SUB-ACUTE HEPATO TOXICITY OF TAMOXIFEN CITRATE

About Author:
Zehira Nazir
Master’s In Clinical Biochemistry
University Of Kashmir
zzehra275@gmail.com

Introduction
There has been increasing public concern that chemicals in the environment are affecting human health by disrupting normal endocrine function. The exposure to these chemicals with steroid-like activity can disrupt normal endocrine function leading to altered reproductive capacity, infertility, endometriosis and breast and uterine cancer. In order to detect and evaluate the safety of such chemicals, several rodent experimental models have been developed. The OECD Enhanced Test Guideline 407 (repeated 28-day oral dose toxicity study) is one of rodent experimental models for detection of endocrine disrupters of these chemicals [1].

In recent years, the development of bioassays to detect chemicals that have the potential to interfere with the endocrine system has become a major task in toxicology. One initiative in this direction that was taken on by the OECD (Organization for Economic Co-operation and Development) was to assess the potential of an enhanced version of the sub acute rodent OECD Test Guideline 407 (OECD 1995) to detect endocrine disrupters acting through various mechanisms of toxicity [2].

REFERENCE ID: PHARMATUTOR-ART-1764

The “war on cancer” is now in its fourth decade, but still the search continues for more selective therapies that destroy tumor cells without harming normal tissues. Cancer is a leading cause of death, with more than 10 million people being diagnosed with the disease annually and it is estimated that by 2020, there will be 15 million new cases every year. Though chemotherapy is successful to some extent, most current anticancer agents do not greatly differentiate between cancerous and normal cells. Thus, in the process of killing cancer cells, chemotherapeutic agents also damage healthy tissues leading to systemic toxicity and adverse side effects. It also greatly limits the maximum allowable dose of the drug. Moreover, large quantities of a drug need to be administered due to rapid elimination and widespread distribution into targeted organs and tissues, which is uneconomical and often results in undesirable toxicity. Thus, the first important step in improving treatment regime is the better utilization of the potency of therapeutic agents by more effectively targeting them to tumor tissues. The therapeutic efficacy of drugs and proteins can be greatly amplified and their toxicity greatly reduced if high concentrations of anticancer agents could selectively be administered to malignant tissues only. Thus, the need to develop novel cancer therapies and drug delivery strategies that provide specific targeting to tumor cells has been continually at the forefront of medical sciences. Nanotechnology, one of the frontier sciences, can pave the way to overcome the numerous barriers to an efficient and safe drug delivery system (DDS).

Tamoxifen:
Tamoxifen, a non-steroidal anti-estrogen that has been used successfully for a decade as post-operative adjuvant therapy for breast cancer, lengthens the disease free interval as well as improves survival [8]. Tamoxifen (Tmx) has a non-steroidal triphenyl ethylene structure and a selective estrogen receptor modulator (SERM) with anti-estrogenic properties in the breast and estrogenic effects in tissues such as bone and the cardiovascular system. These anti-estrogenic properties make Tmx the endocrine treatment of choice for all hormone-sensitive stages of breast cancer [3]. Tamoxifen remains a frontline treatment for hormone-responsive breast cancer, despite its use being associated with a 2–7-fold elevated risk of developing endometrial carcinoma [7]. The US Food and Drug Administration (FDA) approved Tmx for the treatment of advanced breast cancer in late 1998 [48]. Tmx shows its potential effects in patient who possess estrogen receptors (ER) positive cancer cells by competing with estrogen to bind with  estrogen receptor in breast cancer cells [49]. As Tmx therapy is chronic one (3e5 years), oral delivery is the most preferred route of administration and its solubility problem in aqueous milieu has been overcome by forming its salt form, tamoxifen citrate (Tmx citrate). Commercially, Tmx is available only as tablet and oral solution containing Tmx citrate in a daily dose of 10e20 mg. However Tmx citrate also showed the poor oral bioavailability (20e30%) due to its precipitation as free base in the acidic environment of stomach and also due to extensive hepatic and intestinal first pass metabolism, so as to increase its does [50].

Breast cancer:
Breast cancer is the most common cancer in females, the second most common cause of cancer death and the main cause of death in women aged 45–55 in the USA [11]. It is also the most common malignancy among Iranian women with an incidence rate of 18.2/100,000 [51]. Tamoxifen is a partial oestrogen receptor antagonist; it has a10-fold greater anti tumour activity  in breast-cancer patients whose tumours express oestrogen receptors than in those who have low or no levels of expression. Side effects include increased risk of oestrogen-related cardiovascular complications, such as thrombo-embolic phenomena, and development of non-alcoholic fatty liver and non-alcoholic steatohepatitis (NASH) [52,53].Tamoxifen remains a front line treatment for hormone-responsive breast cancer, despite its use being associated with a 2–7-fold elevated risk of developing endometrial carcinoma [7]. Tamoxifen, a non-steroidal anti-estrogen that has been used successfully for a decade as post-operative adjuvant therapy for breast cancer, lengthens the disease free interval as well as improves survival [54]. When used as an adjunct to surgery, the usual dose of tamoxifen is 10 mg twice or three times daily. The tamoxifen dose is typically 10 mg twice daily in post-menopausal women when used in combination with other chemotherapies as an adjunct to surgery, or in the setting of positive axillary lymph nodes [55]. Tamoxifen is generally well tolerated with few side effects [55,56].

Nanotechnology:
Nanotechnology is the understanding and control of matter generally in the 1–100nm dimension range. The application of nanotechnology to medicine, known as nanomedicine,concerns the use of precisely engineered materials at this length scale to develop novel therapeutic and diagnostic modalities.[3,57] Nanomaterials have unique physicochemical properties, such as ultra small size, large surface area to mass ratio, and high reactivity, which are different from bulk materials of the same composition. These properties can be used to overcome some of the limitations found in traditional therapeutic and diagnostic agents.The use of materials in nanoscale provides unparallel freedom to modify fundamental properties such as solubility,diffusivity, blood circulation half-life, drug release characteristics, and immunogenicity. In the last two decades, a number of nano particle-based therapeutic and diagnostic agents have been developed for the treatment of cancer, diabetes, pain,asthma, allergy, infections, and so on.[58,59] These nanoscale agents may provide more effective and/or more convenient routes of administration, lower therapeutic toxicity, extend the product life cycle, and ultimately reduce health-care costs. As therapeutic delivery systems, nanoparticles allow targeted delivery and controlled release. For diagnostic applications, nanoparticles allow detection on the molecular scale: they help identify abnormalities such as fragments of viruses, precancerous cells, and disease markers that cannot be detected with traditional diagnostics. Nanoparticle-based imaging contrast agents have also been shown to improve the sensitivity and specificity of magnetic resonance imaging.Given the vast scope of nanomedicine, we will focus on the therapeutic applications, in particular, drug delivery applications,of nanoparticles.  Many advantages of nanoparticle-based drug delivery have been recognized [60,61].

Advantages:
1. Stability - Polymeric nanoparticles made from natural and synthetic polymers have received the majority of attention due to their stability and ease of surface modi?cation.

2. Controlled release - They can be tailor-made to achieve both controlled drug release and disease-speci?c localization by tuning the polymer characteristics and surface chemistry.

3. Depot effect - It has been established that nanocarriers can become concentrated preferentially  at tumors, in?ammatory sites, and at antigen sampling sites by virtue of the enhanced permeability and retention (EPR) effect of the vasculature. Once accumulated at the target site, hydrophobic biodegradable polymeric nanoparticles can act as a local drug depot depending on the makeup of the carrier, providing a source for a continuous supply of encapsulated therapeutic compound(s) at the disease site, e.g., solid tumors.

4. Small size - Nanoparticles, because of their small size, can extravagate through the endothelium in in?ammatory sites, epithelium (e.g., intestinal tract and liver), tumors, or penetrate micro capillaries. In general, the nanosize of these particles allows for ef?cient uptake by a variety of cell types and selective drug accumulation at target sites.

5. IV Delivery - Nanoparticles have another advantage over larger microparticles because they are better suited for intravenous delivery. The smallest capillaries in the body are 5–6 μm in diameter. The size of particles being distributed into the blood stream must be signi?cantly smaller than 5μm, without forming aggregates, to ensure that the particles do not cause an embolism.

6. Biodegradability - Biodegradable nanoparticles formulated from PLGA and PLA have been developed for drugs with an intracellular target. 

7. Sustained release - Nanoparticles can cross the blood brain barrier (BBB) following the opening of endothelium tight junctions by hyperosmotic mannitol, which may provide sustained delivery of therapeutic agents for dif?cult to treat diseases like brain tumors.

8. Better penetration - Nanoparticles were shown to penetrate throughout the submucosal layers of a rat intestinal loop model, while microparticles were predominantly localized in the epithelial lining.

Nanoparticles as drug delivery systems for cancer therapy
The potential of NPs as putative DDSs is increasingly being realized as these offer numerous advantages as compared with conventional treatment modalities. The polymers used for the formulation of NPs are biocompatible and biodegradable. After, the release of the encapsulated drug, the polymer matrix is degraded into harmless molecules such as hydrogen, nitrogen and water and excreted from the body. An insoluble drug can be made a water soluble formulation by introducing solubilizing moieties into the polymer, thereby improving its bioavailability and biodegradability. These insoluble drugs can also be combined with organic or lipid NPs that keep them in circulation for longer periods. If an efficacious compound has a short half-life in circulation, its stability can be tremendously increased by encasing it within NPs. Moreover, the problem of sustained, controlled release of anticancer drugs needs to be addressed by various nanoparticle formulations. The drug release from NPs can be controlled by modulating the polymer characteristics to achieve the desired therapeutic level in target tissues for required durations for optimal therapeutic efficacy and release of a constant amount of drug per unit time. In the case of central nervous system cancers, many drugs have difficulty in reaching the therapeutic site due to blood brain barrier.

Drug-loaded NPs are able to breach this barrier and have been shown to greatly increase therapeutic concentrations of anticancer drugs in brain tumors. P-glycoprotein (P-gp)-related multi-drug resistance is another major problem that contributes to the resistance of tumors to anticancer agents. NPs with their small size and appropriate surface coating may have the ability to solve the problem of drug resistance. Furthermore, one of the key issues that need to be addressed is targeted drug delivery in cancer therapy. In chemotherapy, pharmacologically active cancer drugs reach the tumor tissues with poor specificity and dose-limiting toxicity, thus resulting in harmful effects to healthy tissues. Targeting cancer cells using NPs loaded with anticancer agents is a promising tactic that could help overcome these challenges. Drug targeting can be achieved by taking advantage of the distinct pathophysiological features of a tumor tissue (passive targeting) or by actively targeting drug carrier, using some target-specific ligands (active targeting)[7].Polycaprolactone is a biodegradable hydrophobic crystalline polymer and is resistant to chemical hydrolysis. It is well suited for colloidal drug delivery due to lack of toxicity and its achiral property [6].PCL is a hydrophobic,semi-crystalline polymer having a glass transition temperature of −60 ?C and melting point ranging between 59 and 64 ?C, dictated by the crystalline nature of PCL which enables easy formability at relativelym low temperatures. The number average molecular weight of PCL samples may generally vary from 3000 to 80,000 g/mol and can be graded according to the molecular weight. PCL is soluble in chloroform, dichloromethane, carbon tetrachloride, benzene, toluene, cyclohexanone and 2-nitropropane at room temperature. It has a low solubility in acetone, 2-butanone, ethyl acetate, dimethylformamide and acetonitrile and is insoluble in alcohol, petroleum ether and diethyl ether [14].

Mouse model:
Mouse models are vital for understanding of the molecular basis and pathogenesis of breast cancer. In addition, they provide valuable information for the development of novel antineoplastic agents with regard to basic pharmacokinetics, pharmacogenomics, and toxicity analysis [16]. Experimental evidence indicates that estrogens are involved in carcinogenic promotion of mouse mammary tumors [17].

Overview of toxicity:

• Toxicity has two main components: the effect caused and the level of exposure (dose) at which the effect is observed. Some tests are designed specifically to detect a particular effect (such as skin and eye irritancy, skin sensitization and mutagenicity studies). Other tests (such as sub-chronic and chronic studies) are designed to detect a wider range of less-specific effects on organs or body systems and the dose range over which the effect develops.

• Information from toxicity tests is first used to provide a classification for a chemical, for example to assign appropriate warning labels for containers, and, where necessary, for selecting measures, such as protective equipment, during manufacture, exposure and use.

• Data from tests that characterize the relationship between dose and toxicological response are integrated with information on human exposure to produce a risk assessment, and to identify control measures necessary to manage and reduce any identified risk. Tests on species such as fish and amphibians are used in a similar way to assess the potential environmental effects of chemicals.

• For pharmaceuticals, results from animal tests are used in combination with data on the efficacy of a potential medicine to decide whether the beneficial effects of the treatment would outweigh the risks of adverse side effects, and to establish a safe dose for use in clinical trials. They may also indicate potential side effects that must be monitored carefully. The prediction of the likely effects of chemical exposure on human health is based primarily on the results of tests involving experimental animals. The number of animals involved in these tests varies.

• A full complement of toxicity tests for a successful pharmaceutical compound that proceeds to the market, involving single dosing, repeat sub-chronic and chronic dosing, reproductive testing, genotoxicity and carcinogenicity testing, can involve between 1,500 and 3,000 animals. The actual numbers required will depend on the need for further tests according to the nature of the test substance and also its toxic properties. The numbers of animals used to test other types of chemical are generally lower, but in some cases, where there is particular controversy about the safety of a chemical, tests may be repeated, with modifications, resulting in the use of even more animals.

• Large numbers of animals are also used in several other tests. For example, a carcinogenicity bioassay generally involves 800 animals in total (400 of each sex) and may be conducted on both rats and mice. Adult animals (typically at least 80 animals of each sex per study), offspring and fetuses are used in reproductive and development studies. Rats and mice are most commonly used (74 percent), but in some cases testing is carried out on other animals such as rabbits (four percent), guinea pigs (three percent), dogs (one percent) or primates (less than one percent).

• The interpretation of the results for assessing human safety depends on a number of assumptions. First, unless there is specific knowledge of species differences in the test response, it is assumed that the effects detected in rodents or other species are the same as those that would be induced in humans. Secondly, it is assumed that the sensitivity of the test animals represents, at best, the average sensitivity of the highly heterogeneous human population and that for some members of the human population the health risk could be much higher [46].

Types of toxicity:
Acute toxicity: The first toxicity test performed on a new chemical is acute toxicity. Acute toxicity occurs almost immediately (hours/days) after an exposure. An acute exposure is usually a single dose or a series of doses received within a 24 hour a single dose exposure within 14 Days of constant observation period. Death is a major concern in cases of acute exposures. The LD50 and other acute toxic effects are determined after one or more routes of administration (one route being oral or the intended route of exposure) in one or more species. The species most often used are the mouse and rat, but sometimes the rabbit and dog are employed. Studies are performed in both adult male and female animals. Food is often withheld the night before dosing. The number of animals that die in a 14-day period after a single dosage is tabulated. In addition to mortality and weight, daily examination of test animals should be conducted for signs of intoxication, lethargy, behavioral modifications, morbidity, food consumption, skin, fur and Attention should be directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma and so on [37].Acute toxicity tests gives

• Quantitative estimate of acute toxicity (LD50) for comparison with other substances,
• Identify target organs and other clinical manifestations of acute toxicity
• Establish the reversibility of the toxic response, and
• Provide dose-ranging guidance for other studies.

Many factors influence toxicity and thus may alter the estimation of the LD50 in any particular study. Factors such as animal strain, age and weight, type of feed, caging, pretrial fasting time, method of administration, volume and type of suspension medium, and duration of observation have all been shown to influence adverse responses to toxic substances. Because of this inherent variability in LD50 estimates, it is now recognized that for most purposes it is only necessary to characterize the LD50 within an order of magnitude range such as 5 to 50 mg/kg, 50 to 500 mg/kg, and so on.

• The acute dermal toxicity test is usually performed in rabbits. The site of application is shaved. The test substance is kept in contact with the skin for 24 h by wrapping the skin with an impervious plastic material. At the end of the exposure period, the wrapping is removed and the skin is wiped to remove any test substance still remaining a single dose exposure within 14 Days of constant observation period and the LD50 is calculated. If no toxicity is evident at 2 g/kg, further acute dermal toxicity testing is usually not performed.

• Acute inhalation studies are performed that are similar to other acute toxicity studies except that the route of exposure is inhalation. Most often, the length of exposure is 4 h. Although by themselves LD50 and LC50 values are of limited significance, acute lethality studies are essential for characterizing the toxic effects of chemicals and their hazard to humans. The most meaningful scientific information derived from acute lethality tests comes from clinical observations and postmortem examination of animals rather than from the specific LD50 value [34].

Sub acute toxicity:
Sub acute toxicity tests are performed to obtain information on the toxicity of a chemical after   repeated administration for a limited period of time and as an aid to establish doses for sub chronic studies.The duration of exposure should normally be 28 days. The test substance is orally administered daily in graduated doses to several groups of experimental animals. At least 10 animals (five female and five male) should be used at each dose level. Clinical chemistry and histopathology are performed [37].

Regulatory Requirement of Toxicity Study
The requirements for the documentation of toxicological investigations of antimicrobial agents basically resemble those available worldwide for other classes of therapeutic drugs. The non-clinical investigations necessary include the following in chronological order:

• Investigations to be performed before clinical trials can be initiated (pre-clinical studies)

• investigations to be performed after clinical studies have been started, but before marketing authorization has been applied for or granted

Investigations possibly to be performed under circumstances to be specified after marketing authorization is applied for or granted (e.g. in the case of conditional approval granted for compounds having an innovative character or special therapeutic value [40].

• The identification of the hazardous properties of a chemical,
• The identification of target organs,
• Characterization of the dose: response relationship,
• Identification of a no-observed-adverse-effect level (NOAEL) or point of departure for establishment of a Benchmark Dose (BMD),
• The prediction of sub acute toxicity effects at human exposure levels,
• Provision of data to test hypotheses regarding mode of action.
• To calculate LD₅₀.
• To predict exact Therapeutic index of a new chemical entity.
• Hazard identification depends on data quality and relevance of the animal model
• Hazard characterization find sensitive period and relevant dose metric, biomarkers, mechanistic basis for interspecies extrapolation
• Dose-response assessment i.e. quantitative relationships, not just administered dose, Human exposure assessment (subpopulations may differ).

Factors Influencing Toxicity
The toxicity of a substance depends on the following:

• Form and innate chemical activity , dosage; especially dose-time relationship, exposure route.
• Species, age, sex
• Ability to be absorbed, metabolism, distribution within the body and excretion
• Presence of other chemicals

The form of a substance may have a profound impact on its toxicity especially for metallic elements. For example, the toxicity of mercury vapor differs greatly from methyl mercury. Another example is chromium(Cr). Cr³⁺ is relatively nontoxic whereas Cr⁶⁺ causes skin or nasal corrosion and lung cancer. The dosage is the most important and critical factor in determining if a substance will be an acute or a chronic toxicant. Virtually all chemicals can be acute toxicants if sufficiently large doses are administered. Often the toxic mechanisms and target organs are different for acute and chronic toxicity. Exposure route is important in determining toxicity. Some chemicals may be highly toxic by one route but not by others. Two major reasons are differences in absorption and distribution within the body. For example:

• Ingested chemicals, when absorbed from the intestine, distribute first to the liver and may be immediately detoxified
• Inhaled toxicants immediately enter the general blood circulation and can distribute throughout the body prior to being detoxified by the liver

Frequently there are different target organs for different routes of exposure. Toxic responses can vary substantially depending on the species. Most species differences are attributable to differences in metabolism. Others may be due to anatomical or physiological differences. For example, rats cannot vomit and expel toxicants before they are absorbed or cause severe irritation, whereas humans and dogs are capable of vomiting.

Mechanism of toxicity [39]
The Mechanism of toxicity illustrates between the relationship of biochemical lesions and tissue injury. Biochemical studies is an important role in determining and conforming safe levels of exposure and may even provide a rational basis for establishing a safe  level of exposure to potential carcinogens. There are different factors affecting for toxicity, these are

Congenital metabolic deficiencies: When particular metabolic pathways are blocked due to congenital deficiencies of specific enzyme, they also indicate the sort of adverse effects which might also cause toxicity .The adverse effect of metabolic overload leads to toxicity due to deficiency of specific enzyme.

Ex- Deficiency of phenyl alanine hydroxylase leading to toxic manifestation of several mental deficiency, neural and dermal lesions  and premature death due to accumulation of  protein, phenyl alanine and its pyruvate,  lactate and acetate in blood and cerebrospinal fluid.

Overload of specific enzyme: Overload of intestinalhydrolases or transport processes involved in absorption is a common cause of GI disturbance of orally administered compounds. This may lead to osmotic diarrhoea due to overload of lactase activity.

Inhibition of specific enzyme: Many toxic compounds exert their toxic effect by inhibition of key enzymes involved in cell function regulation and replication. Such enzyme inhibitors s may themselves be toxic by interfering in essential metabolic processes or, by producing a condition analogue to enzyme deficiency, they may increase the toxicity of other substances to which the organism is exposed.

Ex- Inhibition of choline esterase , toxic effects are observed when the activity is reduced to 50% of normal while  80-90% inhibition death results from respiratory failure due to combination of  Neuromuscular  paralysis and CNS depressant.

Secondary toxic effect: The toxicity of one part leads to rise of specific enzyme or activation of specific receptors which results as secondary toxic effect or influenced toxicity.

Ex- In case of caecal enlargement, calcium absorption is facilitated by non Vit-D regulated process and the resultant hypocalcaemia may be compensated by renal excretion that leads with calcium depositions in kidney causes nephrocalcinosis.

Metabolic activation: Generally xenobiotoics are metabolized by two phases, in first phase involving introduction or unmasking of polar functional group and the second consisting of conjugation reaction with endogenous substrate such as Glucuronate, Sulphate , Amino acid,  or Glutathione. The net result of this reaction is that, the more polar compounds are facilitated by excretion in urine to prevent accumulation in tissue storage site.  In some cases, the metabolite is highly reactive or more toxic than parent compounds. In such cases, the compound converted into a proximate mutagen or carcinogen to produce lethal effect.

Lethal synthesis: In some cases, the metabolite is an electrophilic, highly reactive species can undergo covalent bonding with macromolecules such as nucleic acid and proteins.  In this way of binding frequently introduces the mutagens and carcinogens.

Mutagens and Carcinogens are known to act via direct or indirect effect on genetic or other informational molecule (DNA, RNA, Regulator molecules). Covalent binding to RNA or protein may also play a part in   carcinogenesis ex- Acetylaminofluorene, binding to r-rna correlates more closely with liver tumor development. In some cases, nonmutagenic compounds which don’t form covalent adducts with DNA are associated with an increase in tumor incidence.

Ex- Clofibrate, an antilipidemic agent causes a proliferation of peroxisomes in rat liver and an associated increase in tumor incidence without any evidence of covalent binding of radio labeled compounds; such compounds are detected as mutagenic in Ame’s assay. The peroxisome proliferation leads to an increase in generation of free radicals. , which causes genetic injury.

Biochemical parameters and their Interpretation [45]
Combined with hematology, biochemical profile forms the data base for most diagnostic investigations. Many biochemical parameters tend to have specificity for an organ and/or a limited range of pathological processes. Interpretation of diagnostic biochemical patterns requires an understanding of the pathological implications of each abnormal result. Together with the normal results these form a pattern which reflects one or more underlying disease process. Investigative biochemical profiles are designed to provide all the data necessary for a broad investigation of internal disease. Profiles with limited data are best used for monitoring an established diagnosis for which the results of a more wide ranging profile have already been obtained. Individual biochemical evaluations may be used, for example, for therapeutic drug testing (Phenobarbital, bromide, and digoxin), assessing vitamin status and monitoring liver function (bile acids) and diabetic control (fructosamine).

Serum transaminase activity

Aspartate aminotransferase (AST)
It is present in many tissues and is useful in evaluating muscle and liver damage in small and large animals. AST is not liver specific in any domestic animal species and the reference range in horses is rather broad. Skeletal muscle is the second largest source of AST in animals. It is an absolute prerequisite to eliminate extra hepatic tissue damage as a possible source of serum AST when evaluating the enzyme in relation to the liver.  In combinations with the physical examination and history, the evaluation of other serum enzymes should aid in differentiating the source of increased AST levels. AST is present in both the cytoplasm and mitochondria of hepatocytes (and many other cells) and will elevate in states of altered membrane permeability. In such cases, levels are expected to be less than in states of frank necrosis, when both cytoplasmic and mitochondrial enzymes are released.

Alanine aminotransferase (ALT)
It is considered to be liver specific in small animals. This enzyme is present in high concentrations in the cytoplasm of hepatocytes. Plasma concentrations increase with hepatocellular, damage/necrosis, hepatocyte proliferation, or hepatocellular degeneration. ALT is a cytoplasmic enzyme, and is considered to be liver specific in dogs, primates and some other small animal species. There is little hepatic ALT activity in large domestic animals. Thus, further comments regarding ALT will relate only to dogs and cats. Elevation of serum levels of both AST and ALT can occur with states of altered hepatocellular membrane permeability. Because ALT is located only in the cytoplasm, serum levels tend to be relatively higher than AST, as a result of membrane leakage from the hepatocyte. Mitochondrial enzymes are less likely to be released with most of the conditions which result in increased membrane permeability. Many causes of altered membrane permeability are potentially reversible but some may progress to hepatocellular necrosis which is essentially an irreversible change.

Causes of increased cell membrane permeability include:
Anoxia/circulatory hypoxia
Exposure to toxins
Inflammation
Metabolic disorders
Hepatocyte proliferation

The magnitude of both AST and ALT elevations in serum is generally related to the number of hepatocytes affected. However, the level cannot be used to predict either the type of lesion, or whether cell damage is reversible (leakage) or irreversible (frank necrosis). In fact, focal necrosis may yield a lower concentration of both AST and ALT than would severe, transient hypoxia in which all cells may be affected resulting in a potentially reversible alteration in membrane permeability and diffuse enzyme leakage. Equally increases in ALT and AST may be relatively mild in cases of severe cirrhosis/fibrosis of the liver since there is no ongoing hepatocellular damage.

Alkaline phosphatase (ALP)
The alkaline phosphatases are a group of enzymes which catalyze the hydrolysis of a phosphate group from an organic molecule at an alkaline pH. They are called isoenzymes because they catalyze the same reaction in the same species but have different biochemical properties. ALP is found, to some extent, in all tissues and is relatively stable in serum. However, only a few organs actually contribute to the circulating enzyme level. An elevated alkaline phosphatase concentration is generally due to cholestasis in most adult domestic animals. A mild elevation in immature animals is likely to be the result of normal bone growth. In dogs, when an elevated ALP value is seen, liver disease, Cushing disease, and recent steroid therapy should all be considered. Prolonged steroid therapy resulting in iatrogenic Cushing disease can be diagnosed on the basis of low pre and post cortisol levels. The liver isoenzyme will be elevated in any active liver disease. In acute hepatocellular necrosis, ALT and AST are markedly elevated while ALP is only minimally elevated. Intrahepatic and extra hepatic biliary obstruction causes more dramatic elevations of ALP, which in some cases can be 10-20 times the normal level. This is due to recycling as well as increased synthesis of the liver isoenzyme. Extra hepatic biliary obstruction can be caused if the hepatic or common bile duct is obstructed either partially or completely. Possible causes include tumour, granulomatus inflammation, abscesses, pancreatitis and duodenitis.

Common causes of elevated ALP
Liver disease
Cushing’s disease (dog)
Steroid therapy (dog)
Antiepileptic drugs
Bone growth in young animals
Intestinal damage (horse)
Hyperthyroidism (cats)

Bilirubin
Bilirubin and its components may be helpful when evaluating liver function or haemolysis. These tests may be useful of erythrocytes. It is then carried in the plasma loosely bound in albumin. This bound form is not water soluble and is often referred to as indirect reacting, free, prehepatic, or unconjugated bilirubin. The hepatocyte conjugates the indirect bilirubin with glucuronic acid and it is then referred to as direct or conjugated bilirubin. Direct bilirubin is water soluble. Direct bilirubin is excreted into the intestine via the biliary system. Some of the direct bilirubin is reabsorbed back into the circulation from the intestine. The direct bilirubin is not bound to albumin and is freely filtered by the glomerulae. The renal tubular epithelial cells readily reabsorb the filtered bilirubin in most animals. However, the dog is an exception and small amounts of bilirubin are normal in concentrated urine samples while bilirubinuria in cats is generally considered to be abnormal. An elevation of indirect bilirubin is a rather uncommon finding in small animals, but when it occurs, it is generally the result of acute and severe haemolysis. The haematocrit and red blood cell counts are low when elevated indirect bilirubin is caused by haemolysis. Direct reacting hyperbilirubinaemia occurs as a result of impaired hepatic secretion of bilirubin and/or obstruction to bile flow. Obstruction to bile flow can be intrahepatic, extra hepatic or both. Most jaundiced animals have elevations in both indirect and direct bilirubin. Hemolytic disease may also result in an increase in direct bilirubin since a large proportion of the free bilirubin is conjugated.

Serum Proteins
Almost all proteins in the serum are produced by the liver. Immunoglobulins are the notable exception and they are produced by lymphoid tissue. Serum proteins are relatively short-lived with most having half-lives of about 10 days. The breakdown of these proteins occurs mostly in the liver with some catabolic activity in the intestine and kidney. Animal plasma normally contains 25-35 gm/L of albumin which constitutes 40 -60% of the total protein concentration. Fluid accumulations in body cavities and tissue usually result when albumin levels drop below 10 gm/L. However, fluid may accumulate with higher albumin concentrations in hypertension, and loss of vessel integrity etc. is present. Plasma and serum proteins, act as anions in acid-base balance, take part in coagulation reactions, and serve as carriers for many compounds. In addition to albumin, plasma contains globulins, fibrinogen (removed from serum by the clotting process), glycoproteins, lipoproteins, acute phase proteins and transport proteins. The globulin component is subdivided into important sub fractions identified by electrophoresis as alpha, beta and gamma globulins. The alpha and beta fractions are important carriers of lipids, lipid soluble hormones and vitamins. Gamma globulins are primarily associated with antibodies. Conditions causing inflammation usually cause a measurable increase in serum levels of gamma globulins and often alpha-2 globulins (e.g. αALT). Fibrinogen is a plasma acute phase protein which is utilized in the coagulation process. It is therefore absent in serum. Glycoproteins (carbohydrates bound to protein) and lipoproteins (lipids bound to protein) are the other major plasma proteins. Both of these serve as carriers of the substances bound to them.

Hypoalbuminaemia

• Primary or secondary intestinal malabsorption
• Exocrine pancreatic insufficiency
• Malnutrition, dietary or parasitism
• Chronic liver disease e.g. atrophy or fibrosis
• Glomerulonephropathy resulting in proteinuria
• Acute inflammation (negative acute phase response)
• Severe exudative skin disease or burns

Hypoglobuinaemia

• Immunodeficiency disease, either primary or secondary

Hypoalbuminaemia/hypogiobulinaemia

• External hemorrhage
• Protein-losing enteropathies
• John’s disease

Causes of hyperproteinaemia
Increased albumin

• Dehydration (relative increase)
• Lactation (common in dairy cows)

Increased fibrinogen +/- other acute phase proteins

• Acute inflammation

Increased globulins

• Monoclonal gammopathy, Polyclonal gammopathies, Multiple myeloma, maus inflammation, infection, neoplasia, Ehrlichiosis FIP (OX), Leishmaniasis chronic liver disease.

Measurement of albumin, along with a separation of globulin into its fractions, can be accomplished by serum protein electrophoresis. When placed in an electric field, these proteins migrate at different rates yielding a familiar electrophoretic pattern. Values obtained from measuring serum proteins can provide an accurate reflection of an animal’s health status.

Drug profile

Generic name		Tamoxifen citrate
Class		SERMs
Chemical Formula		C26H29NO
Description		White to off white crystalline powder
Mol. Mass		563.638 g/mol (citrate salt)
Solubility		In water at 37?C is 0.5mg/ml and in 0.02N HCl at 37?C is 0.2mg/ml.Soluble in ethanol, methanol, and acetonitrile. Slightly soluble in acetone and in chloroform
Pharmacokinetic data		Metabolism: extensively metabolized after oral administration. N-desmethyl-Tamoxifen is major metabolite found in plasma. Excretion: fecal excretion Half life: 7 to 14 days.
Route of administration and dose		Administered orally A usual adult dose of 20mg daily.
Uses		Breast cancer, hormone treatment therapy, gynecomastia, angiogenesis
Side effects		Endometrial cancer, bone loss, cardiovascular disorders, CNS disorders

Review of Literature

Literature Review
Extensive literature review was taken from different web portal available on internet to gain insight into the work done by various researchers on nanoparticles.

Greaves et al (1993)[8]-  administered Tamoxifen to Wistar derived alderly park rats and found that alkaline phosphatase activity was about three-fold higher in all treated groups compared with controls.

Nuvaysir et al 1995[18]– Demonstrated the effects of tamoxifen administration on the expression of xenobiotic metabolizing enzymes in F344 rat liver have been investigated. Tamoxifen administration for 7 days produced a dose-dependent increase in enzyme expression similar to that reported to be produced by Phenobarbital

Desai et al (1997) [13]-   reported that Tamoxifen and its metabolite 4-hydroxy Tamoxifen markedly induce cytochrome P450 3A4, a drug-metabolizing enzyme of central importance, in primary cultures of human hepatocytes. Tamoxifen and 4-hydroxy Tamoxifen significantly increased the CYP3A4 expression and activity. Maximal induction was achieved at the 5 _M level. At this level, Tamoxifen and 4-hydroxy Tamoxifen caused a 1.5- to 3.3-fold (mean, 2.1-fold) and 3.4- to 17-fold (mean, 7.5-fold) increase in the CYP3A4 activity, respectively.

Chawla et al (2002) [6]- prepared polycaprolactone (PCL) nanoparticles by solvent displacement method using acetone/water system. Concentration of Tamoxifen in estrogen receptor (ER) positive breast cancer was increased.

Jeong et al. (2005) [28]- prepared Tmx-loaded poly (gamma-benzyl-L glutamate)/poly(ethylene glycol) (PEG) diblock copolymer end capped with galactose moiety (GEG) for liver specific targeting. The specific interaction between asialo glycoprotein receptors (ASGPR) of HepG₂, human hepatoma cell line and galactose moieties of the GEG NPs was confirmed by galactose-specific aggregation test of particles using b-galactose-specific lectin and flow cytometry measurement. It was also concluded from cell cytotoxicity study that HepG₂ cells over expressing ASGPR are more sensitive to TMX-loaded NPs than free TMX as compared with other cell lines. Their studies suggested that specific interaction between HepG₂ cells and GEG of the NPs occurred, which could be exploited for targeting drug-loaded NPs.

Mo and Lim (2005) [26]- developed a novel lectin conjugated isopropyl myristate (IPM)-incorporated poly (D,L-lactic-co-glycolic acid) (PLGA) nanoparticles for the local delivery of Tamoxifen to the lungs. These nanoparticles had a superior in vitro cytotoxicity against A549 and H1299 cells as compared with Tamoxifen loaded nanoparticles without IPM, or Tamoxifen-loaded NPs with only IPM. Thus, their studies showed that these NPs exhibited stronger cell-killing effect because of more efficient cellular uptake via receptor-mediated endocytosis and IPM-facilitated release of Tamoxifen from the nanoparticles.

Shenoy and Amiji (2005) [25]- carried out studies to evaluate and compare the bio distribution profile of Tamoxifen when administered intravenously (i.v.) as a simple solution or when encapsulated in polymeric nanoparticulate formulations, with or without surface-stabilizing agents. Tamoxifen-loaded, polycaprolactone nanoparticles were prepared and in vivo biodistribution studies were carried out in mice bearing a human breast carcinoma xenograft. These nanoparticles when injected in the tumor exhibited a significantly increased level of accumulation of the drug within tumor with time as well as extended their presence in the systemic circulation unlike controls (unmodified nanoparticles or the solution form).

Devalapally et al. (2007)[27]-  developed biodegradable poly(ethylene oxide)-modified poly(1-caprolactone) (PEO–PCL) NPs for the delivery of Tmx and ceramide (CER) to overcome drug resistance in ovarian cancer. Tmx and the apoptotic signaling molecule C (6)-CER were administered intravenously either as a single agent or in combination in aqueous solution and in PEO–PCL NPs to the tumor-bearing mice. There was a significant ( p , 0.05) tumor growth suppression in both wild type SKOV-3 and multi-drug-resistant SKOV-3 (TR) models upon single dose co-administration of Tmx (20 mg/kg) and CER (100 mg/kg) in NP formulations as compared with the individual agents and administration in aqueous solution. The results of their study showed that the combination of TMX and