3.1 Background. Preformulation Studies

Transcription

1 Preformulation Studies 3.1 Background Delivery of any drug requires a suitable dosage form to get optimum therapeutic effects. The development of such dosage forms fundamental properties of the drug molecule either physical or chemical are required to be determined. Pre-formulation is to determination of physicochemical properties of drug molecule prior to the development of suitably designed drug delivery system. The classical preformulation studies require characterization in solid state as well as in solution state. A good pharmacological and toxicological profile of drug alone does not sufficient for product development. These studies for drug molecule and excipients are essential to reduce problems occur during product development. Hence, overall product development cost as well as manufacturing and distribution time to market also reduces. The ultimate goal of preformulation study is to select right physical form of drug molecule, determine physicochemical properties and evaluate stability of drug and excipients under different conditions. All of these results will contribute to develop suitable drug delivery system for effective therapeutic use. This drug delivery system should deliver the molecules to its site of action with minimum side effects at a minimum cost. Preformulation studies are usually provides a tool to select suitable excipients compatible with selected drug and play a key role for development of new formulation. Important preformulation study includes identification of drug and excipients followed by particle size analysis, XRD analysis, DSC analysis, solubility analysis, partition coefficient, solution stability and compatibility studies. These all mentioned properties have their own significant effect on development process of formulation. For example, small size of liposome is very critical to avoid first pass effect. Hence, all excipients should be selected in such a manner that there should be minimum effect on liposomal size. Thus, measurement of particle size of excipients is important to design process for preparation of liposome. To reduce the size of liposome further sonication is performed till its particle size is reduced to a significant extent. For qualitative and quantitative determination of any specified drug the UV spectroscopy is a useful tool. Role of UV spectroscopy in a preformulation study is determination of solubility and dissolution rate. Some of the stability studies are performed with the UV spectroscopy. UV detectors are frequently used with HPLC. In this way UV spectroscopy plays an important role (as being accurate and simple method) in early stages of preformulation. 41

2 Chapter 3 To identify drug molecule by its fingerprint identification pattern IR spectroscopy is used widely. Fourier transform IR (FTIR) spectroscopy is being used as a suitable technique for the physical characterization of pharmaceutical solids. In preformulation, IR can be specially used to determine polymorphism of solid crystals as well as for drug excipients compatibility study. The X-ray diffractometry technique is used to determine substance structure at its atomic level. This technique can be used to differentiate crystalline from noncrystalline materials. This analysis has nondestructive in nature and samples can be dealt in all form like powders, solids, and liquids. Differential scanning calorimetery (DSC) analysis is used determine drug excipients compatibility. To find compatibility between drug and excipients a programmed temperature rise was applied and resulted thermograms were analyzed by comparison with individual thermograms of components. If no interacction observed then excipients find suitable to use with selected drug molecule. The results of the DSC analysis affected by so many variables like heating rate, humidity, pan configuration and particle size. Hence, other supportive techniques like PXRD, FTIR and HPLC should be used to understand the nature of the interactions and determine its relevance in formulation development (Zhang et al ). Solubility analysis should be done for both drug and excipients in different polarity solvents for the formulation of liposome. Solubility of drug plays a major role in the formation of liposomes. The hydrophilic drug molecules usually acquire the aqueous compartment within liposome whereas the hydrophobic and amphiphilic molecules capture the lipid bilayer. Hence, nature of drug is considered critical for development and stability of liposome formulation (Habib et al. 1079). Partition coefficient is another important preformulation parameter. The octanol-water partition (K o/w) coefficient depicts the hydrophilic-lipophilic balance of a drug substance. This ratio in log value is known as log P value, which is used to predict the permeability of drug substance across the biological membrane. In liposomal formulation entrapment of drug is depended on partition coefficient of drug. The delivery of drug is depended on partition coefficient along with nature of the lipid bilayer and size of the drug molecules (Bozzuto and Molinari ). Solution stability of drugs should be evaluated in different preparation media. Because most of the time drug remains in contact with different solvents in preparation process and ultimately 42

3 Preformulation Studies comes into contact with body fluids. If drug shows any sign of degradation then formulation will not provide a desired therapeutic response. Compatibility study is very important stage of preformulation study, because selection of excipients and packaging components are decided on the basis of these studies. To get desired therapeutic response from any dosage form, drug and excipients should be selected very carefully. For development of drug loaded liposomes study of drug-excipients interaction is very critical. Compatibility study between drug and other excipients is necessary to find any possible interaction during manufacturing process or during storage. This kind of interaction may cause degradation of product/s or impurity in final dosage. Sometimes impurity may be toxic that results in unwanted side effects. 3.2 Identification of APIs Physical Appearance The physical appearance of the model drugs i.e. KP, 5-FU and OZ were noted by visual observation as well as by microscopy Melting Point The melting point of KP, 5-FU and OZ were determined in triplicate by placing the sample in sealed capillary which is heated using melting point apparatus till the melting of sample in the capillary and melting temperature was recorded using thermometer (EIE Instruments Pvt. Ltd., Ahmedabad, India) Ultraviolet (UV) Spectra of KP, 5-FU and OZ An accurately weighed amount of drug was dissolved in suitable solvent (phosphate buffer with ph 6.4) and the resultant solution was filtered through 0.45 μ filter (Himedia India) and scanned in the range of nm using UV spectrophotometer (UV 1800, Shimadzu, Tokyo, Japan). The absorption maxima for all the model drugs were similar to the reported absorption maxima which confirmed the identity of selected drugs. 43

4 Chapter Fourier Transformed Infra Red (FTIR) Spectra FTIR spectroscopy is considered as important analytical technique for identification of drug substance as FTIR spectra reveals characteristic peaks, indicating presence of different functional group in drug substance. FTIR spectra of model drugs i.e. KP, 5-FU and OZ were obtained by the method of KBr cell on a FTIR spectrometer (Shimadzu, Tokyo, Japan) with resolution of 2 cm -1. The results of these scans was presented as average data over the range cm -1 as spectra. The FTIR spectra of all the samples were compared with Pharmacopoeial standard spectra to confirm identity of compound. 3.3 Compatibility Study IR spectra were recorded in the wave number region from 4000 to 400 cm -1 using Shimadzu FTIR 8300 Spectrophotometer (Shimadzu, Tokyo, Japan). The procedure consisted of dispersing the sample, either drug alone, excipients (soyphosphatidyl choline (SPC) and/or cholesterol) alone or a physical mixture of drug and the excipients with potassium bromide ( mg) and compressing into discs by applying a 5 ton pressure for 5 min in a hydraulic press. The pellet was then placed in the light path and the spectrum was recorded. 3.4 UV Spectrophotometric Estimation Method for KP, 5-FU and OZ The estimation method of KP, 5-FU and OZ was developed in PBS ph 6.4 using UV spectrophotometric technique. Briefly, for preparation of stock solution, 50 mg drug was weighed and transferred into 100 ml volumetric flask and the volume was made by stated media. From this stock solution, different standard solutions of desired concentration range (μg/ml) were prepared. The absorbance of the standard solution was measured using UV spectrophotometer (UV 1800, Shimadzu, Tokyo, Japan) and the linearity plot of average absorbance (n=3) against KP, 5-FU and OZ concentration was established. 3.5 Result and Discussion Identification of API (a) Physical Appearance KP was found as a white odorless crystalline powder which was practically insoluble in water but freely soluble in ethanol, acetone, chloroform, ether and strong alkali. 44

5 Preformulation Studies Fig. 3.1: Chemical structures of KP 5-FU was found as white to nearly white crystalline powder; practically odorless. It was sparingly soluble in water and slightly soluble in alcohol. It was soluble in DMSO. Fig. 3.2: Chemical structures of 5-FU Gamma oryzanol was found white to off white powder. It was found soluble in acetone, heptane, ether, chloroform, benzene, ethanol (Slightly soluble), and water (very slightly soluble). OZ is a mixture of ferulate esters of different sterols. Important components of OZ are cycloartenyl ferulate, 24 methylene cycloartanyl ferulate and campesteryl ferulate. 45

6 Chapter 3 Fig. 3.3: Chemical structures of OZ components (Cho et al ) (b) Melting Point The melting point of KP, 5-FU and OZ were determined in triplicate and compared with the Pharmacopoeial reported melting point. Comparison of Pharmacopoeial reported melting point and practical observation is reported in Table 3.1. Table 3.1 Melting Point of Different Drugs Drug Observed Melting Point Reference Melting Point KP 94 C C 5-FU 282 C C OZ 135 ºC ºC (c) UV Spectra (i) UV Spectra of KP The UV absorption maxima (λmax) of KP (10 μg/ml) in PBS ph 6.4 was measured and compared with standard λmax as reported in official compendia i.e. Indian Pharmacopoeia 2007 and British Pharmacopoeia It was found at 260 nm. 46

7 Preformulation Studies Fig. 3.4: UV Absorption Maxima of KP (ii) UV Spectra of 5-FU The UV absorption maxima (λmax) of 5-FU (10 μg/ml) in PBS 6.4 was measured and compared with standard λmax as reported in official compendia i.e. Indian Pharmacopoeia 2007 and British Pharmacopoeia It was found at 266 nm. 47

8 Chapter 3 Fig. 3.5: UV Absorption Maxima of 5-FU (ii) UV Spectra of OZ The UV absorption maxima (λmax) of OZ (10 μg/ml) in PBS 6.4 was measured and compared with standard λmax as reported in official compendia i.e. Indian Pharmacopoeia 2007 and British Pharmacopoeia It was found at 327 nm. 48

9 Preformulation Studies Fig. 3.6: UV Absorption Maxima of OZ (d) FTIR Spectra (i) FTIR Spectra of KP The FTIR spectra of KP sample and reference are shown in figure 3.7 and 3.8 respectively. Characteristic peaks of KP were shown in the table 3.2. From this data it was concluded that the FTIR spectra of sample drug resembled with the reference spectra from official compendia i.e. Indian Pharmacopoeia 2010 which confirmed identity and purity of KP sample. Table 3.2 FTIR Bands of Characteristic Functional Groups of KP Wave Number (cm -1 ) Functional Group C=O stretching ketones O-H stretching carboxylic acid , C-O stretching phenolics , , C-H in plane bending Aromatic 49

10 Chapter 3 Fig. 3.7: FTIR Spectra of KP (Sample) Fig.: 3.8 FTIR Spectra of KP (Standard) 50

11 Preformulation Studies (ii) FTIR Spectra of 5-FU The FTIR spectra of 5-FU sample and reference are shown in figure 3.9 and 3.10 respectively. Characteristic peaks of 5-FU were shown in the table 3.3. From this data it was concluded that the FTIR spectra of sample drug resembled with the reference spectra from official compendia i.e. Indian Pharmacopoeia 2010 which confirmed identity and purity of 5-FU sample. Table 3.3 FTIR Bands of Characteristic Functional Groups of 5-FU Wave Number (cm -1 ) Functional Group N-H(Stretching) Free C=O(Stretch) C-N(stretch) C-N(stretch) C-O Fig. 3.9: FTIR Spectra of 5-FU (Sample) 51

12 Chapter 3 Fig. 3.10: FTIR Spectra of 5-FU (Standard) (iii) FTIR Spectra of OZ The FTIR spectra of OZ sample and reference are shown in figure 3.11 and 3.12 respectively. Characteristic peaks of OZ were shown in the table 3.4. From this data it was concluded that the FTIR spectra of sample drug resembled with the reference spectra which confirmed identity and purity of OZ sample. Table 3.4 FTIR Bands of Characteristic Functional Groups of OZ Wave Number (cm -1 ) Functional Group (stretching mode) O-H C-H C= C-C C-O 52

13 Preformulation Studies Fig. 3.11: FTIR Spectra of OZ (Sample) Fig. 3.12: FTIR Spectra of OZ (Standard) 53

14 Chapter Drug excipient compatibility studies: FTIR spectroscopy (i) For 5-FU As a part of the pre-formulation studies, 5-FU was evaluated for compatibility with other excipients, such as the lipids, cholesterol and KP that were intended to be used in the liposomal formulation using FTIR peak matching method. The FTIR spectra of the pure 5-FU and that of the physical mixture (1:1 ratio of 5-FU with the other excipients) were more or less the same with most of the standard 5-FU peaks evident in the spectra of the physical mixture Figure 3.13 A to E. The FTIR spectra of the pure 5-FU and that of the physical mixture (1:1 ratio of 5-FU with the other excipients) were more or less the same with most of the standard 5-FU peaks found also in the spectra of the physical mixture. It was revealed that no chemical interaction occurred between 5-FU and excipients because there was no appearance or disappearance of major peaks in the drug-lipid mixture was found. This study confirmed the compatibility of the drug with the excipients intended to be used in the formulation. Fig. 3.13A: FTIR spectrum of pure SPC 54

15 Preformulation Studies Fig. 3.13B: FTIR spectrum of pure cholesterol Fig. 3.13C: FTIR spectrum of pure KP 55

16 Chapter 3 Fig. 3.13D: FTIR spectrum of pure 5-FU Fig. 3.13E: FTIR spectrum of physical mixture of 5-FU and excipients 56

17 Preformulation Studies (ii) For OZ As a part of the pre-formulation studies, OZ was evaluated for compatibility with other excipients, such as the lipids, cholesterol and KP that were intended to be used in the liposomal formulation using FTIR peak matching method. The FTIR spectra of the pure OZ and that of the physical mixture (1:1 ratio of OZ with the other excipients) were more or less the same with most of the standard OZ peaks evident in the spectra of the physical mixture. However, Fig. 3.14E shows the minor shifting of some peaks in the physical mixture of OZ and excipients compared to pure drug (Fig. 3.14A) and pure excipients (Figs. 3.14B, 3.14C and 3.14D). These minor changes in shifts were due to presence of hydrogen bonds, or existence of some other weak forces like van der Waals forces or dipole moment between the polar functional groups of drug and phospholipid moieties. These factors and forces ultimately lead to formation of double layer sheet of phospholipid, provide stability with slower release of drug (Zhao and Feng ). The FTIR spectra of the pure OZ and that of the physical mixture (1:1 ratio of OZ with the other excipients) were more or less the same with most of the standard OZ peaks found also in the spectra of the physical mixture. It was revealed that no chemical interaction occurred between OZ and excipients because there was no appearance or disappearance of major peaks in the drug-lipid mixture was found. This study confirmed the compatibility of the drug with the excipients intended to be used in the formulation. Fig. 3.14A: FTIR spectrum of pure SPC 57

18 Chapter 3 Fig. 3.14B: FTIR spectrum of pure cholesterol Fig. 3.14C: FTIR spectrum of pure KP 58

19 Preformulation Studies Fig. 3.14D: FTIR spectrum of pure OZ Fig E: FTIR spectrum of physical mixture of OZ and excipients 59

20 Absorbance Chapter Estimation methods (i) Standard curve for KP The UV absorbance of KP standard solutions in the range of 1-10 μg/ml of drug in phosphate buffer ph 6.4 showed linearity at λmax 260 nm. The linearity was plotted for absorbance against concentration with R² value and with the slope equation Y = 0.048X The absorbance values and standard curve are shown in Table 3.5 and Figure Table 3.5: Spectrophotometric estimation of KP at 260 nm Sr.No Concentration (µ g/ml) Absorbance y = x R² = Concentration (ug/ml) Fig. 3.15: Standard curve of KP in phosphate buffer ph 6.4 at 260 nm 60

21 Absorbance Preformulation Studies (ii) Standard curve for 5-FU The UV absorbance of 5-FU standard solutions in the range of 2-20 μg/ml of drug in phosphate buffer ph 6.4 showed linearity at λmax 266 nm. The linearity was plotted for absorbance against concentration with R² value and with the slope equation Y = 0.070X The absorbance values and standard curve are shown in Table 3.6 and Fig Table 3.6: Spectrophotometric estimation of 5-FU at 266 nm Sr.No Concentration (µg/ml) Absorbance y = x R² = Concentration (ug/ml) Fig. 3.16: Standard curve of 5-FU in phosphate buffer ph 6.4 at 266 nm 61

22 Absorbance Chapter 3 (iii) Standard curve for OZ The UV absorbance of OZ standard solutions in the range of 2-20 μg/ml of drug in phosphate buffer ph 6.4 showed linearity at λmax 327 nm. Isoprpyl alcohol was used as co-solvent. The linearity was plotted for absorbance against concentration with R² value and with the slope equation Y = X The absorbance values and standard curve are shown in Table 3.7 and Figure Table 3.7: Spectrophotometric estimation of OZ at 327 nm Sr.No Concentration (µg/ml) Absorbance y = x R² = Concentration (ug/ml) Fig. 3.17: Standard curve of OZ in phosphate buffer ph 6.4 at 327 nm 62