Formulation And Evaluation of Vitamin B 12 Sublingual Spray

I.1 Overview of Methycobalamin

Vitamin B12 is an essential element for the proper growth & reproduction of normal cells, haematopoiesis & formation of nucleoprotein or myelin. Cells which have rapid cell division      need high amount of vitamin B₁₂.Methylecobalamine is linked with fat &carbohydrates metabolism but have major functions in protein synthesis ⁽¹⁾.

I.2 Major functions of vitamin B₁₂

1. Synthesis of DNA in cell & Replication.
2. Haematopoiesis.
3. Nerves protection & Regeneration.
4. Neurotransmitter synthesis.
5. Detoxification.
6. NO stress.

Synthesis of DNA in cell and replication - Vitamin B₁₂ is responsible for the synthesis of DNA and structural stability of centromeres and the sub telomeric DNA, as a methyl donor it contributes in mono carbonic acid metabolic pathway and play critical role in methylation of DNA.DNA methylation is catalysed by DNA methyl transferases that transfer methyl group from Adenosyl methionine to cytosine. Vitamin B₁₂ along with folate and Iron have crucial role in Erythropoiesis, where erythroblast cell requires folate and vitamin B₁₂for proliferation during differentiation by stimulate the formation purine and thymidylate synthesis which ultimately lead to formation of DNA synthesis ⁽²⁾.

Vitamin B₁₂ is important for the proper functioning and development of the brain and nerve cells. They play major role in maintains of the sheaths that cover and protect nerves of CNS and PNS ensuring fast and effective nerve impulse transmission Vitamin B₁₂play significant role in synthesis and maintain of myelin. This vitamin assists an important step in one carbon cycle which is responsible for the synthesis of neurotransmitter ⁽³⁾.

Figure1: Working of Methylcobalamine Inside the body

1. Major Alcohol & drug consumption
   1. Breakdown of alcohol& drug requirement a large amount of vitamin B₁₂& more importantly give high strain the stomach & intestine lining, lead poor development of IF.
2. Diet which has lower vitamin B₁₂ content.
3. Worms/parasite infection, particularly tape down species will consume large amount of vitamin B₁₂.
4. Partial excision of stomach.
5. Age factor- senior citizen develop week stomach lining.
6. Some specific medication ⁽⁵⁾ eq.
   1. Contraception hormones.
   2. Diabetes medication.
   3. High blood pressure medication.
   4. Cardiac arrhythmia medication.
   5. Impotence treatment.
7. Sever disease condition in the liver (storage + transport) problem.

I.4 Vitamin B₁₂ major Deficiencies symptoms

I.5 Absorption of vitamin B₁₂ inside the body^(7,8)

Figure 2: Absorption of vitamin B₁₂ from stomach

Sublingually means “Under the tongue” in which drug placed beneath the tongue this area consists blood vessels, in such a way substance is rapidly absorbed via blood vessel rather than via Digestive tract ^{(9). The} extent of absorption is greatly dependent on lipid solubility, the permeability of the solution, the ionization and molecular weight of the drug molecule⁽¹⁰⁾.Drug can be absorbed by both active & passive mechanism,

Some drug molecule trigger endocytosis via buccal epithelium and drug molecule is engulfed by the cell and deliver into the blood circulation ⁽¹¹⁾.

On the other hand passive absorption takes place by two major pathways⁽¹²⁾.

1.  Inter cellular pathway
2. Intra cellular pathway

Figure3: Mechanism of drug absorption by sublingual route ⁽¹³⁾

FIGURE4: Working of sublingual solution spray to systemic circulation

Diffusion is major process for the transportation of the drug from body cavity to the circulation some of the important factors which affect the diffusion are⁽¹⁷⁾:-

I.8.Criteria for drug selection^(20,21).

Table no:3 Ideal characteristics of drug for sublingual absorption

2.3 Advantages of sublingual sprays.

I.9. Overview of the oral mucosa ^(22,23)

Oral cavity majorly contains    lips – cheek – tongue – hard plate – floor of mouth ⁽²⁴⁾.

1. The buccal, sublingual, mucosal tissue at the ventral surface will cover 60% of oral mucosa, surface area. ⁽²⁵⁾
2. The upper most layer is made up of compact lining of epithelium cell ⁽²⁴⁾, whose major function is to protect the oral mucosa from harmful environment & fluid loss.
3. The next layer is basement membrane made up of lamina propria& sub mucosa.
4. Oral mucosa contains many sensory receptors include taste receptor which is present in the tongue^{. (27)}

Mainly three type of oral mucosa-

1. Lining mucosa- Found in the oral outer vestibule highly elastic & flexible ⁽²⁶⁾.

2. The sublingual mucosa

3. Specialized mucosa - Present on the dorsal surface of the tongue. Mosaic of keratinized & non keratinized epithelium ⁽²⁵⁾

4. Masticatory mucosa On the hard palate & gum comparatively pass flexible. Masticatory epithelium is 25% & specified mucosa in 15% lining mucosa is 60% of total oral lining.⁽²⁷⁾

I.10. Physicochemical property of oral mucosa:

Oral mucosa is different at different region of oral cavity

1. Masticatory mucosa – It is present in those area which involves mechanical process like mastication &speech⁽²⁸⁾. This region is stratified & have keratinized layer on its surface.
2. Lining mucosa – It will cover 60% of oral cavity located in the inner cheeks , floor of mouth , underside of the tongue . The surface is lined by both keratinized & non keratinized epithelium.⁽²⁷⁾

Epithelium is attached to under lying structure which is further connected by connective tissue or lamina propria, separated by buccal lamina ⁽²⁹⁾ . These layers will provide major mechanical support & barrier for penetration of active substance ^(31). _{Detail anatomy of mouth is shown in fig 5.}

Figure 5: Sublingual Anatomy of Human Buccal Cavity

Major barrier is epithelium barrier which property are alter by different mechanism of penetration enhancer. Majorly there are 2 type of penetration enhanceing mechanism –

1. Chemical method
2. Physical method

I.12 Chemical method

They will increase the penetration through membrane without causing any potential damage to oral mucosa⁽³⁰⁾.

 Basic Mechanism of chemical enhancers are-

1. Altering rheology of mucosa membrane^(33).
2. Increasing fluidity of membrane .
3. Temporally altering the lipid bilayer⁽³⁴⁾of membrane .
4. Modification in cellular protein⁽³⁵⁾.
5. Raising thermodynamic activity of the permeate.
6. Fighting enzymatic barrier⁽³⁴⁾.

Chemical enhance can be mixed in a formulation in a combination or alone depending on their efficacy &physicochemical ⁽³⁶⁾

I.13 Physical method ⁽³⁷⁾

Figure 6: Physical Methods increasing buccal permeation

• Sonophoresis- Decreasing the density of lipid domain by ^(40):
• Micromechanical
• Thermal
• Cavitation effect
• Iontophoresis - For delivering water soluble drug & ionized medication a low level of current is used.
• Electro osmosis – The process by which charged particle tend to migrate toward a less charged area⁽⁴¹⁾.
• Electro portion – Large electric pulse will disturb phospholipids bilayer hence allow permeation through tissue⁽⁴²⁾.

II. Material And Methodlogy

This basic study is carried out to gain information about the physical as well as chemical characteristic of drug such as PH, solubility , drug identity and its interaction with other excipients  for the proper designing of drug delivery system . Methylcobalamine was received as a gift sample from Benet Pharmaceutical Ltd 

Methylcobalamine:-

1. Test Performed for identification.
2.  Test Perform for compatibility with other excipients.

Test For the Identification:-

[II.1.A] Organoleptic Character

Colour:- It was determined by physical appearance.

Taste and Odor:- A extremely small amount of drug is used to acquire taste of drug with help of taste buds and smelled to get odour of it.

[II.1.B] Melting point:-

As small amount of drug is taken in a capillary tube which is used from one end and placed in melting point apparatus, gradually the temperature of apparatus is increased the temperature at which drug start melting is recorded.

[II.1.C] IR Spectra

Identification of drug was done by it IR spectra. Similar spectra was obtain from both FTTR ( Jasco 470 plus) and reference given in USP.

[II.1.D] Solubility Profile:-

Solubility of drug was determined in various aquas and organic solvent . A specific amount of drug is allowed to dissolve in different solvent (constant amount ) at room temperature & observed by visual inspection.

Solubility Profile

[II.1.E] Partition Coefficient:-

Partition coefficient is one of the major parameter which determine the liphophilicity of the drug which indirectly show potential penetrating power of the drug through biological membrane for the determine of the partition coefficient water and octanol mixture is taken in 1:1 ratio and mixed thoroughly for 10 min then 10mg of methylcobalamine is allowed to dissolve in thes mixture and shake it well in definite time internal for some time after 24hr separate the 2 layer with the help of separating funnel 2 analysed drug concentration in both phase separately .

[II.1.F] Quantitative Estimation of methylcobalamine:-

Determination of absorption maxima of the drug- Lambda max was determine by taking 10mg of methylcobalamine 10 ml of phosphate buffer of pH-7 in a volumetric flask of 25ml to make 1000 µg/ml .This make stock solution .Them from the above stock solution “A” 1ml is taken & diluted to 10ml by phosphate buffer this make 100mg . Further sub stock solution is prepared of 10 µg/ml .These sample was screened in the range of 400-20nm in simadzu 1700 UV/visible spectrophotometer to find out lambda max of drug.

[II.1.G] Test Performed for compatibility between the drug &adjuvant{ FTIR}

Physicochemical compatibility between the chitosan used as a mucoadhesive agent and the methylcobalamine is carried out by making physical mixture with kBr pellet and allow them to react for 24 hr in dark condition them in this mixture analysed and infrared radiation from 400-4000 cm.    

[II.2.A] Procedure: -

Preparation of concentrate

Mucoadhesive concentrate is prepared by using 1% chitosan solution which was prepared by dissolving 250mg of chitosan in 4% of lactic acid solution and then allow to sonicate for 20 min 5mg of drug is weighed & dissolved in 1ml of water in umber colured container. Further adding appropriate amount of ethyl alcohol in the drug solute to increase it membrane permeability. Finally .9% benzyl alcohol, cyclodextrine citric acid , sodium chloride propylene is added to the solution Both the mucoadhesive mixture & solution is mixed and agitated for 20 min in 50 rpm in mag stirrer In total 1.5gm/ml citric acid is added in finally preparation to protect from microbial growth.

[II.2.B] TABLE OF OPTIMISATI

[II.3.A] Visual inspection

All the prepared formulation are visually inspected for color , clarity , visual dirt particles , precipitate formulation by the ingredient discoloration or cloudiness may indicate microbial contamination.

[II.3.B] pH determination

The pH of all the formulation is evaluated by use pH meter. The pH meter was set at neutral pH-7 in distal water by its adjusting screw and finally placed inside the solution and note the reading where the pH meter stops further fluctuation.

[II.3.C] Viscosity determination

Viscosity of the sample is determined by Oswald broke field viscometer by placing the 84 probe 64 of the instrument in the container containing the sample in appropriate quantity that the probe should get immersed in the sample to avoid error. Note the reading should be taken at which is constant repeat the process for as time and take mean viscosity.

[II.3.D] Determination of methylcobalamine permeation pattern.

Permeation study were out losing vertical glass from diffusion cell, which effective diffusion area 12.56 cm ² donor and receptor compartment is attached to each other by clamp in between both the chamber membrane disk mixed cellulose is placed with [pore size .47µm] Lower chamber is filled with 16ml of phosphate buffer pH 7.4 and temperature is maintained 37± 0.5?C magnetic starring is kept constant at 50rpm to maintain homogeneity , Donor chamber is filled with 1.5ml of test formulation membrane was previously wetted by artificial saliva for 5 minutes. After 30 second the 2ml sample is taken out from the receiver chamber and volume is make up by phosphate buffer to maintain sink condition equal quantity of buffer is place in lower chamber all the sample was analysed in U.V spectro photometer light protected environment

[II.3.E] Determination of total drug content

Assay of accurately weighed amount of formulation were carried out for the determination of net drug content. The weighed sample was dissolved in 10 ml of water distil and further it was filtered. The drug content was estimated Spectrophotometrically at 349nm using standard curve

[II.3.F] Ex vivo drug release study

The in vitro optimized formulation which shows highest permeation is selected for ex vivo study was performed on sublingual membrane of goat, which was preserved at low temperature in krebs solution. sublingual mucosa of 14 cm² was excised carefully and left for 1 hour in artificial saliva to maintain moisture and mimic the condition of sublingual mucosa finally this membrane in mounted of glass vertical franz cell, the donar compartment and receiver compartment with 16ml of phosphate buffer of pH 7.4 at 50 rpm. To maintain sink condition 3ml of sample withdrawn from receiver chamber in equal interval of time and equal amount of phosphate buffer is added into the receiver chamber with constant stirring and temperature is maintained at 37?C±1at last all the sample are diluted with phosphate buffer and analysed by u v photo spectrometer and for the concentration of drug in each sample , permeation pattern of the drug.

[II.3.G] Stability study:-

In assay the percentage drug content was found to be 98% which complied with ICH guideline limit 98-103%. In degradation studies for thermal stress at 50?C,60?C,80?C, was provided for 30 min then store in then change in viscosity, pH, drug content was analysed by U.V. photo spectrophotometer for forced degradation study, 10ml the formulation is taken and mixed with 5% H₂O₂ in a umber colour glass container the resultant solution in allowed to stand for 6hr in a dark room to facilitate oxidation of the drug.To test the sensitivity towards light the proximal formulation is allow to kept in UV light for 6 hour and in dark room for 6 hour and analysed for active drug content.

[II.3.H] Mucoadhesion Testing

Texture analyzer was used to test mucoadhesion strength of the formulation. Initially mucoadhesive rig was cleaned thoroughly then 2 ml of saliva was spreader on the surface of rig and the optimized formulation was placed on it after that rig was tightly closed by upper half and place in a beaked which contain water of temperature 31±?C Then prove is allowed to come in contact with formulation and then reading of mucoadhesion strength was analysed

III. RESULT AND DISCUSSION

[III.1] Preformulation  study:-

(III.I.A)Organoleptic Character

1. IR Spectra

(III.1.D) Partition Coefficient:-

Table No 13: Partition coefficient of drug

(III.1.E) Quantitative Estimation of methylcobalamine:-

Figure 8: Showing maximum absorbing wavelength

(III.1.F) Standard curve in phosphate buffer 7.4 –

Table No 14: Absorbance profile of drug

Figure 9: Standard Curve In Phosphate Buffer 7.4

Physicochemical compatibility between the chitosan used as a mucoadhesive agent and the methylcobalamine is carried out by making physical mixture and sample are compatible not showing any incompatibility.

The spectrum of chitosan showed a broad peak at 3447 cm!1 and a small peak at 2800 cm!1 corresponding to the N-H symmetrical vibration and the typical C-H stretch vibrations, respectively. Amide I and III peaks appeared at 1658 and 1322 cm¹, respectively. Sharp peaks appeared at 1383 cm!1 and 1424 cm!1 corresponding to the CH₃ symmetrical deformation mode. The figure also showed broad peaks at 1030 cm!1 and 1080 cm¹ corresponding to the C-O stretching vibration and other peaks around 893 cm!1 and 1156 cm^!1 corresponding to saccharide

(III.2.A) Visual inspection:- Appearance of all nine batch was tested against white & Black background to check to celerity and turbidity . All the above formulation were checked for 24 hours. The formulation were observed as transparent with reddish tint , no phase separation or precipitate were observed after keeping still for 24 hours.

Table no15: visual inspection of formulation

(III.2.B) pH value of methylcobalmine drug during Sublingual spray:-

pH value of methylcobalmine sublingual spray (F4,F8,F9) were determine using digital pH meter .The stability of concentration is generally affected by pH . the excipient used in the formulation will majorly decided the pH of concentration for example mucosa sublingual absorption pH should be 6-7.5

(III.2.C) Viscosity of the formulation

Viscosity of the solution will show great influence to the formulation if the solution is more viscous it will permeate in more time , hence optimum viscosity of the formulation is required so that it can be easily sprayed through actuator of the spray. Other wise it will block the nozzle of the spray.

Table no 17: viscosity of final formulation

(III.2.D)Determination of drug content

The drug content of methylcobalamine concentration was measured by U.V spectrophototmetric method . The percentages drug content was determine by considering 5 mg of methylcobalamine as a 100% . All the above formulation are within the limits 37% 97% 75% to 99.05% of  drug content.

Table no 18: drug content in final formulation

(III.2.E) DRUG PERMEATION STUDY

In formulation { F1,F2, F3, F4} as the concentration of chitosan increase ,as polymer tend to trap the drug & used to release drug in sustained fasion, only 23% of the drug release in initial  3 minutes. On the other hand when concentration of cyclodextrine increase it will increase partion coefficient of the formulation  as a result increasing the permeation characteristic of the formulation { F5, F6, F7 } release rise up to 36%.  In formulation [F8,F9] as the amount of  PG increases it will increase pore size as a result passage of the formulation is facilitated  to 40%.

Table no 19: Invitro Permiation Data Of Various Formulation

Figure no 9: %CDR of all formulation

(III.2.F) EXVIVO STUDY

Ex vivo study on goat sublingual mucosa shows increase in permeation of drug through mucosa than that of  In vitro condition because chitosan is able to break tight junction hence facilitate the passage of drug through this route .On the other hand PG will increase the pore size of the membrane along with alcohol as a result increase in Para-cellular movement of the drug .Therefore Ex- vivo data  will help to predict In- vivo permeation pattern of the formulation.          

Figure no10: Ex vivo release data

When formulation is allow to expose at higher temperature(50?C,70?C,90?C) the amount of drug content tend to decrease rapidly and the color of the formulation get changed from red to orange This clearly indicate the degradation of methylcobalamine at elevated temperature. Similarly when the formulation is expose to light in presence of oxygen it will degrade rapidely to give aquocobalamine and formaldehyde as a major product , as a result active methylcobalamine concentration reduces in very low time therefore protection from light is primary necessity . table 8.8 to 8.12 show various changes  observed at stress condition

A-Thermal stress

Table no 22: Thermal stability study at 50?C

Table no 23: Thermal stability study at 70?C

Table no 24: Thermal stability study at 90?C

B –photostability study

Table no 26: Photostability stability study

Table no 27: Photostability stability study

(III.2.H) MUCOADHESIVE TESTING

This result of Mucoadhesive testing conclude that formulation is having appropriate mucoadhesive character so that it can show bio adhesion with sublingual mucosa -0.50 value shows good mucoadhesive character due to presence of chitosan matrix in the solution. As the value become more negative it will show more adhesion with the biology membrane.