1.

INTRODUCTION

1.1CANCER Cancer known medically as a malignant neoplasm, is a broad group of various diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors and invade nearby parts of the body. The cancer may also spread to more distant parts of the body through lymphatic system or bloodstream or we can say thatCancer is an abnormal growth of cells caused by multiple changes in gene expression leading to dysregulated balance of cell proliferation and cell death and ultimately evolving into a population of cells that can invade tissues and metastasize to distant sites, causing significant morbidity and, if untreated, death of the host. 1.1 Causes of Cancer Many things are known to increase the risk of cancer, including tobacco use, certain infection, and radiation, lack of physical activity, obesity and environmental pollutants. These can combine with existing genetic faults within cells to cause the disease. A. Chemicals:-Substances that cause DNA mutations are known as mutagens, and mutagens that cause cancers are known as carcinogens. Particular substances have been linked to specific types of cancer. Tobacco smoking is associated with many forms of cancer, and causes 90% of lung cancer. Many mutagens are also carcinogens, but some carcinogens are not mutagens. Alcohol is an example of a chemical carcinogen that is not a mutagen. In Western Europe 10% of cancers in males and 3% of cancers in females are attributed to alcohol. B. Diet and exercise:-Diet, physical inactivity, and obesity are related to approximately 30 35% of cancer deaths.] Physical inactivity is believed to contribute to cancer risk not only through its effect on body weight but also through negative effects on immune system and endocrine system.[20] More than half of the effect from diet is due to overnutrition rather than from eating too few healthful foods.Diets that are very low in vegetables, fruits and whole grains, and high in processed or red meats are linked with a number of cancers. A
1

high-salt diet is linked to gastric cancer, aflatoxin B1, a frequent food contaminate, with liver cancer, and Betel nut chewing with oral cancer. This may partly explain differences in cancer incidence in different countries. For example, gastric cancer is more common in Japan due to its high-salt diet. C. Infection:-Approximately 18% of cancer deaths are related to infectious diseases.A virus that can cause cancer is called an oncovirus. These include human papillomavirus (cervical carcinoma), EpsteinBarr virus (B-cell lymphoproliferative disease and nasopharyngeal carcinoma), Kaposi's sarcoma herpesvirus (Kaposi's sarcoma and primary effusion lymphomas), hepatitis B and hepatitis C viruses (hepatocellular carcinoma), and Human Tcell leukemia virus-1 (T-cell leukemias). Bacterial infection may also increase the risk of cancer, as seen in Helicobacter pylori-induced gastric carcinoma.[24] Parasitic infections strongly associated with cancer include Schistosomahaematobium (squamous cell carcinoma of the bladder) and the liver flukes, Opisthorchisviverrini and Clonorchissinensis (cholangiocarcinoma). D. Radiation:- 10% of invasive cancers are related to radiation exposure, including both ionizing radiation and non-ionizing ultraviolet radiation. Additionally, the vast majority of non-invasive cancers are non-melanoma skin cancers caused by non-ionizing ultraviolet radiation.Sources of ionizing radiation include medical imaging, and radon gas. Radiation can cause cancer in most parts of the body, in all animals, and at any age, although radiationinduced solid tumors usually take 1015 years, and can take up to 40 years, to become clinically manifest, and radiation-induced leukemias typically require 210 years to appear. Some people, such as those with nevoid basal cell carcinoma syndrome or retinoblastoma, are more susceptible than average to developing cancer from radiation exposure. Children and adolescents are twice as likely to develop radiation-induced leukemia as adults; radiation exposure before birth has ten times the effect. Ionizing radiation is not a particularly strong mutagen. Residential exposure to radon gas, for example, has similar cancer risks as passive smoking.Ionizing radiation may be used to treat other cancers, but this may, in some cases, induce a second form of cancer. It is also used in some kinds of medical imaging.Nonionizing radio frequency radiations from mobile phones, electric power transmission, and other similar sources have been described as a possible carcinogen by the World Health

Organization's International Agency for Research on Cancer. However, studies have not found a consistent link between cell phone radiation and cancer risk. E. Heredity:-Hereditary cancers are primarily caused by an inherited genetic defect. Less than 0.3% of the populations are carriers of a genetic mutation which has a large effect on cancer risk and these causes less than 310% of all cancer. Some of these syndromes include: certain inherited mutations in the genes BRCA1 and BRCA2 with a more than 75% risk of breast cancer and ovarian cancer, and hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome) which is present in about 3% of people with colorectal cancer among others. F. Physical agents:-A prominent example of this is prolonged exposure to asbestos, naturally occurring mineral fibers which are a major cause of mesothelioma, which is a cancer of the serous membrane, usually the serous membrane surrounding the lungs. Other substances in this category, including both naturally occurring and synthetic asbestos-like fibers such as wollastonite, attapulgite, glass wool, and rock wool, are believed to have similar effects. Nonfibrous particulate materials that cause cancer include powdered metallic cobalt and nickel, and crystalline silica (quartz, cristobalite, and tridymite). Usually, physical carcinogens must get inside the body (such as through inhaling tiny pieces) and require years of exposure to develop cancer. Physical trauma resulting in cancer is relatively rare. Claims that breaking bone resulted in bone cancer, for example, have never been proven. Similarly, physical trauma is not accepted as a cause for cervical cancer, breast cancer, or brain cancer. G. Hormones:-Some hormones play a role in the development of cancer by promoting cell proliferation. Insulin-like growth factors and their binding proteins play a key role in cancer cell proliferation, differentiation and apoptosis, suggesting possible involvement in carcinogenesis.Hormones are important agents in sex-related cancers such as cancer of the Osteosarcoma may be promoted by growth hormones. Some treatments and prevention approaches leverage this cause by artificially reducing hormone levels, and thus discouraging hormone-sensitive cancersbreast, endometrium, prostate, ovary, and testis, and also of thyroid cancer and bone cancers.

1.3 Facts related to cancer 1. Several factors increase the risk of cancer (officially known as malignant neoplasm), including pollutants, tobacco use, certain infections, radiation, obesity, and lack of physical exercise. 2. An estimated 5 to 10% of cancers are entirely hereditary. Most cancers develop through a combination of hereditary and environmental factors. 3. Smoking causes an estimated 90% of lung cancer. Tobacco has killed 50 million people in the last decade. If trends continue, a billion people will die from tobacco use and exposure this century, which equates to one person every six seconds. 4. Those who sleep less than six hours a night are more likely to develop colon cancer than those who sleep more. 5. Cancer has two main characteristics: abnormal cell growth and the ability to spread to other parts of the body (metastasis). 1.4 Diagnosis of Cancer Most cancers are initially recognized either because of the appearance of signs or symptoms or through screening. Neither of these lead to a definitive diagnosis, which requires the examination of a tissue sample by a pathologist. People with suspected cancer are investigated with medical tests. These commonly include blood tests, X-rays, CT scans and endoscopy. These are done by radiolabelled substances like Tc-99m pertechnate. 1.5 Treatment

1. Surgery:-Surgery is the primary method of treatment of most isolated solid cancers and may play a role in palliation and prolongation of survival. It is typically an important part of making the definitive diagnosis and staging the tumor as biopsies are usually required. In localized cancer surgery typically attempts to remove the entire mass along with, in certain cases, the lymph nodes in the area. For some types of cancer this is all that is needed to eliminate the cancer.
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2. Chemotherapy:-Chemotherapy in addition to surgery has proven useful in a number of different cancer types including: breast cancer, colorectal cancer, pancreatic cancer, osteogenic sarcoma, testicular cancer, ovarian cancer, and certain lung cancers. The effectiveness of chemotherapy is often limited by toxicity to other tissues in the body. 3. Radiation:-Radiation therapy involves the use of ionizing radiation in an attempt to either cure or improve the symptoms of cancer. It is used in about half of all cases and the radiation can be from either internal sources in the form of brachytherapy or external sources. Radiation is typically used in addition to surgery and or chemotherapy but for certain types of cancer such as early head and neck cancer may be used alone. For painful bone metastasis it has been found to be effective in about 70% of people.

CHAPTER-2 LITERATURE SURVEY

2 .LITERATURE SURVEY
2.1 99mTc-Labeled Radiopharmaceuticals
The basic principle of 99mTc-labeling involves reduction of 99mTc7 to an oxidation state that binds to a chelating molecule of interest. In most cases, kits for 99mTc-radiopharmaceuticals are commercially available for routine clinical use. These kits contain the chelating agent of interest and the reducing agent in appropriate quantities. In some kits, suitable stabilizers are added. Limits of volume and activity of Tc-99m that can be added to specific kit vials and expiration time are given in the package inserts provided by the manufacturer. For most 99mTc-radiopharmaceuticals, the expiration time is 6 hr, equal to the physical half-life of 99mTc (t1=2 6 hr). Also included in the package inserts are storage temperatures for the kits before and after the formulation with Tc-99m.(Nuclear Medicine in the new millennium, The Journal of Nuclear Medicine, Jan 2000, 41, 1, 1-4)

2.2 Factors influencing the designing of radiopharmaceuticals :

The following factors need to be considered before, during and after the preparation of a new radiopharmaceutical:1.Compatibility: When a labelled compound is to be prepared, the first criterion to consider is whether the label can be incorporated into the molecule to be labelled. This may be assessed from the knowledge of the chemical properties of the two partners. For example, Tc-99mcan form coordinate covalent bonds, and MDP is a chelating compound containing nitrogen and oxygen atoms with lone pairs of electrons that can be donated to form coordinate bonds. Therefore, when Tc-99mand MDP are mixed together under appropriate physicochemical conditions Tc-99mMDP is formed and remains stable for considerable period of time.
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2. Stoichiometry:In preparing a new radiopharmaceutical, one needs to know the amount of each component to be added. This is particularly important in tracer level chemistry and in Tc-99m chemistry. The concentration of Tc-99min the Tc-99melute is approximately 10-9 M. Although for the reduction of this trace amount of Tc-99monly an equivalent amount of Sn++ is needed ,1000 to 1 million times more of the latter is added to the preparation in order to ensure complete reduction .Similarly ,enough chelating agent such as MDP or Methylene diphosphonate (MDP) , is also added to use all the reduced Tc-99m. An unduly high or low concentration of anyone component may sometime affect the integrity of the preparation. 3. Charge of the molecule: The charge on a radiopharmaceutical determines its solubility in various solvents. The greater the charge, the higher the solubility in aqueous solution. Nonpolar molecules tend to be more soluble in organic solvents and lipids. 4. Size of the molecule: The molecular size of the radiopharmaceutical is an important determinant in its absorption in the biological system. Larger molecule (>~60,000) are not filtered by the glomeruli in the kidney. This information should give some clues as to the range of the molecular weights of the desired radiopharmaceutical that should be chosen for a given study. 5. Protein binding: Almost all drugs, radioactive ornot, bind to plasma protein to variable degrees. The primary candidate for this type of binding is albumin, although many compounds specifically bind to globulins and other proteins as well. Protein binding is greatly influenced by a number of factors, such as the charge on the radiopharmaceutical molecule, the pH,the nature of the protein and the concentration of anions in the plasma. At a lower, pH plasma proteins become more positively charged and therefore anionic drugs bind firmly to them. The nature of a protein, particularly its content of hydroxyl, carbonyl and amino groups and their configuration in the protein structure, determine the strength and extent of its binding to the radiopharmaceutical. Metal chelates can exchange the metal ions with proteins because of the stronger affinity for the metal; such a process is called transchelation and leads to in vivo breakdown of chelates. Protein binding affects the tissue distribution and plasma clearance of the radiopharmaceutical and its uptake by the organ of interest. Therefore determination of extent of plasma protein binding for a new radiopharmaceutical is very important. This can be accomplished by precipitating the protein

with trichloroacetic acid (TCA) from the plasma after administration of radiopharmaceutical and then measuring the activity in the precipitate. 6. Solubility: For injection, radiopharmaceutical should be in an aqueous solution at a pH compatible with blood pH(7.4). The ionic strength and osmolality of the agent should also be appropriate for the blood. In many cases, lipid solubility of a radiopharmaceutical is a determining factor in its localization in an organ:the cell membrane is primarily composed of phospholipids and unless the radiopharmaceutical is lipid soluble, it will hardly diffuse through the cell membrane. The higher the lipid solubility of the radiopharmaceutical, the greater the diffusion through the cell membrane and hence greater the localization in the organ. Protein binding reduces the lipid solubility of the radiopharmaceutical. Ionised drugs are less lipid soluble whereas non-polar drugs are highly soluble in lipid and hence easily diffuse through the cell membrane. Obviously lipid solubility and protein binding of any drugs play a key role in its in vivo distribution and localization. 7. Stability:The stability of a compound is one of the major problems in labelling chemistry. It must be stable both in vivo and in vitro. Temperature, pH and light affect the stability of many compounds and the optimal range of these physicochemical conditions must be established for the preparation and storage of labelled compounds. In vivo breakdown of a radiopharmaceutical results in undesirable biodistribution of radioactivity. 8. Biodistribution: The study of biodistribution of a radiopharmaceutical is most essential in establishing its efficacy and usefulness .This includes tissue distribution, urinary excretion and faecal excretion after administration of radiopharmaceutical . In tissue distribution studies, the radiopharmaceutical is injected into the animals such as mice and rats. The animals are then sacrificed at different time intervals, and different organs are removed. The activities in these organs are measured and compared. The tissue distribution data tell how good the radiopharmaceutical is for imaging the organ of interest .The rate of localization of radiopharmaceutical in an organ is related to its rate of plasma clearance after administration. Urinary and faecal excretions of radiopharmaceutical are important in clinical evaluation. The faster the urinary and faecal excretion, the less the radiation dose. These values can be determined by collecting the urine or faeces at definite time intervals after injection and measuring the activity in the samples.(Fundamentals of nuclear pharmacy, Sixth Edition, Gopal B. Saha).
9

2.3Tc-99mas the label of choice:The reason for such a pre-eminent position of Tc-99min clinical use is its extremely favorable physical and radiation characteristics. Nearly 80% of all the currently used

radiopharmaceuticals are labelled with Tc-99m. The 6.02 hr physical half-life and being a pure -emitter permits the administration of mci amounts of Tc-99mradioactivity without a significant radiation dose to the patient. In other words, technetium shorter physical half-life permits the use of a higher administered dose, which translates to a higher count rate, which will shorten the imaging time. The gamma energy of Tc-99mis optimal for the detector crystal used in the gamma camera. Tc-99mis also readily available and produced daily from a molybdenum generator in most nuclear medicine canters. Moreover, this generator eluted radioisotope is easy to obtain at very cost. The combination of ready availability, excellent imaging properties, favourable dosimetry and high specific activity make Tc-99m a logical choice for labelling (Development of kits for Tc-99m radiopharmaceuticals for infection imaging, IAEA report 2000-2003).

2.4Generation and decay of Tc-99m

Tc-99m is available through the decay of Mo-99. The radionuclide Mo-99 has a high life of 67 hr and decays by emission, 87% of its decay goes ultimately to the metastable state Tc 99m and the remaining 13% to the ground state Tc-99. It has photon transitions of 740 and 780 keV. The radionuclide Tc-99mhas a half-life of 6.02 hr and decays to Tc-99 by isomeric transition or gamma transition of 140 keV. Approximately 10% of these transmissions are via internal conversion. The ground state Tc-99 has half-life of 2.1*105 years and decays to stable Ru-99 by emission. Only 3.7 *10-3% of the decay of Tc-99mleads under -emission directly to stable Ru-99. Because the half-life of Mo-99 and Tc-99mdiffer by a factor of about 11, these two radionuclides tend themselves to the construction of a useful generator (Development of kits for Tc-99m radiopharmaceuticals for infection imaging, IAEA report 2000-2003).

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2.5Reduction of Technetium: a prerequisite for radiolabelling

The chemical form of technetium available from the moly generator is sodium pertechnetate (NaTc99m-04-) is rather a non-reactive species and does not label any compound by direct addition. In Tc-99mlabelling of many compounds, prior reduction of Tc-99mfrom the 7+ state to a lower valence state (3,4 or 5) is required. Various reduction systems that have been used are stannous chloride (SnCl2.2H2O), stannous citrate, stannous tartrate, concentrated Hcl, sodium borohydride (NaBH4), dithionite and ferrous sulphate .Among these stannous chloride in acidic medium is the most commonly used reducing agent in most preparations of Tc-99mlabelled compounds. Stannous salts are preferred due to preparations of Tc99mlabelled compounds. Stannous salts are preferred due to their water solubility, stability, effectiveness at room temperature and low toxicity. The chemical reactions that occur in the reaction of technetium by stannous chloride in the acidic medium can be stated as follows: 3 Sn2+ 3 Sn4+ +6 e2 Tc-99m4- +16 H+ + 6 e-2 Tc-99m4+ + 3 Sn4+ + 8 H2O The above equation indicates that Tc-99m7+ has been reduced to Tc-99m4+. Other reduced states such as Tc-99m3+ and Tc-99m5+ may be formed under different physiochemical conditions. The amount of Tc-99matoms in the Tc-99meluate is very small (~10-9M) and therefore only a minimal amount of Sn2+ is required for reduction of such a small quantity of Tc-99; however enough Sn2+ is added to ensure the complete reduction. The ratio of Sn2+ ions to Tc-99matoms may be as large as 106.

The reduced Tc-99mspecies are chemically reactive and combine with a wide variety of chelating compounds. A schematic reaction would be: Reduced Tc-99m+ chelating agent Tc-99m-chelate (Nuclear Imaging methods for non-invasive drug monitoring. Advanced Drug
11

Delivery, BhatnagarAnish, Hustnix Roland, Alavi Abass, 2000, 41, 41-54).

12

CHAPTER-3 AIMS AND OBJECTIVES

13

AIMS AND OBJECTIVES

1. Evaluation of given drug formulation using Tc-99m as a radiotracer. 2. Determination of labelling efficiency and its stability study. 3. Lipophilicity and serum protein binding. 4. Blood kinetics in rabbits. 5. Isolation of murine peritoneal macrophage 6. Extraction of peritoneal macrophage by histopaque 1077. 7. Biodistribution study in tumor bearing mice. 8. Scintigraphy

14

CHAPTER-4 MATERIALS AND METHODS

15

4.1 MATERIALS AND EQUIPMENTS

1. Analytical weighing balance 2. Micropipettes(10,000 & 1000~t1) 3. Sterile disposable syringes(1,2& 5 ml) 4. Centrifuge 5. Vortex mixer 6. Dose calibrator 7. Millipore filter 8. Gamma Counter 9. Gamma camera 10. Macrophage media 11. Syringes 5ml, 10ml 12. 23-G needle 13. Eppendrof 5810R

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4.2 CHEMICALS REQUIRED

1. Drug formulation INM-08 2. Stannous chloride 3. pH strips 4 .ITLC-SG strips Sigma Glaxo Gellman, USA Sd fine chemicals ltd Merck, India Merck, India Qualigens Analar, glaxo laboratories Merck, India Merck, India Sigma Brit. Sd fine chemicals

4. Acetone 5. Sodium hydroxide 6. Sodium chloride 7. Sodium bicarbonate 8. Acetic acid 9. Trichloroacetic acid 10. Dichloromethane 11. Pyridine 12. Tc-99mpertechnate 13. Chloroform limited 14. Isoflurane 15. Trypan blue 16. Histopaque 1077

17.Brewer thioglycollate medium

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4.3METHODS
The given drug was labelled with Tc-99mby simple reduction method using Sncl2.2H2O as the reducing agent. 1-1.5 mg of drug in 1.0ml of distilled water, and 100g of Sncl2.2H2O was added. The ph of the solution was 6.5. The contents were filtered through 200nm membrane Millipore filter. Subsequently 0.5 ml Tc-99m-pertechnate solution(2mCi) was added to the filtrate, shaken gently and incubated at room temperature for 15 min. Then the complex was subjected to quality control.

Quality control parameters

Evaluation of radiolabelling efficiency: The radiolabelling efficiency was evaluated using silica gel impregnated instant thin layer chromatography (ITLC-SG) strips as stationary phase and 100% acetone and 0.9% Nacl as the mobile phases. The ITLC-SG strips were cut into narrow strips of 1 cm width and 10 cm length. 2l sample was applied on one end of the strip, 1cm above the base. This strip was then allowed to run in the solvent chamber containing 100% acetone/0.9% Nacl in ascending mode. When the solvent migrated about 2/3rd of the strip length the experiment was discontinued and strips were removed and air dried. This was followed by cutting the strip into two halves and each half was counted for the radioactivity retained in it. The free technetium moves with the solvent front in both the solvents and the reduced or hydrolysed Tc, if any, along with the labelled stays at the base. The radioactive count left with lower half of the strip w.r.t the total count thus provided the estimate of the %age of drug that was added. This procedure was repeated at time intervals of 1hr, 2hr, 4hr and 24hr for the determination of in vitro Tc-99mdrug stability.

Lipophilicity: The lipophilicity of the labelled drug was measured by experimental evaluation of its organic/aqueous partition coefficient. An aliquot of 0.1ml of the labelled complex was mixed with 1.9ml of physiological saline and 2ml of distribution equilibrium. The two layers were allowed to separate by leaving the contents undisturbed for 15mins at

18

room temperature. Both the fractions were collected separately and radioactivity was measured in 10 111 of each fraction.

%lipophilicity = counts in organic (lower)fraction * 100 Total counts

Serum protein binding: In vitro serum protein binding of Tc-99mdrug was carried out in human serum by protein precipitation with 10% trichloroacetic acid (TCA). To 900l of fresh human serum, 100l of the labelled complex was added, mixed and incubated for 1hr at 370C. After adding approximately equal volume of 10% TCA the mixture was centrifuged at 3000rpm for 5 minutes. The supernatant was collected, the pellet resuspended in 1ml of 5% TCA(w/v), centrifuged as earlier and the supernatant was collected in the same tube during each washing. The pellet was then dissolved in 1% NAOH solution to make the volume equal to the supernatant fraction. Radioactivity was measured in both the precipitate and the supernatant fractions in a well type gamma counter. Protein binding of the complex was expressed as the fraction of radioactivity associated with protein (pellet) with reference to the total radioactivity.

%protein binding = counts in pellet fraction*100 Total counts

Its protein binding was found to be 37-39%. Blood kinetics: The blood clearance study was performed in three rabbits weighing about 2.5kg after administration 300l of the labelled product into the dorsal ear vein of each rabbit. At different time intervals blood samples from the veins of the other ear of the animal were withdrawn. The samples were weighed and radioactivity was measured using a gamma counter that was calibrated for Tc-99menergy. The data was expressed as percent
19

administered dose at each time point considering the whole body blood as 7% of the body weight. 2l of the sample was spotted on the ITLC-SG strip at each time point and run in acetone to check the in vivo stability of the complex. Macrophage: These are the type of WBC that are part of body defence mechanism and made in bone marrow, distributed throughout the body. When a foreign invader like bacteria enter blood stream, macrophage secrete certain substance in a battle to help kill the bacteria i.e when a macrophage encounters something foreign it tends to surrounds it and destroy it. Histopaque 1077: Solution of polysucrose and sodium diatrizote adjusted to a density of 1.077+-0.001g/ml. Suitable for multiple purposes after isolation including studing cellmediated lympholysis. During centrifugation erythrocytes and granulocytes are aggregated by polysucrose and other mononuclear cells remain at the interface. Trypan Blue: Vital stain used to selectively color dead tissues or cells blue. Based on the principles that live cells possess intact cell membrane that excludes certain dyes such as trypan blue, Eosin or propidium whereas dead cells do not. Reactivity of trypan blue is based on the fact that the chromopore is negatively charged and does not interact with the cells unless the membrane is damaged. Therefore, all the cells exclude the dye are viable. Biodistribution: Biodistribution of the radiolabelled was carried out in tumor bearing mice. 100l of the complex was injected intravenously into the tail vein of each mouse. The mice were sacrificed at1hr, 4hr, and 24hr post administration of the complex. Various organs were removed, made free from adhering tissue and weighed. The radioactivity was measured in each organ and expressed as percent injected dose per whole organ.

% injected dose = counts in kcpm * 100 Weight of organ * total standard count injected Total standard count injected = (standard count * dilution factor) - tail count

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 Scintigraphy:The tumor bearing mice were administered 100l of the labelled complex intravenously through the tail vein and imaged at different time intervals under a gamma camera. Isolation of murine peritoneal macrophage Procedure: 1. a For resident peritoneal cells: euthanize untreated mice by rapid cervical dislocation. Euthanasia should be performed by skilled technicians to avoid excessive bleeding into the peritoneal cavity. Co2 euthanasia can also be used with good effect. 1.b For elicited peritoneal cells:- fill a 5ml syringe with 3% brewer thioglycollate medium. Attach 23-G needle and inject 1ml of the solution /mouse into the peritoneal cavity. Be sure to avoid the bladder. Allow inflammatory response to proceed for 4 days, then euthanize in step 1 a. 2. Soak the abdomen of each mouse with 70% alcohol and then make a small incision along the midline with sterile scissors. Retract the abdominal skin manually to expose the intact peritoneal wall. 3. Fill a 10ml syringe with cold harvest medium with the bevelled end of a 20-G needle

facing inward, insert needle through peritoneal wall along the mouse left side (spleen side) and inject 10ml of the cold harvest medium into each mouse. 4. Using same syringe and needle, aspirate fluid from peritoneum. Move needle away from the viscera to cause tenting of the peritoneal wall and withdraw peritoneal fluid slowly. Expect fluid recovery of ~8ml/mice. 5. Remove needle from syringes and dispense peritoneal fluid into a 50ml conical

polypropylene centrifuge tube on ice. Cells should be kept cold throughout the procedure. 3-5 mice can be euthanized at the same time and the procedure is performed sequentially using 35 10ml syringes. For large scale isolation, several successive repetition of this procedure can be performed using same syringe and needles.

(~1000 rpm in eppendrof 5810R), 40C. Discard supernatant and resuspend cell pellet in cold
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DMEM/F-12 by gently tapping the bottom of the tube and pipetting up and down. A good general rule of thumb is to resuspend macrophages in a volume of 1ml/5 mice. 7. Count cells by hemacytometer:-1st diluting 20l of cell. Suspension into 180l of

DMEM/F-12(final dilution of 10x) and then applying 10l to a hemacytometer. 8. Adjust cell concentration in harvest medium to ~1-

keep on ice.

Macrophage extraction by using histopaque 1077 Procedure:1. Obtain all material autoclave instruments. Prepare media as specified and keep at

370C. Obtain mice. 2. Fill 10ml syringes with sterile PBS 1x and cap with 20 gauge needles. Mark each

mice with identification. Place on ice. NOTE:-Aliquot PBS into a 50ml falcon tube when filling syringes, so there is less change of contaminationof entire PBS bottles. Fill syringes from 50ml falcon tube. 3. Anesthetize mouse in anesthetizing chamber:Add a small amount of isoflurane to tissue in bottom of chamber. Place mice in chamber. Watch for breathing of mice and take out when it slows down. If mice die from

anesthetizing in this step, it will not a problem. 4. Euthanize mouse by cervical dislocation. Hold mice by nape of neck firmly and pull

tail with other hand until feel a pop and a separation between cranium and backbone. 5. Sterilize mice:Place mice in a beaker with 70% ethanol.
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 6.

Place mice in tissue culture hood immediately. Laporotomy incision:Make a small incision in abdominal skin of mice(use 1 set of forceps and scissors). Open up mice by pulling skin with both hands to each side of mice. Do this firmly so

that skin is completely pulled down, but gently so that not open up intraperitoneal cavity or break any limbs. Spray peritoneum with 70% ethanol. 7. Inject cold PBS:Uncap needle inside hood and squirt out a small amount of PBS to get rid of any

bubbles. Inject needle into the mice in lower abdominal area, preferably in a region where

there is fat. Insert needle gently and not too deeply so do not perforate any organs. Immediately after inserting the needle, begin injecting PBS into intraperitoneal cavity

forcefully. Take needle out slowly so that fat not clog the hole (if take out too fast the PBS will

come out of the hole). 8. Wash mice:Hold mice by tail and swish around to wash IP cavity with the PBS that has been

injected 9. Macrophage extraction:Using same syringe, go to upper part of abdomen and insert needle horizontally. Make sure do not perforate any organs. Hold needle inside peritoneal cavity in a horizontal position with the needle hole- side

down and extract the PBS containing macrophages. Do this slowly, if done too fast then aspirate fat and clog needle.
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Try to get at least 7ml of fluid. If needle insert again in another part lifting a tent and extracting the fluid. Cap needle

again and place back on ice. Repeat this process for each mice. 10. Now add histopaque:To a 15 ml conical cfgtube,add 3.0 ml histopaque-1077 and bring to room

temperature. Carefully layer 3.0 ml whole blood onto the histopaque-1077.cfg at 400*g for exactly

30 min at room temp.cfg at lower temperature, such as 40c,may result in cell clumping and poor recovery. After cfg carefully aspirate with a Pasteur pipet the upper layer to within 0.5 cm of the

opaque interface containing mononuclear cells. Discard upper layer. tube. Add to this tube 10ml isotonic phosphate buffered saline solution and mix by gentle Carefully transfer the opaque interface, with a Pasteur pipet into a clean conical cfg

aspiration. 11. 12. slide. Cfg at 250*g for 10 min. Aspirate the supernatant and discard. Add the supernatant microscopic slide. Then put a drop of trypan blue on one of the slide and do not add this dye to the other

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fig 3:- absence of macrophage

fig 4:-presence of macrophage

fig 5:- interaction between liposomes and macrophage

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BIODISTRIBUTION STUDIES:

Preparation of Tc-99m labeled drug formulation was administered to patient animals sacrificed at different time intervals. Activity is measured by gamma counter. Plot of % radioactivity Vs organ gives the tissue distribution chart of labelled formulation.

ORGAN Blood 1.32 Heart 0.09 Lungs 1.74 Liver 10.99 Spleen Kidney Stomach Intestine Tumor Muscle

% I.D per gram organ at 4h pi

2.09 0.51 0.04 0.09 0.86 0.05

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BIODISTRIBUTION STUDIES:

Series 1
12 10 8 6 4 2 0 Series 1

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SCINTIGRAPHY: Animal with radiolabeled drug was imaged at specific interval at gamma camera. The scintigraphy is shown below:

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CHAPTER-5 RESULT AND DISCUSSION

29

5. RESULT AND DISCUSSION:

5.1Determination of labelling efficiency: Various parameters were optimized for the radiolabelling of formulation INM-08 with Tc99m. We varied one parameter at a time and keeping other factors constant. The parameters affecting labelling efficiency are:1. SnCl2 2. pH 3. Incubation time The first parameter The concentration of SnCl2 is varied and measured for the % labelling efficiency.

pH 5.5 6 6.5 7 7.5 8 86.7 89.1 95.5 98.7 96.7 91

% L.E

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100 98 96 94 92 90 88 86 84 82 80 5.5 6 6.5 7 7.5 8

pH Fig 1:- shows graph between pH and Labelling efficiency.

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SnCl2 50 75 100 125 150 175

%L.E 81.3 91.4 98.4 86.7 81.4 78

120

100

80

60

40

20

0 50 75 100 125 150 175

SnCl2 Fig:- shows variation to see labelling efficiency.

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Blood kinetics: Blood clearance kinetics was performed in albino New Zealand rabbits. Drug formulation was radiolabelled with Tc-99m as explained before 0.2ml of the radiolabelled formulation was injected into the dorsal auditory vein of left ear and care was taken that rabbit did not bleed. After the interval of 15min, 30min, 1hr, 2hr, 4hr, 6hr, 24hr from the time of injection blood was withdrawn. COUNTS WERE MEASURED WITH THE GAMMA COUNTER.

33

Vary to see the labelling efficiency.

SnCl2 50 75 100 125 150 175

% L.E 81.3 91.4 98.4 86.7 81.4 78

Incubation time (Hr) 0 86.7

%L.E

0.25 92.6 0.5 98.5

Thus from the above data we can say that labelling efficiency is maximum when pH is 7, concentration of SnCl2 is 100g/l and incubation time is around half an hour. Therefore the drug is radiolabelled and optimized for the excellent labelling efficiency with the above set parameters.

34

After determination of labelling efficiency of the radiolabel, the in-vivo stability of the complex was checked up to 24 hrs.

Time (Hr) 0.5 1 2 4 6 24

%L.E 99 98.7 97.6 96.4 96.1 95.1

Blood kinetics: Blood clearance kinetics was performed in albino New Zealand rabbits. Drug formulation was radiolabelled with Tc-99m as explained before 0.2ml of the radiolabelled formulation was injected into the dorsal auditory vein of left ear and care was taken that rabbit did not bleed. After the interval of 15min, 30min, 1hr, 2hr, 4hr, 6hr, 24hr from the time of injection blood was withdrawn. COUNTS WERE MEASURED WITH THE GAMMA COUNTER. Plot of % radioactivity Vs time pharmacokinetic parameters was drawn as shown.

35

Time (hr) 0.25 0.5 1 2 4 6 24

% radioactivity 74.5 46.9 34.8 28.7 18.1 14 4.9

Isolation of murine peritoneal macrophage Procedure:

1. a For resident peritoneal cells: euthanize untreated mice by rapid cervical dislocation. Euthanasia should be performed by skilled technicians to avoid excessive bleeding into the peritoneal cavity. Co2 euthanasia can also be used with good effect. 1.b For elicited peritoneal cells:- fill a 5ml syringe with 3% brewer thioglycollate medium. Attach 23-G needle and inject 1ml of the solution /mouse into the peritoneal cavity. Be sure to avoid the bladder. Allow inflammatory response to proceed for 4 days, then euthanize in step 1 a. 2. Soak the abdomen of each mouse with 70% alcohol and then make a small incision along the midline with sterile scissors. Retract the abdominal skin manually to expose the intact peritoneal wall.

36

3.

Fill a 10ml syringe with cold harvest medium with the bevelled end of a 20-G needle

facing inward, insert needle through peritoneal wall along the mouse left side (spleen side) and inject 10ml of the cold harvest medium into each mouse. 4. Using same syringe and needle, aspirate fluid from peritoneum. Move needle away from the viscera to cause tenting of the peritoneal wall and withdraw peritoneal fluid slowly. Expect fluid recovery of ~8ml/mice. 5. Remove needle from syringes and dispense peritoneal fluid into a 50ml conical

polypropylene centrifuge tube on ice. Cells should be kept cold throughout the procedure. 3-5 mice can be euthanized at the same time and the procedure is performed sequentially using 35 10ml syringes. For large scale isolation, several successive repetition of this procedure can be performed using same syringe and needles. 6. Centrifuge the peritoneal exudate cells (PEC) in a refrigerated centrifuge 10m (~1000 rpm in eppendrof 5810R), 40C. Discard supernatant and resuspend cell pellet in cold DMEM/F-12 by gently tapping the bottom of the tube and pipetting up and down. A good general rule of thumb is to resuspend macrophages in a volume of 1ml/5 mice. 7. Count cells by hemacytometer:-1st diluting 20l of cell. Suspension into 180l of

DMEM/F-12(final dilution of 10x) and then applying 10l to a hemacytometer. 8. Adjust cell concentration in harvest medium to ~1s/ml and

keep on ice. Macrophage extraction by using histopaque 1077 Procedure:1. Obtain all material autoclave instruments. Prepare media as specified and keep at

370C. Obtain mice. 2. Fill 10ml syringes with sterile PBS 1x and cap with 20 gauge needles. Mark each

mice with identification. Place on ice. NOTE:-Aliquot PBS into a 50ml falcon tube when filling syringes, so there is less change of contaminationof entire PBS bottles. Fill syringes from 50ml falcon tube. 3. Anesthetize mouse in anesthetizing chamber:37

Add a small amount of isoflurane to tissue in bottom of chamber. Place mice in chamber. Watch for breathing of mice and take out when it slows down. If mice die from

anesthetizing in this step, it will not a problem. 4. Euthanize mouse by cervical dislocation. Hold mice by nape of neck firmly and pull

tail with other hand until feel a pop and a separation between cranium and backbone. 5. 6. Sterilize mice:Place mice in a beaker with 70% ethanol. Place mice in tissue culture hood immediately. Laporotomy incision:Make a small incision in abdominal skin of mice(use 1 set of forceps and scissors). Open up mice by pulling skin with both hands to each side of mice. Do this firmly so

that skin is completely pulled down, but gently so that not open up intraperitoneal cavity or break any limbs. Spray peritoneum with 70% ethanol. 7. Inject cold PBS:Uncap needle inside hood and squirt out a small amount of PBS to get rid of any

bubbles. Inject needle into the mice in lower abdominal area, preferably in a region where

there is fat. Insert needle gently and not too deeply so do not perforate any organs. Immediately after inserting the needle, begin injecting PBS into intraperitoneal cavity

forcefully. Take needle out slowly so that fat not clog the hole (if take out too fast the PBS will

come out of the hole).

38

8.

Wash mice:Hold mice by tail and swish around to wash IP cavity with the PBS that has been

injected 9. Macrophage extraction:Using same syringe, go to upper part of abdomen and insert needle horizontally. Make sure do not perforate any organs. Hold needle inside peritoneal cavity in a horizontal position with the needle hole- side

down and extract the PBS containing macrophages. Do this slowly, if done too fast then aspirate fat and clog needle. Try to get at least 7ml of fluid. If needle insert again in another part lifting a tent and extracting the fluid. Cap needle

again and place back on ice. Repeat this process for each mice. 10. Now add histopaque:To a 15 ml conical cfgtube,add 3.0 ml histopaque-1077 and bring to room

temperature. Carefully layer 3.0 ml whole blood onto the histopaque-1077.cfg at 400*g for exactly

30 min at room temp.cfg at lower temperature, such as 40c,may result in cell clumping and poor recovery. After cfg carefully aspirate with a Pasteur pipet the upper layer to within 0.5 cm of the

opaque interface containing mononuclear cells. Discard upper layer. tube. Add to this tube 10ml isotonic phosphate buffered saline solution and mix by gentle Carefully transfer the opaque interface, with a Pasteur pipet into a clean conical cfg

aspiration. Cfg at 250*g for 10 min. Aspirate the supernatant and discard.
39

11. 12. slide.

Add the supernatant microscopic slide. Then put a drop of trypan blue on one of the slide and do not add this dye to the other

BIODISTRIBUTION STUDIES:
Preparation of Tc-99m labeled drug formulation was administered to patient animals sacrificed at different time intervals. Activity is measured by gamma counter. Plot of % radioactivity Vs organ gives the tissue distribution chart of labelled formulation.

ORGAN Blood Heart Lungs Liver Spleen Kidney Stomach Intestine Tumor Muscle

% I.D per gram organ at 4h pi 1.32 0.09 1.74 10.99 2.09 0.51 0.04 0.09 0.86 0.05

40

CHAPTER-6 CONCLUSION

41

Drug was labelled with Tc-99m resulting in more than 95% labelling efficiency. The complex was sufficiently stable at in vitro conditions as observed up to 24hr. Biodistribution studies in tumor bearing mice showed that the major accumulation of the activity was in kidney followed by liver in 1 hr showing that the renal as well as hepatobiliary were the main routes of excretion. Since, the scintigraphic scan post injection fairly correlates with the blood kinetic and biodistribution data. This study substantiates the potential of the given formulation for tumor using Tc-99m as a radiotracer.

42

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1.

Fundamentals of nuclear pharmacy,Sixth Edition, Gopal B. Saha.

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Textbook of Radio pharmacy -theory & practice, Charles B. Sampson.

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Nuclear Medicine in the new millennium, The Journal of Nuclear Medicine, Jan 2000,

41, 1, 1-4.

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Development of kits for Tc-99m radiopharmaceuticals for infection imaging, IAEA

report 2000-2003.

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Nuclear Medical Imaging, European Nuclear Society, Deconinck F, 2006, 14, 1-10.

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Nuclear Imaging methods for non-invasive drug monitoring. Advanced Drug

Delivery, BhatnagarAnish, Hustnix Roland, AlaviAbass, 2000, 41, 41-54.

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Scintigraphic detection of infection and inflammation: new developments with special

emphasis on receptor interaction European Journal of Nuclear Medicine, Van Der Laken, C.J. boerman O.C, Oyen W.J.G et al,1998, 25, 535-546.

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Characterization and potential biomedical applications, designed peptides as model

self-assemblingnanosystems, Jiban J Panda, Ankurkaul, Shadabalam, Anil Delivery, FEB 2011, VOL 2,193-204.
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9.

Intranasal clobazan delivery in the treatment of status epilepticus, Kiruba Florence, Babbar Anil kumar, KaulAnkur, Mishra anilkumar, (journal of

LalanManisha,

pharmaceutical sciences,Feb 2011,vol100, issue 2 692-703).

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Evaluation of iscom matrices clearance from rabbit nasal cavity by gamma

scintigraphy, Ravi S pandey Anil k babbar ankur kaul anil k mishra, vinod k dixit.

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