish liver oil, egg yolk, milk and butter. It is involved with the proper utilization of calcium and phosphate and normal calcification of bone and its deficiency leads to the development of rickets.
E.Mellanby in 1919 proved that cod-liver oil and other fats rich in vitamin A were also anti-rachitic. Mc Collum, however, showed that this anti-rachitic property of cod-liver oil, etc, was distinct from the growth promoting vitamin A, for spinach which is especially rich in the latter vitamin was strong growth promoting and anti-ophthalmic but had no effects on rickets .
Once again, it was known for some time that rickets could be cured by ultra-violet light. But the exact manner in which ultra-violet light produced this effect was not known until it was shown that ultra-violet irradiation could force anti-rachitic activity to foods. The substance which was thus activated by ultra-violet light was known to be ergosterol, a vegetable sterol the beneficial effects of ultra-violet light in human rickets are believed to be due to the activation of 7-dehydrocholesterol, an animal sterol present in the fat under the skin.
Different between Cholecalciferol and Ergocalciferol
Cholecalciferol is very similar to Ergocalciferol, but has valuable biological differences. It occurs in cod-liver and other fish-liver oils, and may be produced synthetically from cholesterol by way of dehydrocholestrol. Cholecalciferol is used as a reference standard for assays of anti rachitic compounds, it is equally potent for mammalian and avian species, while Ergocalciferol is equally active as Cholecalciferol mammalians species but has only about one-fiftieth of the activity for avian species.
Physical and Chemical Properties:
Cholecalciferol is also called as activated 7-dehydrocholestrol or vitamin D3. Their chemical formula is C27H44O and molecular weight is 384.7. Its melting point is 84°C to 88°C.
It is white odorless crystals which are affected by air and light.
Cholecalciferol is insoluble in water, soluble in alcohol, acetone, chloroform, ether and fixed oils.
International Standard Unit Value:
Cholecalciferol contains 40 thousand units of anti -rachitic activity in 1 mg.
Stability of Cholecalciferol:
It is oxidized and inactivated by moist air with in a few days. Deterioration of pure cryst Cholecalciferol is negligible after storage of 1 year in amber evacuated ampoules at refrigerator temps, Vitamin D2 may be kept for 9 months under the same conditions. Normally Cholecalciferol is considered more stable then Ergocalciferol
Production of Cholecalciferol:
The naturally produced Cholecalciferol, is the form obtained from animal sources in the diet, or formed in the skin by the action of ultra violet light from sun light on 7-dehydrocholestrol and
Artificially produced form D2 or Ergocalciferol, is the form manufactured in the laboratory by irradiating the plant sterol, ergo sterol and it is the form most readily available for pharmaceutical use
UV Lightâ†’â†’ â†“
Cholecalciferol (Vitamin D3)
Figure2: The formation of Cholecalciferol in the body.
Cholecalciferol found in and is isolated from fish-liver oils. Methods of separation vitamin D3 include chromatography, molecular distillation, esterification, and fractionation of the esters.
Accelerated stability studies.
Table 1: Division of climatic zones for Stability testing
Accelerated Stability studies Conditions:
The stability storage conditions for Injections or Tabs are as mentioned below:
Table 2: Table illustrating Accelerated Stability Storage conditions
Stability Test Program:
The stability test program is as given below:
Table 3: Table illustrating Accelerated Stability test program
T â†’ Analysis to be performed at these intervals
N.A â†’ Not applicable
15 Packs of product is required to perform the complete analysis as per the accelerated stability specification. The details of the samples kept along with additional packs are mentioned below:
Table 4: Table illustrating Accelerated Stability Samples Specifications
As & when a significant process modification takes place or a new critical equipment is added in the manufacturing process, accelerated stability study of three batches, manufactured after incorporating changes, will be performed . Samples of these batches will be packed in simulated market container & stored at a temperature of 40 + 2oC & relative humidity of 75 + 5%. Samples will be kept for a period of 6 months.
Pathways of Chemical Degradation during Stability Studies Of Products:
Pathways of Chemical degradation include;
Isomerization and Racemization.
Decarboxylation and Elimination.
Drug-Excipient and Drug-Drug Interactions.
It is a well known chemical degradation pathway for Cholecalciferol. Oxygen, which participates in most oxidation reactions, is abundant in the environment to which Cholecalciferol is exposed, during either processing or long term storage.
Oxidation mechanisms for drug substances (Cholecalciferol) depend on the chemical structure of the drug and the presence of reactive oxygen species or their oxidants.
Photodegradation procedure has been reported for large number of drug substances. The mechanisms for these reactions are generally very complicated. It is often accompanied by oxidation in the presence of oxygen. Thus, drug substance such as Cholecalciferol, whose oxidation was described above, are degraded to different products in the presence and absence of light.
Temperature and Relative humidity universally affect the drug molecule but if we protect the drug from light from the start of manufacturing e.g., As Cholecalciferol (Raw material) is packed in aluminium container, while during drug manufacturing it is protected from light, then it is not necessary to check light as critical parameter during ASS of Cholecalciferol.
Official Storage Condition:
Official storage conditions are as given below;
Freezer = -10OC to -20OC
Cold = NMT 8OC (Refrigerator)
Cool = 8OC- 15OC
Room Temperature = 15OC- 30OC
Warm = 30OC- 40OC
Excessive heat = above 40OC
Conditions Specific to Drug Products:
Table 7: Table illustrating Storage Conditions for Refrigerator drug Products
In the absence of accelerated study, data from elevated temperature (e.g., 5°C ± 3°C or 25°C ± 2°C) on a single batch should be conducted for an appropriate time period to address the effect of short-term excursions outside of the proposed storage condition.
Light Sensitive Drug Product:
Photostability testing should be conducted on at least one primary batch of the drug product.
The standard conditions for Photostability testing are according to ICH Q1B.
Evaluation of Stability Data:
A stability report is generated detailing the protocol design as well as results and conclusions. Results of testing are recorded at the time of manufacture and at different time intervals.
Stability of Cholecalciferol (Vitamin D3):
Cholecalciferol stability is affected by light, temperature, oxygen and relative humidity. It is quite stable at room temperature but at 40°C or higher temperature and higher humidity than 45% it is quite unstable. Its formulated drug products are stabilized by adding antioxidants and solubilizers. Cholecalciferol is more stable than ergocalciferol.
Degradation review related to Cholecalciferol:
A review of the available literature on degradation of Cholecalciferol during storage has not led to any literature specifically about degradation products of this compound. There are some articles that describe the stability studies of Cholecalciferol under various conditions. However, the authors do not mention which compound is formed, when Cholecalciferol is degraded.
Hoffmann-La Roche, states that,
The nature of Cholecalciferol's (Vitamin D3) degradation products is unknown. Therefore, the stability is only examined by carrying out the assay for Cholecalciferol after storage under the chosen conditions.
Identity and Purity Requirements:
A pharmaceutical analyst initially interfaces and interacts with various department and professionals from several disciplines to provide and give input that assure that the new chemical entity (NCE) or new molecular entity (NME) indeed has the proposed structure and defined requisite purity. The new drug product development process can be accelerated by the use of combinatorial chemistry and high-throughput screening.
Assay of Active Ingredients:
Assay is defined as; Quantitative analysis of active ingredients. Assay for pure compound is a simple but for finished dosage form have more detail steps necessary for the extraction of drug in a solvent followed by separation.
Non-specific assay methods are frequently used during analysis of products. This applies specially in the case of acid-alkali titrations for bases and acids. Many inorganic salts, also, are determined normally on the content of one of the ions present; thus sodium sulphate is assayed on its sulphate content by precipitation as barium sulphate.Even when modern newly physical methods are used, as for example the measurement of ultraviolet absorption, this may be by no means specific. For example, most simple aromatic substances show absorption, which can form the basis of assay, in the region 260-300nm; this absorption is quality of aromatic ring. When a coloured complex of a compound is produced or the compound is inherently coloured, its determination can be made in the visible region 380-780nm.
CHAPTER NO 2
Bone Mineral Disorders and Cholecalciferol:
Those disorders which are produced or arised due to change in regulation of calcium and phosphate homeostasis in bone are called as Bone mineral disorders.
Calcium and phosphorus, the 2 major elements of bone, are essential not only for the mechanical strength of the skeleton but also for the normal function of many other cells in the body.
Accordingly, a complicated regulatory mechanism has evolved to tightly regulate calcium and phosphate homeostasis. Parathyroid hormone (PTH) and vitamin D are primary regulators, where as calcitonin, glucocorticoids, and estrogens (regulators) play secondary roles. These hormones or drugs that mimic or quash their actions are used in the treatment of bone mineral disorders, as are called regulators of bone mineral homeostasis. In those regulators, one of the active drugs is Cholecalciferol.
a. Rickets: The essential and important disturbance of the skeleton in rickets lies in failure of deposition of calcium phosphate at the growing ends of the bones. This might be due to reduction in the calcium and phosphorus of the serum or to failure in the mechanism at the bone ends by which calcium and phosphorus are retracted from the blood and deposited in the osteoid tissue. The blood in rickets patient shows decreased inorganic phosphate common with normal but in some cases low serum calcium. Deficiency of phosphorus has more importance in the causation of rickets than that calcium.
b. Osteomalacia: This is due to defective absorption of vitamin D and calcium and phosphorus as a result of chronic diarrheas', particularly sprue, celiac disease, biliary fistula and chronic pancreatitis. In Osteomalacia, decalcification found throughout the skeleton without any change in the epiphyseal disks, as they are already ossified.
c. Osteoporosis: In Osteoporosis, characteristic sites of fracture include vertebral bodies, the distal radius, and the proximal femur, but osteoporotic individuals have generalized skeletal fragility, and fractures at other sites, such as ribs and long bones, also are common. It may be more appropriate to consider osteoporosis as the result of multiple physical, hormonal, and nutritional factors acting alone or in combined.
d. Paget's disease: The characteristic feature is increased resorption of bone followed by an increase in bone formation. In early stage, bone resorption predominates and bones are very vascular. This has been termed the osteoporotic, osteolytic, or destructive stage of disease. Body calcium balance may be negative. Commonly, the higher resorption is followed closely by formation of new pagetic bone. As the activity decreases, the resorptive rate may decline progressively as compare to formation, eventually leading to development of hard, dense, less vascular bone and a positive calcium balance. Increased generation and over activity of osteoclasts are considered the major abnormality in Paget's disease. In Paget's disease, the osteoclasts are larger than normal and contain multiple pleomorphic nuclei. Increased numbers of osteoclast like multinucleated cells are produced from hematopoietic precursors in long-term marrow culture from individuals with Paget's disease.
Some of the clinical features of bone mineral disorders are given below;
a.Osteomalacia: A condition of abnormal mineralization of adult bone secondary to nutritional deficiency of vitamin D or inherited defects in the formation or action of active vitamin D metabolites is called as Osteomalacia.
b.Osteoporosis: Osteoporosis is abnormal loss of bone with increased risk of fractures, spinal deformities, and loss of stature; remaining bone histologically normal.
c.Paget's disease: A bone disorder, of unknown origin, characterized by excessive bone destruction and disorganized repair is known as Paget's disease. It has complications like skeletal deformity, musculoskeletal pain, kidney stones and organ dysfunction secondary to pressure from bony overgrowth.
d. Rickets': As compared to Osteomalacia, it occurs in the growing skeleton.
Figure3: Figure illustrating effects of active metabolites of Cholecalciferol (D3), PTH and Calcitonin on Calcium and Phosphorus homeostasis.
a. Rickets: In rickets', the diagnostic features are enlarged epiphyses, delayed dentition, after the age of two, the presence of rickety rosary, mild spasmophilia or convulsions.
b. Osteomalacia: The diagnosis is confirmed in Osteomalacia by X-Ray examination of the bones, which look more translucent as compared to normal bones. Examination of the blood may show normal or lowered serum phosphorus with diminished serum calcium and slightly higher phosphatase. It should be suspected in all cases of vague aches and pains in the limbs and back in women who give history of insufficient diet, inadequate exposure to sunlight and multiple pregnancies etc.
c. Paget's Disease: It is difficult to determine or diagnose because the disease is often asymptomatic. It is frequently diagnosed when roentgenograms are obtained for other reasons or because of a high level of alkaline phosphatase on routine blood screening.
a. Rickets: The important principle in the treatment of rickets is the removal of the causative factor, which is responsible for the defective absorption and utilization of calcium and phosphorus. In the common infantile, adequate dosage should not be less than 1500 units daily.
b. Osteomalacia: The diet should be generous and rich in calcium and Cholecalciferol as well as other nutrients, because subjects of Osteomalacia may also suffer from deficiency of protein, iron and other vitamins. Cholecalciferol should be given in dose of at least 5000 units daily. c. Osteoporosis: Regular physical activity of reasonable intensity is stated at all ages. For children and adolescents, enough and proper dietary calcium is important if peak bone mass is to reach the level approximate for genetic endowment. Cholecalciferol should be given of at least 5000 IU daily to patient.
a. Osteoporosis: The typical feature of osteoporosis is a loss of bone, which tend to be most conspicuous in parts of the skeleton containing abundant trabecular bone. The bony trabecular are thinner and more widely divided than usual, resulting in an increased susceptibility to fractures.
b. Paget's disease: It may present as a solitary lesion (monostatic) or may occur at many sites in the skeleton (polyostotic) with marked variation at each location.
c. Rickets and Osteomalacia:The basic change is defective bone mineralization resulting in over abundant non-mineralized osteoid.This phenomenon contrasts with osteoporosis , where the mineral content of the remaining bone is normal, but the total bone mass is decreased.
Figure 4: The figure shows biosynthesis of Cholecalciferol follows alternate routes, forming the highly active 1, 25-(OH)2 D3 in the presence of PTH.
Sign and Symptoms:
Deficiency of Cholecalciferol results in improper absorption of Ca2+ and phosphate. The consequent decrease in plasma Ca2+ stimulates PTH secretion, which acts to restore plasma Ca2+ at the expense of bone; plasma concentrations of phosphate remain subnormal due to the phosphaturic effect of increased circulating PTH. In children, it causes rickets, while in adults, Osteomalacia will be found. Muscle weakness, hypophosphatemia and osteoporosis occur after inadequate intake of Cholecalciferol.
Absorption, Metabolism and Excretion:
Cholecalciferol is absorbed from the gastrointestinal tract. Absorption is reduced in impaired hepatic and biliary function. Before Cholecalciferol can exert its physiological action it is converted into one or more metabolites, one of which is 25-hydroxycholecalciferol.Cholecalciferol and its metabolites are mainly excreted in the bile and only small amounts are eliminated in the urine.
Figure 5: Figure shows sites of formation of Cholecalciferol (vitamin D3) to its metabolically active form 1, 25 dihydroxy Cholecalciferol.
Cholecalciferol is used for the treatment of following diseases;
d. Paget's disease.
Cholecalciferol should be given with care to patients with impaired renal function. Special care should be taken to ensure proper dosage in infants.
When Cholecalciferol is given in very large doses, it produces loss of appetite and the animal becomes lethargic and drowsy. There is progressive loss of weight with diarrhea and if the Cholecalciferol is not stopped, death occurs in about two weeks.
Other side effects are pallor, lassitude, and persistent nausea with or without vomiting, diarrhea, sweating, and headache and in some frequency of micturation.
Treatment of Toxic effects:
If symptoms of over doses arise Cholecalciferol administration should be discontinued temporarily and high quantities of fluid and electrolytes given. The patient should be placed or put on a low calcium diet. In overdoses, exposure to sunlight should be avoided.
Sodium sulphate should be administered by mouth to reduce absorption of calcium and injection of magnesium sulphate reduces the blood- calcium concentration.