The Concept of Killing Cancer Tumours with the Assistance of Transdermal Patches And Nano Aptamer Bioconjugates Author: K.M.Devraj* , Vishnu Institute of Pharmaceutical Education and Research, Narsapur,Medak(A.P),India. Co-Author: K.V.Subhaskar Reddy, Vishnu Institute of Pharmaceutical Education and Research,Narsapur, Medak(A.P),India.

ABSTRACT: The number of medications and the ways in which they can be administered have expanded dramatically over the years. Skin is an effective medium from which absorption of the drug takes place and enters the circulatory system through Transdermal patches. This concept explains the involvement of nanosized aptamer bioconjugates that binds the anti-cancer drug irrespective of the nature (lipophilic or hydrophilic) and introduced into the subject and site of action occur which reduces many risks like alopecia, anemia, severe nausea, ulcers and bone marrow damages. This is a manipulated process that depends on the case. This concept is only restricted to cancer tumours in the benign and the pre-malignant stage. Cancer is the most deadliest disease today in the world, the drugs used in chemo-therapy are non-specific and circulates through out the body in the bloodstream when injested. Meanwhile it damages the body systems and kills the cancererous tumours too. But the healthy life is not guaranteed. Keeping this in mind we developed a concept that includes Transdermal patches as the drug delivery system , an aptamer- Bio conjugate binder that binds the anti cancer drug irrespective of its permeable nature and several other aids that are used in the effective permea-bility and absorption of the drug through the skin where beneath the tumours are subjected. Hence though the drug is non-specific it would act on site and lump size: quantity ratio would be applied to avoid further complications As far no work is carried out done on this. Work on this conception will bring a revolution in the drug delivery systems used in cancer treatments. .

INTRODUCTION Cancers are caused by abnormalities in the genetic material of the transformed cells. These abnormalities may be due to the effects of carcinogens, such as tobacco smoke, radiation, chemicals, or infectious agents. Other cancer-promoting genetic abnormalities may randomly occur through errors in DNA replication, or are inherited, and thus present in all cells from birth. The heritability of cancers is usually affected by complex interactions between carcinogens and the host's genome.Cancers are caused by a series of mutations. Each mutation alters the behavior of the cell somewhat. Cancer is fundamentally a disease of regulation of tissue growth. In order for a normal cell to transform into a cancer cell, genes which regulate cell growth and differentiation must be altered.]Genetic changes can occur at many levels, from gain or loss of entire chromosomes to a mutation affect-ing a single DNA nucleotide. There are two broad categories of genes which are affected by these changes. Oncogenes may be normal genes which are expressed at inappropriately high levels, or altered genes which have novel properties. In either case, expression of these genes promotes the malig-nant phenotype of cancer cells. Tumor suppressor genes are genes which inhibit cell division, survival, or other properties of cancer cells. Tumor suppressor genes are often disabled by cancer-promoting genetic changes. Typically, changes in many genes are required to transform a normal cell into a cancer cell.

Again cancers are of two types benign and malignant. The benign tumours are the cancers cells from the symptomatic stage to initiation stage and malignant tumours are those which are rapidly mutated with a firm root. The treatment for the benign tumours are much satisfactory that can avoid major risks if diagnosed in the proper time and treated , where as malignant tumours are fatal, there are less chances for the treatment using chemo-therapy and radiation therapy using harmful rays , if imagined that a person is survived after treatment , its not guaranteed that health is assumable and leads a painful life , all time taking measures to recover his body and its mechanisms. Such is the adverse nature of the chemotherapeutic drugs and radiation therapy. The Illustrations below sow the benign and malignant stages of cancer.

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CHEMOTHERAPY:

Chemotherapy, in its most general sense, is the treatment of disease by chemicals especially by killing micro-organisms or cancerous cells. In popu-lar usage, it refers to antineoplastic drugs used to treat cancer or the combination of these drugs into a cytotoxic standardized treatment regimen. In its non-ontological use, the term may also refer to antibiotics (antibacterial chemotherapy). Most commonly, chemotherapy acts by killing cells that divide rapidly, one of the main properties of cancer cells. This means that it also harms cells that divide rapidly under normal circumstances: cells in the bone marrow, digestive tract and hair follicles; this results in the most common side effects of chemotherapy�myelosuppression (decreased production of blood cells), mucositis (inflammation of the lining of the digestive tract) and alopecia (hair loss). Other uses of cytostatic chemothera-py agents are the treatment of autoimmune diseases such as multiple sclerosis, Dermatomyositis, Polymyositis, Lupus, rheumatoid arthritis and the suppression of transplant rejections . Newer anticancer drugs act directly against abnormal proteins in cancer cells; this is termed therapy. Hence chemotherapy is the most painful treatment and the problem arises with these neoplastic agents because of its non-specificity in the action. Its kills the tumours but also the healthy normally mutating cells. Hence drugs taken orally gets in to the whole body circulation and goes on damaging the systems it passes through and kills the cancerous tumours too.Therefore it takes a long time to recover the body systems.So,targeted drug delivery systems are most effective dosage forms that show a specific site action.

Dosage of chemotherapy can be difficult: If the dose is too low, it will be ineffective against the tumor, whereas, at excessive doses, the toxicity (side effects, neutropenia) will be intolerable to the patient. This has led to the formation of detailed "dosing schemes" in most hospitals, which give guid-ance on the correct dose and adjustment in case of toxicity. In immunotherapy, they are in principle used in smaller dosages than in the treatment of malignant diseases.In most cases, the dose is adjusted for the patient's body surface area, a measure that correlates with blood volume. The BSA is usually calculated with a mathematical formula or a nomogram, using a patient's weight and height, rather than by direct measurement.

Most chemotherapy is delivered intravenously, although a number of agents can be administered orally (e.g., melphalan, busulfan, capecitabine). In some cases, isolated limb perfusion (often used in melanoma), or isolated infusion of chemotherapy into the liver or the lung have been used. The main purpose of these approaches is to deliver a very high dose of chemotherapy to tumour sites without causing overwhelming systemic dam-age.Depending on the patient, the cancer, the stage of cancer, the type of chemotherapy, and the dosage, intravenous chemotherapy may be given on either an inpatient or an outpatient basis. For continuous, frequent or prolonged intravenous chemotherapy administration, various systems may be surgically inserted into the vasculature to maintain access. Harmful and lethal toxicity from chemotherapy limits the dosage of chemotherapy that can be given. Some tumours can be destroyed by sufficiently high doses of chemotherapeutic agents. However, these high doses cannot be given because they would be fatal to the patient.

Targeted delivery mechanisms

Specially targeted delivery vehicles aim to increase effective levels of chemotherapy for tumor cells while reducing effective levels for other cells. This should result in an increased tumor kill and/or reduced toxicity.

Specially targeted delivery vehicles have a differentially higher affinity for tumor cells by interacting with tumor-specific or tumour-associated anti-gens.

In addition to their targeting component, they also carry a payload - whether this is a traditional chemotherapeutic agent, or a radioisotope or an im-mune stimulating factor. Specially targeted delivery vehicles vary in their stability, selectivity, and choice of target, but, in essence, they all aim to increase the maximum effective dose that can be delivered to the tumor cells. Reduced systemic toxicity means that they can also be used in sicker patients, and that they can carry new chemotherapeutic agents that would have been far too toxic to deliver via traditional systemic approaches.

Adverse effects

Chemotherapeutic techniques have a range of side effects that depend on the type of medications used. The most common medications mainly affect the fast-dividing cells of the body, such as blood cells and the cells lining the mouth, stomach, and intestines. Common side effects include :

Depression of the immune system, which can result in potentially fatal infections. Although patients are encouraged to wash their hands, avoid sick people, and to take other infection-reducing steps, about 85% of infections are due to naturally occurring microorganisms in the pa-tient's own gut and skin. This may manifest as systemic infections, such as sepsis, or as localized outbreaks, such as shingles. Sometimes, chemotherapy treatments are postponed because the immune system is suppressed to a critically low level.

The treatment can be physically exhausting for the patient, who might already be very tired from cancer-related fatigue. It may produce mild to severe anemia. Treatments to mitigate anemia include hormones to boost blood production (erythropoietin), iron supplements, and blood transfusions.

Tendency to bleed easily. Medications that kill rapidly dividing cells or blood cells are likely to reduce the number of platelets in the blood, which can result in bruises and bleeding. Extremely low platelet counts may be temporarily boosted through platelet transfusions. Sometimes, chemotherapy treatments are postponed to allow platelet counts to recover.

Gastrointestinal distress. Nausea and vomiting are common side effects of chemotherapeutic medications that kill fast-dividing cells. This can also produce diarrhea or constipation. Malnutrition and dehydration can result when the patient doesn't eat or drink enough, or when the pa-tient vomits frequently, because of gastrointestinal damage. This can result in rapid weight loss, or occasionally in weight gain, if the patient eats too much in an effort to allay nausea or heartburn. Weight gain can also be caused by some steroid medications. These side effects can frequently be reduced or eliminated with antiemetic drugs. Self-care measures, such as eating frequent small meals and drinking clear liquids or ginger tea, are often recommended. This is a temporary effect, and frequently resolves within a week of finishing treatment.

Hair loss. Some medications that kill rapidly dividing cells cause dramatic hair loss; other medications may cause hair to thin. These are tem-porary effects: hair usually starts growing back a few weeks after the last treatment, sometimes with a tendency to curl that may be called a "chemo perm".

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Damage to specific organs may occur, with resultant symptoms:

Cardiotoxicity (heart damage) Hepatotoxicity (liver damage) Nephrotoxicity (kidney damage) Ototoxicity (damage to the inner ear), producing vertigo.

Nausea and vomiting

Chemotherapy-induced nausea and vomiting (CINV) is common, but use of less emetogenic chemotherapy and better antiemetics have reduced the risks in recent times. Stimulation of the vomiting center in the brain results in the coordination of responses from the diaphragm, salivary glands, cra-nial nerves, and gastrointestinal muscles to produce the interruption of respiration and forced expulsion of stomach contents known as retching and vomiting. The vomiting center is stimulated directly by afferent input from the vagal and splanchnic nerves, the pharynx, the cerebral cortex, cholin-ergic and histamine stimulation from the vestibular system, and efferent input from the chemoreceptor trigger zone (CTZ). The CTZ is in the area postrema, outside the blood-brain barrier, and is thus susceptible to stimulation by substances present in the blood or cerebral spinal fluid. The neuro-transmitters dopamine and serotonin stimulate the vomiting center indirectly via stimulation of the CTZ.

The 5-HT3 inhibitors are the most effective antiemetics and constitute the single greatest advance in the management of nausea and vomiting in pa-tients with cancer. These drugs are designed to block one or more of the signals that cause nausea and vomiting. The most sensitive signal during the first 24 hours after chemotherapy appears to be 5-HT3. Blocking the 5-HT3 signal is one approach to preventing acute emesis (vomiting), or emesis that is severe, but relatively short-lived. Approved 5-HT3 inhibitors include Dolasetron (Anzemet), Granisetron (Kytril, Sancuso), and Ondansetron (Zofran). The newest 5-HT3 inhibitor, palonosetron (Aloxi), also prevents delayed nausea and vomiting, which occurs during the 2�5 days after treatment. A granisetron transdermal patch (Sancuso) was approved by the FDA in September 2008. The patch is applied 24�48 hours before chemo-therapy and can be worn for up to 7 days depending on the duration of the chemotherapy regimen.

Another drug to control nausea in cancer patients became available in 2005. The substance P inhibitor aprepitant (marketed as Emend) has been shown to be effective in controlling the nausea of cancer chemotherapy. The results of two large controlled trials were published in 2005, describing the efficacy of this medication in over 1,000 patients.

Some studies and patient groups claim that the use of cannabinoids derived from marijuana during chemotherapy greatly reduces the associated nau-sea and vomiting, and enables the patient to eat. Some synthetic derivatives of the active substance in marijuana (Tetrahydrocannabinol or THC) such as Marinol may be practical for this application. Natural marijuana, known as medical cannabis is also used and recommended by some oncolo-gists, though its use is regulated and not legal everywhere.

Other side effects

In particularly large tumors, such as large lymphomas, some patients develop tumor lysis syndrome from the rapid breakdown of malignant cells. Although prophylaxis is available and is often initiated in patients with large tumors, this is a dangerous side effect that can lead to death if left un-treated.Less common side effects include pain, red skin (erythema), dry skin, damaged fingernails, a dry mouth (xerostomia), water retention, and sexual impotence. Some medications can trigger allergic or pseudoallergic reactions.Some patients report fatigue or non-specific neurocognitive problems, such as an inability to concentrate; this is sometimes called post-chemotherapy cognitive impairment, referred to as "chemo brain" by pa-tients' groups.Specific chemotherapeutic agents are associated with organ-specific toxicities, including cardiovascular disease (e.g., doxorubicin), in-terstitial lung disease (e.g., bleomycin) and occasionally secondary neoplasm (e.g., MOPP therapy for Hodgkin's disease).

So, this was a clear introduction about cancer , chemotherapeutic drugs and its adverse effects.

After taking a glance of all the considerations in cancers , Now it becomes a responsibility of a pharmacist to reduce the side effects and propose an optimized way of delivering these chemotherapeutic drugs in the most effective way that reduces all the side effects and a novel drug delivery sys-tem, which in the future may not panic the mankind as what the present situations are threatening today.

What is a localized therapy?

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This is a traditional procedure of diagnosing and killing the cancer cells, Hence the localized therapy involves the usage of chemicals and radiation.

The systemic diagnosis of a tumour using ultra microscopy.

Strange, But yes chemotherapeutic drugs in transdermal patches:

Our aim is to induce the drug in the most novel way. Because Skin is an effective medium from which absorption of the drug takes place and enters the circulatory system. Various types of transdermal patches are used to incorporate the active ingredients into the circulatory system via skin. The patches have been proved effective because of its large advantages over other controlled drug delivery systems.

A TRANSDERMAL PATCH

The number of medications and the ways in which they can be administered have expanded dramatically over the years. Transdermal drug delivery system has been in existence for a long time. In the past, the most commonly applied systems were topically applied creams and ointments for dermatological disorders. The occurrence of systemic side-effects with some of these formulations is indicative of absorption through the skin. A number of drugs have been applied to the skin for systemic treatment. In a broad sense, the term transdermal delivery system includes all topically administered drug formulations intended to deliver the active ingredient into the general circulation. Transdermal therapeutic systems have been de-signed to provide controlled continuous delivery of drugs via the skin to the systemic circulation. Moreover, it over comes various side effects like painful delivery of the drugs and the first pass metabolism of the drug occurred by other means of drug delivery systems. So, this Transdermal Drug Delivery System has been a great field of interest in the recent time. Many drugs which can be injected directly into the blood stream via skin have been formulated. The main advantages of this system are that there is controlled release of the drug and the medication is painless. The drug is main-ly delivered to the skin with the help of a transdermal patch which adheres to the skin. A Transdermal Patch has several components like liners, ad-herents, drug reservoirs, drug release membrane etc. which play a vital role in the release of the drug via skin. Various types of patches along with various methods of applications have been discovered to delivery the drug from the transdermal patch. Because of its great advantages, it has become one of the highly research field among the various drug delivery system. Here, a general view over the transdermal patch has been discussed along with its advantages, disadvantages, methods of applying, care taken while applying, types and applications of transdermal patch and recent advances along with recent patents and market products.

TRANDERMAL DRUG DELIVERY TECHNOLOGY

A transdermal patch or skin patch is a medicated adhesive patch that is placed on the skin to deliver a specific dose of medication through the skin and into the bloodstream.

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The first commercially available prescription patch was approved by the U.S. Food and Drug Administration in December 1979, which adminis-tered scopolamine for motion sickness.

Components of Transdermal Patch

Liner - Protects the patch during storage. The liner is removed prior to use.

Drug - Drug solution in direct contact with release liner.

Adhesive - Serves to adhere the components of the patch together along with adhering the patch to the skin.

Membrane - Controls the release of the drug from the reservoir and multi-layer patches.

Backing - Protects the patch from the outer environment.

Conditions in which Transdermal Patches are used

Transdermal patch is used when:

(1)When the patient has intolerable side effects (including constipation) and who is unable to take oral medication (dysphagia) and is requesting an alternative method of drug delivery.

(2) Where the pain control might be improved by reliable administration. This might be useful in patients with cognitive impairment or those who for other reasons are not able to self-medicate with their analgesia.

(3) It can be used in combination with other enhancement strategies to produce synergistic effects.

Conditions in which Transdermal Patches are not used

The use of transdermal patch is not suitable when:

(1)Cure for acute pain is required.

(2) Where rapid dose titration is required.

(3) Where requirement of dose is equal to or less then 30 mg/24 hrs.

Two major factors affect the bioavaibility of the drug via transdermal routes:

(1) Physiological factors (2) Formulation factors

Physiological factors include

(1) Stratum corneum layer of the skin (2) Anatomic site of application on the body (3) Skin condition and disease (4) Age of the patient (5) Skin me-tabolism (6) Desquamation (peeling or flaking of the surface of the skin) (7) Skin irritation and sensitization (7) Race

Formulation factors include

(1)Physical chemistry of transport (2) Vehicles and membrane used (3) Penetration enhancers used (4) Method of application (5) Device used

Care taken while applying transdermal patch

(1)The part of the skin where the patch is to be applied should be properly cleaned. (2) Patch should not be cut because cutting the patch destroys the drug delivery system. (3) Before applying a new patch it should me made sure that the old patch is removed from the site. (4) Care should be taken while applying or removing the patch because anyone handling the patch can absorb the drug from the patch. (5) The patch should be applied accu-rately to the site of administration.

Mechanism of Action of Transdermal Patch

The application of the transdermal patch and the flow of the active drug constituent from the patch to the circulatory system via skin occur through various methods.

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1. Iontophoresis

Iontophoresis passes a few milliamperes of current to a few square centimeters of skin through the electrode placed in contact with the formulation, which facilitates drug delivery across the barrier. Mainly used of pilocarpine delivery to induce sweating as part of cystic fibrosis diagnostic test. Ion-tophoretic delivery of lidocaine appears to be a promising approach for rapid onset of anesthesia.

2.Electroporation

Electroporation is a method of application of short, high-voltage electrical pulses to the skin. After electroporation, the permeability of the skin for diffusion of drugs is increased by 4 orders of magnitude. The electrical pulses are believed to form transient aqueous pores in the stratum corneum, through which drug transport occurs. It is safe and the electrical pulses can be administered painlessly using closely spaced electrodes to constrain the electric field within the nerve-free stratum corneum.

3.Application by ultrasound

Application of ultrasound, particularly low frequency ultrasound, has been shown to enhance transdermal transport of various drugs including mac-romolecules. It is also known as sonophoresis. Katz et al. reported on the use of low-frequency sonophoresis for topical delivery of EMLA cream.

4.Use of microscopic projection

Transdermal patches with microscopic projections called microneedles were used to facilitate transdermal drug transport. Needles ranging from ap-proximately 10-100 µm in length are arranged in arrays. When pressed into the skin, the arrays make microscopic punctures that are large enough to deliver macromolecules, but small enough that the patient does not feel the penetration or pain. The drug is surface coated on the microneedles to aid in rapid absorption. They are used in development of cutaneous vaccines for tetanus and influenza.

Various other methods are also used for the application of the transdermal patches like thermal poration, magnetophoresis, and photomechanical waves. However, these methods are in their early stage of development and required further detail studying.

Types of Transdermal Patch

1. Single-layer Drug-in-Adhesive

The adhesive layer of this system also contains the drug. In this type of patch the adhesive layer not only serves to adhere the various layers together, along with the entire system to the skin, but is also responsible for the releasing of the drug. The adhesive layer is surrounded by a temporary liner and a backing.

2. Multi-layer Drug-in-Adhesive

The multi-layer drug-in adhesive patch is similar to the single-layer system in that both adhesive layers are also responsible for the releasing of the drug. The multi-layer system is different however that it adds another layer of drug-in-adhesive, usually separated by a membrane (but not in all cas-es). This patch also has a temporary liner-layer and a permanent backing.

3. Reservoir

Unlike the Single-layer and Multi-layer Drug-in-adhesive systems the reservoir transdermal system has a separate drug layer. The drug layer is a liq-uid compartment containing a drug solution or suspension separated by the adhesive layer. This patch is also backed by the backing layer. In this type of system the rate of release is zero order.

4. Matrix

The Matrix system has a drug layer of a semisolid matrix containing a drug solution or suspension. The adhesive layer in this patch surrounds the drug layer partially overlaying it.

5. Vapour Patch

In this type of patch the adhesive layer not only serves to adhere the various layers together but also to release vapour. The vapour patches are new on the market and they release essential oils for up to 6 hours. The vapours patches release essential oils and are used in cases of decongestion mainly. Other vapour patches on the market are controller vapour patches that improve the quality of sleep. Vapour patches that reduce the quantity of ciga-rettes that one smokes in a month are also available on the market.

Recent research done in the field:

Many research works have been and are few are going on in this field. Few of the latest research done in the field of transdermal patches are stated below:

Pain-free diabetic monitoring using transdermal patches

The first prototype patch measures about 1cm2 and is made using polymers and thin metallic films. The 5×5 sampling array can be clearly seen, as well as their metallic interconnections. When the seal is compromised, the interstitial fluid, and the biomolecules contained therein, becomes accessi-

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ble on the skin surface. Utilizing micro-heating elements integrated into the structural layer of the patch closest to the skin surface, a high-temperature heat pulse can be applied locally, breaching the stratum corneum. During this ablation process, the skin surface experiences temperatures of 130°C for 30ms duration. The temperature diminishes rapidly from the skin surface and neither the living tissue nor the nerve endings are affected. This painless and bloodless process results in disruption of a 40�50ìm diameter region of the dead skin layer, approximately the size of a hair folli-cle, allowing the interstitial fluid to interact with the patch's electrode sites.

Testosterone Transdermal Patch System in Young Women with Spontaneous Premature Ovarian Failure

In premenopausal women, the daily testosterone production is approximately 300 µg, of which approximately half is derived from the ovaries and half from the adrenal glands. Young women with spontaneous premature ovarian failure (sPOF) may have lower androgen levels, compared with normal ovulatory women. Testosterone transdermal patch (TTP) was designed to deliver the normal ovarian production rate of testosterone. The ad-dition of TTP to cyclic E2/MPA therapy in women with sPOF produced mean free testosterone levels that approximate the upper limit of normal.

Transdermal Patch of Oxybutynin used in overactive Bladder

The product is a transdermal patch containing Oxybutynin HCl and is approved in US under the brand name of Oxytrol and in Europe under the brand name of Kentera. OXYTROL is a thin, flexible and clear patch that is applied to the abdomen, hip or buttock twice weekly and provides con-tinuous and consistent delivery of oxybutynin over a three to four day interval. OXYTROL offers OAB patient�s continuous effective bladder control with some of the side effects, such as dry mouth and constipation encountered with and oral formulation. In most patients these side effects however are not a troublesome.

Transdermal Patch (Ortho Evra�)

The patch is 4.5 square centimeters in size and has three layers: the inner release liner which should be removed before application, a layer containing hormones, and an outer polyester protective layer. The patch contains 6 milligram of progestin, Norelgestromin 0.75 milligram of Ethinyle Estradiol. The patch is applied on the skin through which the hormones are absorbed in order to provide continuous flow of hormones during menstrual cycle. The patch is marketed by Ortho McNeil Pharmaceutical with the brand name Ortho Evra.

Rotigotine transdermal patch

The rotigotine transdermal patch is used for symptom control in Parkinson�s disease. The patches are effective in reducing the symptoms of early Parkinson�s disease, and in reducing �off� time in advanced Parkinson�s disease. It is available in market under the brand name of NeuproR.

Before these patches go into the market, they have to be carefully studied. One way to study these patches are through the use of Franz Diffusion Cell systems. This system is used to study the effects of temperature on the permeated amount of a specific drug on a certain type of membrane, which in this case would be the membrane that is used in the patches. A Franz Diffusion Cell system is composed of a receptor and a donor cell. In many of these research studies the following procedure is used. The donor cell is set at a specific temperature (the temperature of the body), while the receptor cell is set at different one (temperature of the environment).

Different runs are performed using different temperatures to study the impact of temperature on the release of a certain medicament through a certain type of membrane. Although different concentrations of the medicament are used in this study, they do not affect the amount permeated through the membrane (the process is constant). From Chemical kinetics it�s concluded that these studies are zero order, since the concentration plays no role in

the permeated amount through the membrane.

Some pharmaceuticals must be combined with substances, such as alcohol, within the patch to increase their ability to penetrate the skin in order to be used in a transdermal patch. Others can overwhelm the body if applied in only one place, and are often cut into sections and applied to different parts of the body to avoid this, such as nitroglycerin. Many molecules, however, such as insulin, are too large to pass through the skin.

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A lot of progress has been done in the field of Transdermal Patches. Due to large advantages of the Transdermal Drug Delivery System, this system interests a lot of researchers. Many new researches are going on in the present day to incorpo-rate newer drugs via this system. Various devices which help in increasing the rate of absorption and penetration of the drug are also being studied. However, in the present time due to certain disadvantages like large drug molecules cannot be delivered, large dose cannot be given, the rate of absorption of the drug is less, skin irritation, and etc. the use of the Transdermal Drug Delivery System has been limited. But, with the invention of the new devices and new drugs which can be incorporated via this system, it used is increasing rapidly in the present time.

DRUG ACTION

For many years, the clinical pharmacology of anticancer drugs was poorly understood due primarily to the lack of sensitive and specific assays for measuring the concentration of these compounds in biologic fluids. The development and widespread application of high-performance liquid chroma-tography and other sophisticated analytical tools allows measurements of plasma drug (and metabolite) concentrations to be performed with a high degree of precision and efficiency. Clinical pharmacokinetic studies of anticancer drugs, particularly new agents, are performed routinely in the con-text of phase I and II clinical trials. Although the pharmacokinetic characteristics of many drugs have been well defined, the application of this in-formation to the clinical care of individual patients still lags far behind other therapeutic areas in medicine. Plasma concentrations of digoxin, theo-phylline, aminoglycosides, phenytoin, and many other drugs are monitored routinely to optimize efficacy and reduce toxicity; yet the measurement of doxorubicin or 5-fluorouracil (5-FU) concentrations in plasma is virtually meaningless, since there are no established relationships (of proven thera-peutic utility) between pharmacokinetics and clinical effects for these or most other commonly used anticancer drugs. A notable exception is metho-trexate where delayed clearance is known to be related to an increased risk of severe toxicity.

Pharmacokinetic-pharmacodynamic relationships are difficult to develop for many reasons. For most antineoplastic agents, there is a delay of days to weeks between measurement of drug concentrations and clinical effect. It is therefore necessary to observe patients frequently following chemother-apy administration to accurately assess the drug effect. The maximum observed effect may be significantly less than the true maximum effect, unless patients are seen daily. Although the desired effect of cancer chemotherapy is a reduction in tumor volume, usually optimized by maximizing the dose, the narrow therapeutic index of cancer chemotherapy drugs requires that most dosing strategies focus on minimizing toxicity rather than on op-timizing efficacy. Despite these difficulties, significant progress has recently been made in understanding the clinical pharmacodynamics of anti-cancer drugs, and further studies in this area will no doubt lead to more rational administration of cancer chemotherapy.

This chapter will focus on the principles of clinical pharmacology as they apply to cancer chemotherapy and will attempt to illustrate how an under-standing of clinical pharmacokinetics and pharmacodynamics can optimize the therapeutic index of cancer chemotherapy.

General Mechanisms of Drug Action

The initial requirement for drug action is adequate drug delivery to the target site. This depends largely on blood flow in the tumor bed and the diffu-sion characteristics of the drug in tissue. However, delivery may also be influenced by the extent of plasma protein-binding and, for orally adminis-tered drugs, by absorption, first-pass metabolism in the liver, and the requirement for activation by various mechanisms. Blood flow across a capil-lary bed is directly proportional to the arteriovenous pressure difference and inversely proportional to the geometric and viscous resistances. The ge-ometric resistance to blood flow increases with increasing tumor size, a factor that may limit drug and oxygen delivery to large tumors and thereby diminish the effectiveness of treatment with chemotherapy or radiation.

The most common route of drug administration for treatment of both localized and disseminated disease is by intravenous infusion, which, by defini-tion, makes 100% of the drug available in the blood. Drugs may be administered by a number of routes in addition to intravenous infusions, however, to achieve specialized pharmacologic and therapeutic goals. Regional administration may be employed to more directly target the drug to the princi-pal tumor site and to achieve a higher drug concentration in the vicinity of the tumor. Intraperitoneal infusion of cisplatin for ovarian cancers, intra-pleural administration of bleomycin in the treatment of solid tumors, and intrathecal administration of cytarabine (ara-C) as a means of treating leu-kemias are examples of intracavitary drug delivery. Alternatively, intravascular administrations such as intra-arterial infusion of fluorodeoxyuridine into the hepatic artery for treatment of liver diseases has been used to achieve a pharmacologic advantage. Although oral administration is the most convenient and least expensive route of drug administration, it is associated with problems of inconsistent drug bioavailability among and within pa-tients. More consistent pharmacokinetics are achieved with subcutaneous or intramuscular drug injections.

Delivery of the drug to the target cell is also dependent on the rate of removal from the blood. Excretion, either by the kidneys or by the biliary route, constitutes a major clearance mechanism. In addition, many drugs are cleared by metabolism to less effective or inactive metabolites as the blood passes through large body organs. Drug binding to plasma proteins can also effectively lower the concentration of free drug that is available for entry into target cells to a small fraction of the total concentration in blood.

Membrane Transport

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In order to produce cytotoxicity, most anticancer drugs require uptake into the cell. A number of mechanisms exist for the passage of drugs across the plasma membrane including passive diffusion, facilitated diffusion, and active transport systems. Passive diffusion of drugs through the lipid bilayer structure of the plasma membrane is a function of the size, lipid solubility, and charge of the drug molecule. If the extracellular drug concentration is constant, then drug accumulation by the cell will continue until the rate of drug uptake from the extracellular space is equal to the rate of drug efflux from the cell. At this point, a dynamic equilibrium is reached and intracellular and extracellular drug concentrations are equal. As drug is cleared from the extracellular space, intracellular drug levels will decline if the drug is not bound or metabolized intracellularly. An important feature of the passive diffusion process is that it does not saturate. That is, as the extracellular drug concentration increases, influx into the cell increases propor-tionally and high intracellular drug levels can be achieved. Passive diffusion, however, is a highly inefficient and nonspecific process, although it may be a particularly important mechanism of drug uptake when carrier-mediated processes are nonfunctional, such as occurs in some cases of meth-otrexate (MTX) resistance.

The passage of physiologically important hydrophilic compounds across the plasma membrane is usually mediated by a specific receptor, or carrier, in the plasma membrane that facilitates the translocation of the substance into or out of the cell. Carrier-mediated transport systems are distinguished from passive diffusion by having a higher degree of specificity and by being saturable at high extracellular drug concentrations due to the presence of a finite number of receptor molecules within the membrane. Once all carrier sites become occupied, further increases in extracellular drug concentra-tion will not produce further increments in drug influx unless a component of passive diffusion comes into play. The affinity of the carrier for the substrate can be estimated from the Michaelis constant (Km), the drug concentration at which the influx rate is one half maximal; the lower the Km, the higher the carrier affinity. Although carrier-mediated systems enhance the rate of influx into the cell, not all carriers are able to translocate com-pounds against electrochemical forces and ultimately develop gradients such that the intracellular concentration exceeds the extracellular drug level. To do so requires the expenditure of energy and the coupling of carrier-mediated transport to an energy-requiring reaction, usually hydrolysis of adenosine triphosphate.

Many antineoplastic drugs, particularly those that are structural analogs of natural compounds, gain entry into the cell by carrier mediated mecha-nisms. The functional and physiologic characteristics of several human nucleoside transporters have been characterized. However, substantial addi-tional information is rapidly emerging as more of these molecules are cloned and their specificities are revealed. Naturally occurring nucleosides are transported by both facilitated diffusion (equilibrative) and by concentrative mechanisms. Nucleoside analogs that are important in cancer therapy al-so use these transporters, but some specificity is emerging. For instance, ara-C, floxidine, and pentostatin appear to use equilibrative transporters, whereas fludarabine, gemcitabine, and cladribine appear to be substrates for concentrative transport systems in addition to equilibrative pathways. Nucleobase transporters have also been identified, but their role in the entry of useful antimetabolites such as thiopurines and 5-FU into the cell has not been established. Transport of reduced folates and methotrexate is an active energy-dependent process that can be mediated by two distinct mechanisms: a membrane carrier system capable of the rapid transport of reduced folates and of 4-amino analogs of folic acid and a group of mem-brane-bound folate receptors termed the folate binding proteins, which are brought into the cell by endocytosis to release ligand before recycling back to the membrane. Candidate cDNAs for this function have now been identified. Altered MTX transport features have been described in acute lym-phoblastic leukemia blasts and in osteosarcoma as a mechanism of acquired resistance. l-phenylalanine mustard uses at least two amino acid transport systems and its influx can be inhibited by the amino acid substrates specific for these transport carriers.

The importance of transmembrane movement of a drug to its pharmacologic effect depends on several factors, including the rate of drug delivery to the tissue, the affinity of the transport process, and the nature of the intracellular biochemical events required for drug action. Although membrane transport can be the rate-limiting step in drug action if it governs the rate at which the drug reaches intracellular targets, this is not always the case. If drug delivery to a cell is slow relative to the influx rate, then the drug effect will be limited primarily by extracellular concentration (i.e., blood flow and diffusion of the drug). Similarly, if a drug requires intracellular activation, such as phosphorylation of nucleoside analogs or polyglutamylation of methotrexate, before it can exert a cytotoxic effect, then the rate-limiting step in drug action could be activation rather than transport if the rate of ac-tivation is slow relative to the rate of influx into the cell.

Finally, it is important to recognize that membrane transport is frequently bidirectional with the final drug concentration in the cell representing the balance between drug influx and drug efflux. These processes may use different carrier systems and operate at different rates. Several efflux systems that appear to have importance in cancer chemotherapy are the systems that mediate various forms of multidrug resistance.

Activation of Anticancer Drugs

Activation Reaction Drug Aquation Cisplatin

Hydrolysis Irinotecan

Polyglutamylation Methotrexate

Phosphorylation Cytarabine

Fludarabine

Cladribine

Phosphoribosylation 5-Fluorouracil

6-Mercaptopurine

6-Thioguanine

Microsomal oxidation Cyclophosphamide

Ifosfamide

Procarbazine

Microsomal reduction Bleomycin

Demethylation Dacarbazine

Hexamethylmelamine

Acetylation Amonafide

Many anticancer drugs require activation before they are able to exert a cytotoxic effect. The activation process may involve chemical or enzymatic reactions in either normal or tumor tissues. Intracellular activation by tumor cells is a critical determinant of effect for virtually all antimetabolites. Nucleoside antimetabolites such as ara-C, fludarabine, gemcitabine, and cladribine require phosphorylation to active nucleotide triphosphate forms and incorporation into DNA before they are able to exert a cytotoxic effect. Nucleobase analogs such as 6-mercaptopurine and 6-thioguanine undergo phosphoribosylation to the nucleoside monophosphate forms, which are active inhibitors of de novo purine nucleotide synthesis. Amination of 6-mercaptopurine to thioguanine monophosphate followed by phosphorylation, reduction to the deoxynucleotide, and a subsequent phosphorylation re-sults in 2'-deoxythioguanine triphosphate, which is a substrate for incorporation into DNA. Phosphoribosylation also converts 5-FU to the mono-

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phosphate, which is then phosphorylated to the diphosphate, reduced to the deoxynucleotide, and dephosphorylated to the active monophosphate F-dUMP, which inhibits thymidylate synthase. Additionally, the drug may be cytotoxic after incorporation of either the ribosyl or deoxyribosyl triphos-phate, respectively, into RNA or DNA. Although methotrexate is an effective enzyme inhibitor in its native form, intracellular conversion of the drug to polyglutamate metabolites significantly increases its potency and facilitates its binding to a number of enzymatic sites. Consistent with this is the finding of a more favorable clinical outcome in ALL patients whose blasts accumulated higher levels of MTX polyglutamates. It is important to note that phosphorylation of nucleic acid analogs and polyglutamylation of MTX produces charged molecules that are unlikely to diffuse or to be trans-ported out of cells.

The rate of formation of the activated drug species in the cell depends on the rate of transmembrane influx of the drug, the amount and affinity of the activating enzyme(s) in the cell, the extent of competition by the naturally occurring substrates of the activating enzymes, and the rate of degradation of the activated drug by catabolic enzymes. For many antimetabolites, membrane transport is rapid relative to enzymatic activation and is therefore not rate limiting. Once inside the cell, antimetabolites must compete with the natural enzyme substrates for binding and activation. Finally, the acti-vated drug then becomes a substrate for catabolic enzymes in the cell that tend to degrade it to the parent compound or to an inactive metabolite. Thus, the concentration of the active cytotoxic drug in the cell is the result of all of these processes.

The pyrimidine nucleoside analog, ara-C, provides an excellent example of these processes. Ara-C gains entry to the cell by a high-capacity equili-brative nucleoside transport system; transport velocity is nearly proportional to ara-C concentration up to 100 mM. This process may limit ara-C acti-vation in cells at plasma ara-C concentrations < 1 ìM achieved by standard dose rates (< 20 mg/m

2/h). However, at higher dose rates that achieve > 10 ìM ara-C in plasma (250 mg/m2/h), the transport system provides cellular concentrations of ara-C that saturate the rate of ara-C phosphorylation. After gaining entry to the cell, ara-C is metabolized in three successive phosphorylation reactions to ara-C triphosphate (ara-CTP), which, after its in-corporation into replicating or repairing DNA by various DNA polymerases, is inhibitory to cell growth. The initial activating enzyme, deoxycytidine kinase, is present at the lowest specific activities in human leukemic blasts and is believed to be the rate-limiting step in the formation of ara-CTP and probably for incorporation of the drug into DNA. At each phosphorylation step, ara-C and its metabolites compete with endogenous deoxycytidine and its nucleotides for enzyme binding. Biochemical modulation strategies that reduce dCTP and thereby activate dCyd kinase result in increased ara-CTP formation and improved clinical response. Opposing the activation of ara-C are cytidine deaminase and dCMP deaminase, which convert ara-C and ara-CMP, respectively, to inactive uracil derivatives. In addition, the activity of phosphatases such as 5'-nucleotidase, the activities of which differ among cell types, may be important determinants of the steady-state ara-CTP concentrations and the rate of elimination of the triphos-phate at the end of an ara-C infusion. The response of patients with acute leukemia treated with single-drug ara-C, either on an intermittent schedule or by continuous infusion, was strongly correlated with the ability of cells to retain ara-CTP or with the steady-state ara-CTP concentrations in blasts during therapy. These findings validate the importance of favorable pharmacokinetic characteristics for response to ara-C in particular and provide a basis for pharmacologic modulation strategies with other drugs.Loss or diminished affinity of an activating enzyme or enhanced activity of a catabol-ic enzyme may be responsible for drug resistance. Although molecular reagents are now available that have permitted the discovery of dCyd kinase deficiencies in selected clinical samples, this does not appear to be a major cause of clinical resistance to ara-C because the blasts of patients with re-sistant disease accumulate ara-CTP levels similar to those of responders.

Drug Targets

Although cytotoxic anticancer drugs have traditionally been classified based on their mechanisms of action or their origins, they can also be grouped based on the target of drug action. There are essentially four potential targets of drug action: nucleic acids, specific enzymes, microtubules, and hor-mone/growth factor receptors. When nucleic acids are the target, it is generally DNA rather than RNA that is presumed to cause cell death. There are several mechanisms by which drugs can bind DNA, the most well understood being alkylation of nucleophilic sites within the double helix. Most clinically effective alkylating agents have two moieties capable of developing a charged carbon that binds covalently to negatively charged sites on DNA such as the O6 or N7 positions of guanine. The cross-linking of the two strands of DNA produced by the bifunctional alkylating agents prevents the use of that DNA as a template for further DNA and RNA synthesis leading to inhibition of DNA replication and cell death. Although alkylating agents are among the most widely used drugs in clinical oncology, the relationship of pharmacologic parameters to clinical effects has not been well defined for these agents. In part, this has been due to the lack of sensitive and specific techniques to detect drug-DNA binding in clinical specimens. Studies of chlorambucil-DNA binding in the tumor cells of patients with chronic lymphocytic leukemia have demonstrated considerable heterogenei-ty in drug-DNA binding among patient samples, but no clear correlations between amount of drug bound and disease stage or sensitivity to treatment have been shown, although the drug clearly targets purines. In contrast, the formation of cisplatin adducts to DNA has been shown to correlate with cell kill in mammalian tumor cell lines. Immunologic methods have been used to quantitate platinum-DNA adduct formation in either peripheral white blood cells after cisplatin therapy or in buccal cells of patients receiving cisplatin with carboplatin chemotherapy. A subsequent study that used atomic absorption spectroscopy to quantitate total cell platinum in lymphocytes indicated a relationship between the adduct levels after the first dose of either single-drug cisplatin or carboplatin and clinical response in 49 patients with 24 different tumor types. Although adduct formation in these surrogate cell types was correlated with the response of the tumor to chemotherapy in previously untreated patients, it is difficult to imagine that such determinations will continue to reflect response as the originally platinum-sensitive tumor becomes resistant to treatment. A second mechanism of drug binding to nucleic acids is intercalation, the insertion of a planar ring structure between two adjacent nucleotide bases of DNA. This mechanism is characteristic of many antitumor antibiotics. The antibiotic molecule is non-covalently, although firmly, bound to DNA and distorts the shape of the double helix, resulting in inhibition of RNA or DNA synthesis. Many agents capable of classical intercalation, such as doxorubicin and mitoxan-trone, are also inhibitors of topoisomerase II and may produce DNA strand breaks by inhibition of the reannealing function of this enzyme. Indeed, a direct correlation has been noted between DNA topoisomerase II activity and cytotoxicity in doxorubicin-sensitive and -resistant P388 leukemia cells. A third mechanism of nucleic acid damage is illustrated by the anticancer drug bleomycin. The amino terminal tripeptide of the bleomycin molecule appears to intercalate between guanine-cytosine base pairs of DNA. The opposite end of the bleomycin peptide binds Fe (II) and serves as a ferrous oxidase, able to catalyze the reduction of molecular oxygen to superoxide or hydroxyl radicals that produce DNA strand scission. Predictably, the levels of antioxidant enzymes such as catalase, peroxidases, and super oxide dismutase in plasma and blood are inversely correlated with chromo-somal damage.

Enzymes represent the second general category of targets for chemotherapeutic agents. Antimetabolites function as inhibitors of key enzymes in the purine or pyrimidine biosynthetic pathways or as inhibitors of DNA polymerases. The triphosphate of fludarabine, for instance, is known to inhibit both ribonucleotide reductase and DNA ligase I. After incorporation into DNA, it not only inhibits the function of multiple DNA polymerases, DNA primase, DNA ligase I, but it is also resistant to removal by the proof-reading exonuclease activities associated with DNA polymerases. Since these enzymes are highly active during DNA replication, antimetabolites tend to be cytotoxic only when present in sufficient concentration during the vul-nerable S phase of the cell cycle; these drugs are thus frequently referred to as S phase-specific. Nevertheless, because these enzymes are also re-quired for repair of damaged DNA, it is likely that antimetabolites that inhibit these enzymes will be synergistic with agents that elicit an incision DNA repair response that requires resynthesis of a DNA patch, regardless of cell cycle stage.

The effectiveness of enzyme inhibitors also depends on the amount of the target enzyme, its affinity for the inhibitor, and on the extent of competi-tion by natural substrates for enzyme binding. For example, complete saturation of all dihydrofolate reductase binding sites is required before the en-zyme is effectively inhibited. As MTX inhibits enzymatic activity, dihydrofolate, the natural substrate, accumulates behind the metabolic block and is able to effectively compete with MTX for further enzyme binding. Thus, large amounts of MTX, well in excess of the enzyme binding capacity, are required to effectively inhibit dihydrofolate reductase activity. Similarly, in the case of 5-FU, the dUMP/F-dUMP ratio may be an important determi-nant of optimal inhibition of the target enzyme thymidylate synthase, and high ratios have been associated with lack of tumor response. Similarly, the

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amount of thymidylate synthase expression or activity is an important determinant of 5-FU activity and correlates with therapeutic response. In addi-tion, high basal levels of thymidine phosphorylase have recently been associated with lack of response to 5-FU.

In addition to the enzymes required for purine and pyrimidine biosynthesis, the topoisomerases are important targets of several antineoplastic agents. Topoisomerase I and II catalyze the passage of DNA strands through single- or double-strand breaks in the DNA molecule, respectively, by nicking and then reannealing the DNA strands. Topoisomerase inhibitors bind to the enzyme and stabilize the reaction intermediate enzyme-DNA cleavable complex. This interference with the DNA breakage-resealing process, which is necessary for both DNA replication or RNA transcription, results in DNA strand breaks that are lethal to the cell. The epipodophyllotoxins, etoposide and teniposide, are potent inhibitors of topoisomerase II, as are a number of DNA intercalating agents including doxorubicin, actinomycin D, and amsacrine.

Two topoisomerase I inhibitors, topotecan and irinotecan, have recently been approved for broad clinical use. Whereas topotecan interacts with topoisomerase I directly, irinotecan requires activation by carboxylesterases for SN-38 in order to affect the target. It is not yet clear whether iri-notecan�s antitumor effects are due primarily to intratumoral or intrahepatic activation, although there is some evidence that higher enzyme activity results in a greater cytotoxic effect.

The microtubule spindle structure provides a third target for chemotherapeutic agents, classically the Vinca alkaloids, vincristine and vinblastine, but more recently vinorelbine. The Vinca alkaloids exert their cytotoxic effects by binding to specific sites on tubulin, inhibiting assembly of tubulin into microtubules and ultimately dissolution of the mitotic spindle structure. The microtubule system in cells performs a variety of other important func-tions, including transport of solutes, cell movement, and chromosomal separation, and provides structural integrity, any one of which could potential-ly be disrupted by tubulin binding agents. The taxanes are a newer class of agents, consisting of the natural plant alkaloid paclitaxel and a semisyn-thetic derivative docetaxel. These novel plant alkaloids inhibit cell division by stimulating tubulin polymerization, thus enhancing the formation and stability of microtubules. Paclitaxel-treated cells accumulate large numbers of microtubules, free and in bundles, that disrupt microtubule function and that ultimately cause cell death. Although docetaxel appears to be more potent than paclitaxel, the drugs appear to have similar toxicity profiles.

The search for specific small molecule inhibitors of hormone and growth factor receptors has been ongoing since the demonstration that antiestrogens can be effective treatment for breast cancers that contain the estrogen receptor. Antiandrogens, such as flutamide and bicalutamide, are also important in the treatment of prostate cancer. The rapid advances in our understanding of cancer biology has led to an explosion in new targets and potential new drugs. Drugs targeted against angiogenesis and matrix metalloproteinases have completed phase I testing, and many other new agents are in late preclinical or early clinical evaluation.

But our work had a complication , we were successful in introducing the drug into transdermal patches and after studying the drug action and the drug action mechanism�s.We were stuck up the absorption with the drug because most of the drugs(cancer

drugs) are LIPOPHILIC and where as its much notable that a drug must be a HYDROPHILIC if has to permeate into the skin through perspiration pathway, if permeability is once possible then absorption of the drug is readily possible.

So, we introduced a new particle called APTAMER of nano sized and it is a bio conjugate.

NANO APTAMER BIOCONJUGATES

The combination of targeted delivery and controlled drug release are potentially desirable properties when treating oncologic diseases where it is de-sirable that a cytotoxic dose of the drug is delivered to cancer cells over an extended period of time without killing the surrounding noncancerous tis-sue. Critical to achieving this goal is the engineering of specialized vehicles that encapsulate chemotherapeutic drugs for controlled release, and the targeting of these vehicles to cancer cells with ligands that recognize tumor-specific or tumor-associated antigens . A wide variety of targeting mole-cules have been assessed, with varying degrees of success, for their potential application in cancer therapy, including humanized antibodies and sin-gle-chain Fv generated from murine hybridoma or phage display, minibodies ,and peptides . Interestingly, nucleic acid ligands, also called aptamers , have emerged as a novel class of ligands that rival antibodies in their potential for therapeutic and diagnostic applications . Aptamers are RNA or DNA oligonucleotides that fold by intramolecular interaction into unique three-dimensional conformations capable of binding to target antigens with high affinity and specificity. Considering the many favorable characteristics of aptamers, including small size, lack of immunogenicity, and ease of isolation, which together has resulted in their rapidprogress into clinical trials , we became interested in examining these molecules for targeted de-livery of controlled release polymer drug delivery vehicles.

As proof of concept we used RNA aptamers that bind to the prostate-specific membrane antigen a well-known transmembrane protein that is over expressed on prostate cancer epithelial cells , to develop specialized nanoparticle-aptamerbioconjugates for targeted delivery to prostate cancer cells. Prostate cancer is the single most common form of non-skin malignancyin men in the United States , and vehicles that target this disease for therapy may have a role in the management of this disease . We used the following criteria forthe development of our delivery vehicles: first, we were inter-ested in developing drug encapsulated particles with a polymer system with components that were biocompatible, biodegradable, and approved by the Food and Drug Administration for a prior clinicaluse. Second, we were interested in developing particles that could be efficiently linked to the negatively charged nucleic acid aptamers using simple chemistry with minimal to no adverse effect on the three-dimensional conformation and lig-and recognition properties of aptamers. Third, we were interested in developing delivery vehicles that demonstrate differentially high uptake efficien-cy by the targeted cells. Fourth, we were interested in developing vehicles with extended circulating half-life to increase the likelihood of their effec-tiveness in future therapeutic applications . We used rhodamine-labeled dextran (as a model drug) and developed drug encapsulated pegylated PLA nanoparticles with a negative surface charge. Using the PSMA aptamer, we developed nanoparticle-aptamer bioconjugates and then examined whether the targeting and uptake of these vehicles by prostate epithelial cells, which express the PSMA protein, could be achieved.

Nanoparticles Drug encapsulated nanoparticles were prepared using the water-in-oil-in-water solvent evaporation procedure (double emulsion method) as described previously . The size (nm) and surface charge ( potentialin mV) of nanoparticles were evaluated by Quasi-elastic laser light scattering with a Zeta PALS dynamic light scattering detector(Brookhaven Instruments Corporation, Holtsville, NY; 15 mW laser, incident beam = 676 nm). Surface mor-phology and size were also determined by high-resolution scanning electron microscopy (JEOL6320FV).

Nanoparticle-Aptamer Conjugation Fifty microliters of PLA-PEG-COOH nanoparticle or microparticle suspension ( 10 µg/µL in DNase RNase-free water)was incubated with 200 µL

of 400 mmol/L 1-(3-dimethylaminopropyl)-3-ethylcarbodimidehydrochloride (EDC) and 200 µL of 100 mmol/L N-hydroxysuccinimide(NHS) for 15 minutes at room temperature with gentle stirring.The resulting NHS-activated particles were covalently linkedto 50 µL of 3'-NH2-modified A10

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PSMA aptamer 3'-NH2 and5'-FITC-modified A10 PSMA aptamer where indicated. The resulting aptamer-nanoparticle bioconjugates were washed, resuspended,and preserved in suspension form in DNase RNase-free water. The conjugation of 5'-FITC and 3'-NH2-modified A10 PSMA aptamer to PLA-PEG-COOH nanoparticles and microparticles (0.5 mg/mL)was analyzed with the FACScan flow cytometer and fluorescent microscopy, respec-tively.

Development of Controlled Release Polymer Drug Delivery Vehicles Suitable for Conjugation to Aptamers. We chose to generate particles with PLA and its derivatives. Particles that are generated from PLA are expected to have a neutral to slightly negative surface charge, a desirable characteristic because particles with a positive surface charge may non specifically interact with the negatively charged aptamers and diminish their binding characteristics. We hypothesized that by modifying the terminal ends of polymers with a hydrophilic carboxylic acid functional group, we may provide additional negative charge on particles that could repel the similarly charged aptamers and minimize charge interaction between the aptamers and particle surface. In addition, the presence of the carboxylic acid group on the particle surface would allow an easy conversion to NH Sester for covalent linkage to NH2-modified aptamers. We also decided to incorporate PEG in generating pegylated nanopar-ticle-aptamerbioconjugates. PEG has been shown to prolong nanoparticle circulating half-life , presumably because of the ability of PEG to reduce nonspecific adsorption of bio macromolecules such as proteins and nucleic acid. This latter characteristic may also contribute to minimizing nonspe-cific interaction of conjugated aptamers with the nanoparticle surface.

Development of Nanoparticle-Aptamer Bioconjugate for Targeted Drug Delivery to Prostate Cancer Cells. Two RNA aptamers were previously selected against the extra cellular region of the PSMA protein . One aptamer, A10, has 2'-fluoro-modifiedribose on all pyrimidines and a 3'-inverted deoxythymidine cap, which together confer significant nuclease resistance to this molecule. Because we are in-terested in developing a methodology for using aptamers to target the delivery of controlled released polymer particles to specific cells together with the fact that PSMA represents a well-characterized target of significant importance in prostate cancer, we used the A10 PSMA aptamer to generate our nanoparticle-aptamer bioconjugates. We demonstrated that consistent with the original report , the binding activity of A10 PSMA aptamer is re-stricted to LNCaP cells, which express the PSMA but not PC3 cells, which do not express the PSMA protein(data not shown). We then demonstrated that the A10 aptamer binds to the acinar epithelial cells of prostate cancer tissue consistent with the expected pattern of PSMA expression in the pros-tate gland .

Formalin-fixed, paraffin-embedded prostate cancer slides were stained in the absence (�PSMA Apt) or presence (+PSMA Apt) of biotinylated A10 PSMA aptamer for 30 minutes. Slides were washed with PBS twice, incubated with streptavidin-horseradish peroxidase for 10 minutes, washed with PBS twice, incubated with the peroxidase substrate, washed with PBS twice, mounted, and analyzed by light microscopy. Cells which express the PSMA protein are stained in brown. Using the PLA-PEG-COOH nano- and microparticles and the A10PSMA aptamer, we generated nanoparticle-aptamer and microparticle-aptamer bi-oconjugates for the assessment of our conjugation strategy. To examine the presence of the aptamers on particle surface, we used A10 PSMA ap-tamer with a 5'-FITC-label and a 3'-NH2 modification to yield fluorescent nanoparticle-aptamer and microparticle-aptamer bioconjugates. The acid group on the particle surface was first converted to NHS ester in the presence of EDC and subsequently was covalently coupled to the amine-modified aptamer. To assess the specificity of aptamer interaction with the particle surface, we incubated aptamers with nanoparticles and micropar-ticles without the conversion of carboxylic acid to NHS ester (i.e., absence of EDC); thus, any interaction would be nonspecific (i.e., charge or hy-drogen bond interaction).The microparticle-aptamer bioconjugates were characterized by microscopy and given the size limitations, the nanoparticle-aptamerbioconjugates were characterized by flow cytometry .These data demonstrate the specificity of our conjugation reaction.

A, schematic diagram of the conjugation reaction of PLA-PEG-COOH nanoparticles or microparticles with 3'-NH2-modified, 5'-FITC-labeled PSMA aptamers. B, conjugation of microparticles with aptamers. The acid group on the surface of PLA-PEG-COOH microparticles (MP) were left untreated (�EDC) or converted to NHS ester (+EDC) and particles were incubated with NH2-modified FITC-labeled PSMA aptamers. After the wash, the mi-croparticles resulting after nonspecific interaction with aptamers (�EDC) or after covalent linkage with aptamers (+EDC) were visualized by trans-mission light microscopy (top row) and fluorescent microscopy (bottom row). C, conjugation of nanoparticles with aptamers. The acid group on the surface of PLA-PEG-COOH nanoparticles were left untreated (�EDC) or converted to NHS ester (+EDC), and particles were examined without ap-tamers (NP, black curve) or incubated with NH2-modified FITC-labeled aptamers (+Apt). The bioconjugates resulting from the covalent linkage of aptamers and nanoparticles [NP+Apt (+EDC, green curve)] demonstrated an approximate 7-fold increase in fluorescence when compared with nano-particles that were incubated but had no covalent linkage to aptamers [NP+Apt (�EDC, blue curve)].

Nanoparticle-Aptamer Bioconjugates Selectively and Efficiently Deliver Drugs to Targeted Cells Through time course studies, we next demonstrated that the binding of pegylated nanoparticle-aptamer bioconjugates to LNCaP cells was significant-ly enhanced when compared with control pegylated nanoparticles lacking the A10 PSMA aptamer .In the case of PC3 prostate epithelial cells, which do not express the PSMA protein, no measurable difference in binding was observed between the bioconjugate and the control group (Fig. 4A , bot-tom).The number of nanoparticles attaching to representative cells after 75 minutes incubation of bioconjugates or control nanoparticles with LNCaP or PC3 cells was quantified by fluorescent microscopy .The data demonstrates a 77-fold enhancement in the binding of bioconjugates versus the con-

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trol group in LNCaP cells (13.25± 7.56 versus 0.17 ± 0.45 nanoparticles per cell for bioconjugates versus control group, respectively; mean ±SD, n = 150, P> 0.001). A notable observation was a remarkably low binding efficiency of nanoparticles in non targeted PC3 cells(0.03 ± 0.18 versus 0.30 ±

0.62 nanoparticles per cell for bioconjugates versus control group; mean ±SD, n = 150), presumably attributed to the presence of the PEG group . The binding of bioconjugates to LNCaP cells was clearly detectable at the early time points; however, by16 hours, the differential binding of nano-particle-aptamer bioconjugates versus control group became markedly pronounced.

A, binding of nanoparticle-aptamer bioconjugates to prostate epithelial cells. LNCaP cells and PC3 cells were grown on chamber slides and incubated in culture medium supplemented with 50 µg of rhodamine-labeled dextran encapsulated pegylated nanoparticles, or 50 µg of rhodamine-labeled dex-tran encapsulated pegylated nanoparticle-aptamer bioconjugates (NP-Apt) for 2 hours (left panel) or 16 hours (right panel). Cells were washed in PBS three times, fixed, and permeabilized, stained with 4',6'-diamidino-2-phenylindole (nuclei) and Alexa-Flour Phalloidin (cytoskeleton), washed, and analyzed by light transmission or fluorescent microscopy. The rhodamine-dextran encapsulated nanoparticles or nanoparticle-aptamer bioconju-gates are shown in red. B, evaluation of nanoparticle-aptamer bioconjugates uptake by LNCaP cells. LNCaP cells were grown on chamber slides and incubated in culture medium supplemented with rhodamine-dextran encapsulated nanoparticle-aptamer bioconjugates (50 µg) for 2 hours, washed,

and analyzed at 100x magnification along their z-axis at 0.1 µm intervals by fluorescent microscopy. Approximately 150 individual images were

combined to reconstruct a three-dimensional image of the cell. A through E represent the same LNCaP cell being rotated at 45-degree intervals. This rotation is schematically represented below each panel to show the position of cell and the axis of rotation. F represents a single image through mid z-axis point of cell demonstrating particles in mid cross-section of cell and confirming the intracellular position of particles. The cell nuclei and the actin cytoskeleton are stained with blue (4',6-diamidino-2-phenylindole) and green (Alexa-Flour Phalloidin), respectively. The intracellular rhoda-mine-dextran encapsulated nanoparticle-aptamer bioconjugates are shown in red.

An antibody-mediated enhancement in the rate of PSMA endocytosisin LNCaP cells had previously been reported , and we were able to detect a measurable but a relatively modest enhancement in the rate of PSMA endocytosis in response to the A10 PSMA aptamer binding to LNCaP cells. We next examined if the binding of bioconjugates to LNCaP cells results in particle uptake .Using z-axis fluorescent microscopy and three-dimensional image reconstruction, we studied the localization of the nanoparticle-aptamerbioconjugates after incubation with LNCaP cells. The data demonstrate that even at 2 hours, the particles were largely internalized into cells. This differential uptake of bioconjugates by LNCaP versus PC3 cells was re-producibly observed with different passages of cells and nanoparticle-aptamer bioconjugate preparations.

In summary, aptamers are quickly emerging as a powerful class of ligands with utility in therapeutic applications. Specialized delivery vehicles that use these molecules for targeted delivery will likely have a role in future therapeutic modalities. In this article, we have developed controlled release polymer drug delivery vehicles suitable for conjugation to aptamers and developed, as proof of concept, nanoparticle-aptamer bioconjugates ,which target and are taken up by prostate cancer epithelial cells. These vehicles are potentially suitable for efficient and specific targeted delivery of chemo-therapeutic drugs to prostate cancer cells, and additional in vivo studies are needed to determine the bio distribution of these vehicles after systemic administration. Once these vehicles are optimized, we postulate that a similar strategy may be used to develop nanoparticle aptamer bioconjugates for targeted drug delivery to a myriad of important human diseases.

Aptamers: multifunctional tools for target validation and drug discovery Aptamers are synthetic nucleic acid ligands capable of specifically binding a wide variety of target proteins. They are rapidly iso-lated from combinatorial libraries by automate in vitro selection techniques. Their high inhibitory potential and straight-forward chemical modification qualify them as excellent tools for the detection and functional validation of targets from genomic and pro-teomic research. Recent develments have also demonstrated their potential as substitute inhibitors to streamline identification of small molecule leads in HTS programs, thus linking target validation with drug discovery.

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These are some of the obtained microscopic results of nano aptamer bioconjugates in a schematic study.

OUR AIM

Our aim is to introduce the chemotherapeutic drugs in reduced nano sized and bind inside the nano aptamer bio-conjugates and then replace them in the drug reservoir of transdermal patch, which is well known for its con-trolled release and novel concern.

For an effective mechanism we proposed a formula called DQTD (Drug Quantity to Tumour Destruction)

This formula categorises the lump size : quantity ratio.

Here the size of the lump is measured and an approximate number of cancer cells are estimated, this estimation can be done by mo-lecular X-ray technique and a calculation is carried out in such a way that, the quantity of the drug is estimated to kill one tumour and calculated quantity for the total number of tumours in the lump , Now the transdermal patch is administered on the lump , here the controlled release of drug occurs and the drug acts only on the lump and as the required quantity of drug is available, it further do not lead to over dose or toxicity. Therefore only the tumour in the lump is been killed and assures a quick recovery.

Discussing about other complexities alopecia ,anemia ,nausea are particularly avoid-ed because the drug is never circulated in the whole blood of circulation, the following is illustrated.

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Breast cancer is most common in the women, the above picture shows the tumour in the benign stage .

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How is the formula applied ?

This calculation further optimizes the way of treating the cancer !

In which cases the formula is not applicable ?...........In malignant stages.

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Breast cancer in malignancy

Is�nt that seems to be a novel drug delivery technology !

No work is yet carried out on such hypothesis.Hypothesis may not be the correct denotion because it can be practically applied and its evidential, But need an proper assetion to be carried out .Its a manipulation process because the procedure changes from individual to individual because all the subjects may not suffer from the same kind of cancer. Hence work is under progress.

Why this technology ?

Today�s human being can neglect anything but he may never neglect health when in danger. The chemotherapy is the most painful as well an expensive process and a time consuming too. So, our technology is the most novel when compared to other therapy�s and its an expensive too because every batch is to be prepared for the particular patient as demanded. This is the first step to such drug delivery process but yes, we assure that if seriously focused on this work , it may gradually lead to an evolution in the drug delivery systems and the application of transdermal patches will be extensively used in the treatment of most diseases because of its robustness and most of the side effects like toxicity and incompatibilities can be avoided.

Conclusions :

Our conception is to introduce the nanosized aptamer bioconjugates that binds the Anti-cancer drug irrespective of its nature (lipophilic or hydrophilic) and permeate into the biological layers and release the drug at the site of action. Asusual the Transdermal patch is applied on the surface region where the tumors are located at a distance and drug quantity is specific, the whole process is a manipulation form and an optimized way of killing the cancer tumours avoid-ing the major health risks that the mankind is been facing from the past .As far no work is carried out on this. Work on this conception will bring a revolution in the drug delivery systems used in cancer treatments.

*HOPE FOR CURE*

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- K.M.DEVRAJ

VISHNU INSTITUTE OF PHARMACEUTICAL EDUCATION AND RESEARCH

Narsapur , Medak , Andhra Pradesh , India

e-mail : devrajbuck@gmail.com

Contact: 9533105173

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