FINAL PROJECT LEVOFLOXACIN

NEW DRUG DEVELOPMENTAL &

APPROVAL PROCESS

L

EVOMAX

(Levofloxacin)

Submitted By:

Zohaib Ahmad(Roll#14)

Jalwaz Tihami(Roll#20)

Azeem Imam(Roll#25)

Rizwan Rashid(Roll#43)

Ali Tariq(Roll#136)

Submitted to:

Sir Ikram Ullah Khan

B-Pharm, M.Phil,

COLLEGE OF PHARMACY

GC UNIVERSITY

FAISALABAD

MS (TQM), R.Ph.

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POST MARKETING SURVIELLANCE

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CHAPTER#01

INTRODUCTION OF DRUG DISCOVERY AND

DEVELOPMENT

INTRODUCTION:

Discovering and bringing one new drug to the public typically costs a pharmaceutical or biotechnology company nearly $900 million and takes an average of 10 to 12 years. In special circumstances, such as the search for effective drugs to treat AIDS, the U.S. Food and Drug Administration (FDA) has encouraged an abbreviated process for drug testing and approval called fast-tracking. The drug discovery and drug development process is designed to ensure that only those pharmaceutical products that are both safe and effective are brought to market. PPD provides a broad array of drug discovery and development services and products to pharmaceutical, biotechnology and medical device companies to expedite drug development, from drug discovery through clinical studies and post-approval support.

Drug development is a blanket term used to define the entire process of bringing a

new drug or device to the Market. It includes Drug discovery / product development, pre-clinical research (microorganisms/animals) and Clinical trials (on humans). Few people still refer to the drug development as mere preclinical development.

HOW ARE NEW DRUGS DISCOVERED?

New drugs begin in the laboratory with scientists, including chemists and pharmacologists, who identify cellular and genetic factors that play a role in specific diseases. They search for chemical and biological substances that target these biological markers and are likely to have drug-like effects. Out of every 5,000 new compounds identified during the discovery process, only five are considered safe for testing in human volunteers after preclinical evaluations. After three to six years of further clinical testing in patients, only one of these compounds is ultimately approved as a marketed drug for treatment. The following sequence of research activities begins the process that results in development of new medicines:

• Target Identification. Drugs usually act on either cellular or genetic

chemicals in the body, known as targets, which are believed to be associated with disease. Scientists use a variety of techniques to identify and isolate

individual targets to learn more about their functions and how they influence disease. Compounds are then identified that have various interactions with the drug targets that might be helpful in treatment of a specific disease.

• Target Prioritization/Validation. To select targets most likely to be useful in

the development of new treatments for disease, researchers analyze and compare each drug target to others based on their association with a specific disease and their ability to regulate biological and chemical compounds in the body. Tests are conducted to confirm that interactions with the drug target are associated with a desired change in the behavior of diseased cells. Research scientists can then identify compounds that have an effect on the target selected.

• Lead Identification. A lead compound or substance is one that is believed to

have potential to treat disease. Laboratory scientists can compare known substances with new compounds to determine their likelihood of success. Leads are sometimes developed as collections, or libraries, of individual molecules that possess properties needed in a new drug. Testing is then done on each of these molecules to confirm its effect on the drug target.

• Lead Optimization. Lead optimization compares the properties of various

lead compounds and provides information to help biopharmaceutical companies select the compound or compounds with the greatest potential to be developed into safe and effective medicines. Often during this same stage of development, lead prioritization studies are conducted in living organisms (in vivo) and in cells in the test tube (in vitro) to compare various lead compounds and how they are metabolized and affect the body.

WHAT IS REQUIRED BEFORE AN INVESTIGATIONAL DRUG CAN BE TESTED IN HUMAN VOLUNTEERS?

In the preclinical stage of drug development, an investigational drug must be tested extensively in the laboratory to ensure it will be safe to administer to humans. Testing at this stage can take from one to five years and must provide information about the pharmaceutical composition of the drug, its safety, how the drug will be formulated and manufactured, and how it will be administered to the first human subjects.

• Preclinical Technology. During the preclinical development of a drug,

laboratory tests document the effect of the investigational drug in living organisms (in vivo) and in cells in the test tube (in vitro).

• Chemistry Manufacturing and Controls (CMC)/Pharmaceutics. The

results of preclinical testing are used by experts in pharmaceutical methods to determine how to best formulate the drug for its intended clinical use. For example, a drug that is intended to act on the sinuses may be formulated as a time-release capsule or as a nasal spray. Regulatory agencies require testing that documents the characteristics -- chemical composition, purity, quality and potency -- of the drug's active ingredient and of the formulated drug.

• Pharmacology/Toxicology. Pharmacological testing determines effects of the

candidate drug on the body. Toxicology studies are conducted to identify potential risks to humans.

Results of all testing must be provided to the FDA in the United States and/or other appropriate regulatory agencies in order to obtain permission to begin clinical testing in humans. Regulatory agencies review the specific tests and documentation that are required to proceed to the next stage of development.

HOW ARE INVESTIGATIONAL DRUGS TESTED IN HUMANS?

Testing of an investigational new drug begins with submission of information about the drug and application for permission to begin administration to healthy volunteers or patients.

Investigational New Drug (IND)/Clinical Trial Exception (CTX)/Clinical Trial Authorization (CTA) Applications. INDs (in the U.S.), CTXs (in the U.K.) and

CTAs (in Australia) are examples of requests submitted to appropriate regulatory authorities for permission to conduct investigational research. This research can include testing of a new dosage form or new use of a drug already approved to be marketed.

In addition to obtaining permission from appropriate regulatory authorities, an institutional or independent review board (IRB) or ethical advisory board must approve the protocol for testing as well as the informed consent documents that

volunteers sign prior to participating in a clinical study. An IRB is an independent committee of physicians, community advocates and others that ensures a clinical trial is ethical and the rights of study participants are protected.

Clinical testing is usually described as consisting of Phase I, Phase II and Phase III clinical studies. In each successive phase, increasing numbers of patients are tested.

• Phase I Clinical Studies. Phase I studies are designed to verify safety and

tolerability of the candidate drug in humans and typically take six to nine months. These are the first studies conducted in humans. A small number of subjects, usually from 20 to 100 healthy volunteers, take the investigational drug for short periods of time. Testing includes observation and careful documentation of how the drug acts in the body -- how it is absorbed, distributed, metabolized and excreted.

• Phase II Clinical Studies. Phase II studies are designed to determine

effectiveness and further study the safety of the candidate drug in humans. Depending upon the type of investigational drug and the condition it treats, this phase of development generally takes from six months up to three years. Testing is conducted with up to several hundred patients suffering from the condition the investigational drug is designed to treat. This testing determines safety and effectiveness of the drug in treating the condition and establishes the minimum and maximum effective dose. Most Phase II clinical trials are randomized, or randomly divided into groups, one of which receives the investigational drug, one of which gets a placebo containing no medication and sometimes a third group that receives a current standard treatment to which the new investigational drug will be compared. In addition, most Phase II studies are double-blinded, meaning that neither patients nor researchers evaluating the compound know who is receiving the investigational drug or placebo.

• Phase III Clinical Studies. Phase III studies provide expanded testing of

effectiveness and safety of an investigational drug, usually in randomized and blinded clinical trials. Depending upon the type of drug candidate and the condition it treats, this phase usually requires one to four years of testing. In Phase III, safety and efficacy testing is conducted with several hundred to

thousands of volunteer patients suffering from the condition the investigational drug treats.

New Drug Application (NDA)/Marketing Authorization Application (MAA):

NDAs (in the U.S.) and MAAs (in the U.K.) are examples of applications to market a new drug. Such applications document safety and efficacy of the investigational drug and contain all the information collected during the drug development process. At the conclusion of successful preclinical and clinical testing, this series of documents is submitted to the FDA in the U.S. or to the applicable regulatory authorities in other countries. The application must present substantial evidence that the drug will have the effect it is represented to have when people use it or under the conditions for which it is prescribed, recommended or suggested in the labeling. Obtaining approval to market a new drug frequently takes between six months and two years.

DOES TESTING CONTINUE AFTER A NEW DRUG IS APPROVED?

After the FDA (or other regulatory agency for drugs marketed outside the U.S.) approves a new drug, pharmaceutical companies may conduct additional studies, including Phase IIIb and Phase IV studies. Late-stage drug development studies of approved, marketed drugs may continue for several months to several years.

• Phase IIIb/IV Studies. Phase IIIb trials, which often begin before approval,

may supplement or complete earlier trials by providing additional safety data or they may test the approved drug for additional conditions for which it may prove useful. Phase IV studies expand testing of a proven drug to broader patient populations and compare the long-term effectiveness and/or cost of the drug to other marketed drugs available to treat the same condition.

• Post-Approval Studies. Post-approval studies test a marketed drug in new

age groups or patient types. Some studies focus on previously unknown side effects or related risk factors. As with all stages of drug development testing, the purpose is to ensure the safety and effectiveness of marketed drugs

STEPS IN NEW DRUG DEVELOPMENT TILL NDA IS

FILED

CHAPTER#02

FLOW CHART FOR NEW DRUG

DEVELOPMENT

NEW CHEMICAL ENTITY

• Organic Synthesis • Molecular Modification

• Isolation from Plants

PRECLINICAL STUDIES • Chemistry • Physical Properties • Biological Properties ADME Toxicology • Preformulation

INVESTIGATIONAL NEW DRUG APPLICATION (IND) • Submission • FDA Review CLINICAL TRIALS • Phase І • Phase І І • Phase І І І

PRECLINICAL STUDIES (Continued)

• Long Term Animal Toxicity • Product Formulation

• Manufacturing and Controls

• Package and Label Design

NEW DRUG APPLICATION (NDA)

• Submission • FDA Review

• Preapproval Plant Inspection

• FDA Action

POST MARKETING SURVEILLANCE

• Phase 4 clinical studies • Additional Indications • Adverse Drug Reporting • Product Defect Reporting

CHAPTER#03

SYNTHESIS OF LEVOFLOXACIN

Levofloxacin is a synthetic compound and is synthesized as follow;

Procedure:

1,2-Cyclic sulfamidates undergo efficient and regiospecific nucleophilic cleavage with 2-bromophenols (and related anilines and thiophenols), followed by Pd(0)-mediated amination to provide an entry to substituted and enantiomerically pure 1,4-benzoxazines (and quinoxalines and 1,4-benzothiazines). This chemistry provides a short and efficient entry to (3S)-3-methyl-1,4-benzoxazine 19, a late stage intermediate in the synthesis of levofloxacin. This intermediate, through a series of steps, is than converted into levofloxacin.

CHAPTER#04

PRECLINICAL STUDIES

™ Chemistry

™ Physical Properties

™ Biological Properties

• Pharmacology

• Pharmacokinetics

• Toxicity

UNIT-1:

CHEMISTRY OF LEVOFLOXACIN

IUPAC-Name: (S)-7-fluoro-6-(4-methylpiperazin-1-yl)-10-oxo-4-thia-1-azatricyclo

[7.3.1.05,13] trideca-5(13),6,8,11-tetraene-11-carboxylic acid

Chemical Formula: (-) - (S)- 9 fluoro- 2, 3- dihydro -3- methyl- 10- (4- methyl-

1-piperazinyl) –7 – oxo -7H – pyrido [1, 2, 3 -de]- 1, 4 benzoxazine- 6- carboxylic acid hemihydrate.

Empirical Formula: C18H20FN3O4 ½H2O

Routes: Oral, IV, ophthalmic

Molecular Weight: 370.38.

Stable Coordination Compounds:

Levofloxacin has the potential to form stable coordination compounds with many metal ions. This in vitro chelation potential has the following formation order: Al+3_>Cu+2_>Zn+2_>Mg+2_>Ca+2_.

Structural Formula of Levofloxacin

PHYSICAL PROPERTIES

Appearance: Levofloxacin is a light yellowish-white to yellow-white crystal or

crystalline powder.

Stability: Stable under ordinary conditions

Melting Point: 218 ºC (http://www.chemblink.com/products/100986-85-4.htm) Solubility: Insoluble in water

The data demonstrate that from pH 0.6 to 5.8, the solubility of levofloxacin is essentially constant (approximately 100 mg/mL). Levofloxacin is considered soluble to freely soluble in this pH range, as defined by USP nomenclature. Above pH 5.8, the solubility increases rapidly to its maximum at pH 6.7 (272 mg/mL) and is considered freely soluble in this range. Above pH 6.7, the solubility decreases and reaches a minimum value (about 50 mg/mL) at a pH of approximately 6.9.

Physical State: crystalline powder Odour: Odourless

BIOLOGICAL PROPERTIES

These biological properties are based on pre-clinical studies that are carried out in animals.

(A) PHARMACOLOGY:

Pharmacotherapeutic Group: Quinolone Antibacterials, Fluoroquinolones

Levofloxacin is a synthetic antibacterial agent of the fluoroquinolone class and is the S (-) enantiomer of the racemic drug substance ofloxacin.

Mechanism of action:(Chemical Basis)

Levofloxacin is a broad-spectrum antibiotic that is active against both Gram-positive and Gram-negative bacteria. It functions by inhibiting DNA gyrase, a type II topoisomerase, and topoisomerase iv, which is an enzyme necessary to separate replicated DNA, thereby inhibiting cell division.

The fluoroquinolones interfere with DNA replication by inhibiting an enzyme complex called DNA gyrase. In particular, some congeners of this drug family display high activity not only against bacterial topoisomerases but also against eukaryotic topoisomerases, and are toxic to cultured cells and in vivo tumor models. Although the quinolone is highly toxic to mammalian cells in culture, its mechanism of cytotoxic action is not known. Quinolone-induced DNA damage was first reported in 1986.

P

Bioavailability

Approximately 99%.

Rapidly absorbed from GI tract.Peak plasma concentrations usually attained 1-2 hours after an oral dose. Steady-state plasma concentrations attained within 48 hours with once-daily regimen.

Distribution:

Extent

Widely distributed into body tissues and fluids, including skin, blister fluid, and lungs. It is also distributed into CSF.

Plasma Protein Binding

24-38% bound to serum proteins.

Elimination:

Metabolism

Undergoes limited metabolism to inactive metabolites.

Elimination Route

Eliminated principally as unchanged drug in urine. Approximately 87% of an oral dose eliminated in urine and <4% eliminated in feces.

Half-life

Terminal elimination half-life approximately 6-8 hours after oral administration.

Levofloxacin, when administered orally to rats at a dose of 20 mg/kg, was absorbed primarily from the small intestine, and the maximum serum concentration (2.5 ug/ml) was reached 0.5 hours after administration, except for the level, in the central nervous system and fat, levofloxacin concentration in almost all tissues of the body were higher than the serum level, demonstrating the good transference to tissues. Drug concentrations in the main organs were high in the kidneys and liver and lowest in the brain.

Repeated dose toxicity:

Studies of one and six month’s duration by gavage have been carried out in the rat and monkey. Doses were 50, 200, 800 mg/kg/day and 20, 80, 320 mg/kg/day for 1 and 6 months in the rat and 10, 30, 100 mg/kg/day and 10, 25, 62.5 mg/kg/day for 1 and 6 months in the monkey.

Signs of reactions to treatment were minor in the rat with slight effects principally at 200 mg/kg/day and above in reducing food consumption and slightly altering haematological and biochemical parameters. The “No Observed Adverse Effect Levels” (NOEL) in these studies were concluded to be 200 and 20 mg/kg/day after one-and six months, respectively.

Toxicity after oral dosing in the monkey was minimal with reduced body weight at 100 mg/kg/day together with salivation, diarrhoea and decreased urinary pH in some animals at this dose. No toxicity was seen in the 6-months study. The NOELs were concluded to be 30 and 62.5 mg/kg/day after 1 and 6 months respectively.

The “ No Observed Adverse Effect Levels” (NOEL) in the six-month studies were concluded to be 20 and 62.5 mg/kg/day in the rat and monkey respectively.

Subacute Toxicity:

Following 4-weeks oral administration to rats, no toxicological changes in clinical signs, hematology, blood chemistry urinalysis and histopathology were observed in the 50 mg/kg and 200 mg/kg administered groups. At a dose of 800 mg/kg, however, increased M/E ratio of bone marrow cells, decreased neutrophil count and slight degeneration of the articular cartilage of limb joint were observed. Following 4 weeks oral administration to cynomolgus monkeys, no toxicological changes were observed

at doses of 10 and 30 mg/kg, but salivation, diarrhea, slight inhibition of body weight gain and decrease in urine pH were observed at 100 mg/kg.

CHAPTER#05

PREFORMULATION

™ Bulk Characterization

™ Solubility Analysis

™ Stability Analysis

UNIT-1:

BULK CHARACTERIZATION

Molecular Weight: 370.38.

Method: By Mass Spectroscopic Method Particle Size: Large

Method: By sieving method particle size is determined. it is large in size

Melting Point: 218 ºC (http://www.chemblink.com/products/100986-85-4.htm) Method: By Hot Stage Microscopy

The melting point of a drug can be measured using three techniques: ¾ Capillary melting

¾ Hot stage microscopy

¾ Differential scanning calorimetry or thermal analysis.

Capillary Melting

Capillary melting (the observation of melting in a capillary tube in a heated metal block) gives information about the melting range but it is difficult to assign an accurate melting point.

Hot stage Microscopy

This is the visual observation of melting under a microscope equipped with a heated and lagged sample stage. The heating rate is controllable and up to three transitions can be registered. It is more precise as the phase transitions (first melt, 50% melt and completion) can be registered on a recorder as the melting proceeds, and because of the high magnification the values are more accurate.

Differential Scanning Calorimetry and Thermal Analysis:

Neither of the previous methods is as versatile as either differential thermal analysis (DTA) or differential scanning calorimetry (DSC). An additional advantage is that the sample size required is only 2-5 mg. DTA measures the temperature difference between the sample and a reference as a function of temperature or time when heating

at a constant rate. DSC is similar to DTA, except that the instrument measures the amount of energy required to keep the sample at the same temperature as the reference, i.e. it measures the enthalpy of transition. When no physical or chemical change occurs

within the sample then there is neither a temperature change nor input energy to maintain an isotherm. However, when phase changes occur then latent heat suppresses a temperature change and the isothermal energy required registers as an electrical signal generated by thermocouples. Crystalline transitions, fusion, evaporation and sublimation are obvious changes in state which can be quantified (Fig. 8.3). The major concern in preformulation is polymorphism, and the measurement of melting point and other phase changes is the primary diagnostic tool. Confirmation by IR spectroscopy and X-ray diffraction

is usually required.

Crystal Habit: Microcrystalline

Technique: By SEM, Crystal Habit is observed Microscopy:

The microscope has two major applications in pharmaceutical preformulation:

¾ Basic crystallography, to determine crystal morphology (structure and habit), polymorphism and solvates

¾ Particle size analysis

Most pharmaceutical powders have crystals in the range 0.5-300 /Jim. However, the distributions are often smaller, typically 0.5-50 /mi, to ensure good blend homogeneity and rapid dissolution. These are the major reasons for particle size control. A lamp-illuminated mono-objective microscope fitted with polarizing filters above and below the stage is more than adequate. For most preformulation work a 10 x eyepiece and a 10-x objective are ideal, although occasionally, with micronized powders and when following solid-solid and liquid-liquid transitions in polymorphism, 10 x 20 may be required.

Thermal analysis has been widely used as a method of purity determination and the USP includes an appendix describing the methods. This is particularly pertinent at the preformulation stage, because early samples of a new drug are inevitably 'dirty' while Improvements in synthetic route are made. Thermal analysis is rapid and will discriminate 0.002% of impurity.

Hygroscopicity: Hygroscopic

A substance that absorbs sufficient moisture from the atmosphere to dissolve itself is deliquescent is called hygroscopic. A substance that loses water to form a lower hydrate or becomes anhydrous is termed efflorescent. These are extreme cases, and most pharmaceutical compounds are usually either impassive to the water available in the surrounding atmosphere or lose or gain water from the atmosphere, depending on the relative humidity (RH). Materials unaffected by RH are termed non-hygroscopic, whereas those in dynamic equilibrium with water in the atmosphere are hygro-scopic. Ambient RH (0% poles and desert, 55% temperate and 87% tropics) can vary widely and continually depending on the weather and air temperature, and these cyclic changes lead to constant variations in the moisture content of unprotected bulk drug and excipients. The constant sinusoidal change in day and night temperatures is the major influence. For this reason pharmaceutical air conditioning is usually set below 50% RH, and very hygroscopic products, e.g. effervescents, which are particularly moisture sensitive, are stored and made below 40% RH.

Crystal Morphology

Crystals are characterized by repetition of atoms or molecules in a regular three-dimensional structure, which is absent in glasses and some polymers. There are six crystal systems (cubic, tetragonal, orthorhombic, monoclinic, triclinic and hexagonal), which have different internal structures and spatial arrangements. Although not changing their internal structure, which occurs with polymorphism, crystals can adopt different external structures. This is known as crystal habit, of which five types are recognized:

¾ Tabular: moderate expansion of two parallel faces ¾ Platy: plates

¾ Prismatic: columns ¾ Acicular: needle-like

¾ Bladed: flat acicular.

These occur in all six-crystal systems.

Conditions during crystallization will contribute to changes in crystal habit and may be encountered in early batches of a new drug substance until the synthetic route has been optimized. Crystal habit can be modified by:

¾ Excessive supersaturation, which tends to transform a prism or isodiametric (granular) crystals to a needle shape.

¾ Cooling rate and agitation, which changes habit as it changes the degree of supersaturation. Naphthalene gives thin plates (platy) if rapidly recrystallized in cold ethanol or methanol, whereas slow evaporation yields prisms.

¾ The crystallizing solvent affects habit by preferential absorption on to certain faces, inhibiting their growth. Resorcinol produces needles from benzene and squat prisms from butyl acetate.

The addition of cosolvents or other solutes and ions which change habit by poisoning crystal growth in one or more directions. Sodium chloride is usually cubic, but urea produces an octahedral habit.

Powder Flow Properties: Flow Ability: Good

Technique: Flow ability is determined by calculating angle of repose. Angle of Repose: 22º

Explanation:

A static heap of powder, with only gravity acting upon it, will tend to form a conical mound. One limitation exists: the angle to the horizontal cannot exceed a certain value, and this is known as the angle of repose (0). If any particle temporarily lies outside this limiting angle, it will slide down the adjacent surface under the influence of gravity until the gravitational pull is balanced by the friction caused by interparticulate forces. Accordingly, there is an empirical relationship between 6 and the ability of the powder to flow. However, the exact value for angle of repose does depend on the method of measurement. The angles of repose given in Table 8.14 may be used as a guide to flow.

A simple relationship between angle of repose, Carr's index and the expected powder flow is shown in Figure 8.6. When only small quantities of powder are available, an alternative is to determine the 'angle of spatula' by picking up a quantity of powder on

a Table 8.14 Angle of repose as an indication of powder flow propertiesspatula and estimating the angle of the triangular section of the powder heap viewed from the end of the spatula. This is obviously crude but is useful during preformulation, when only small quantities of drug are available.

Of primary importance when handling a drug powder is flow. When limited amounts of drug are available this can be evaluated by measurements of bulk density and angle of repose. These are extremely useful derived parameters to assess the impact of changes in drug powder properties as new batches become available. Changes in particle size and shape are generally very apparent; an increase in crystal size or a more uniform shape will lead to a smaller angle of repose and a smaller Carr's index.

Bulk density

A simple test has been developed to evaluate the flowability of a powder by comparing the poured (fluff) density (pBmin) and tapped density (psmax) of a powder and the rate at which it packed down. A useful empirical guide is given by Carr's compressibility index ('Compressibility' is a misnomer, as compression is not involved):

This is a simple index that can be determined on small quantities of powder and may

be interpreted as

A similar index has been defined by Hausner (1967):

Values less than 1.25 indicate good flow (= 20% Carr), whereas greater than 1.25 indicates poor flow (= 33% Carr). Between 1.25 and 1.5, added glidant normally improves flow. Carr's index is a one-point determination and does not always reflect the ease or speed with which the powder consolidates. Indeed, some materials have a high index (suggesting poor flow) but may consolidate rapidly. Rapid consolidation is essential for uniform filling on tablet machines, when the powder flows at pBmin into

the die and consolidates, approaching pBmaxJ at compression. An empirical linear relationship exists between the change in bulk density and the log number of taps in a jolting volumeter. Non-linearity occurs up to two taps and after 30 taps when the bed consolidates more slowly. The slope is a measure of the speed of consolidation and is useful for assessing powders or blends with similar Carr's indices and the benefit of glidants.

Polymorphic Forms:

Three polymorphic forms (anhydrous α, β, γ) and two pseudopolymorphic forms (hemihydrate and monohydrate) of levofloxacin are present. Hemihydrate and monohydrate forms are mentioned in EP 0444 678 B1 and in U.S. Patent No. 5,545,737. These two patents are directed toward processes for the preparation of hemihydrate form free of monohydrate and for the preparation of monohydrate free of hemihydrate.

Transformation Kinetics:

Heating the hemihydrate form resulted in a removal of the hydrated water to give anhydrous form γ. Further heating resulted in the formation of anhydrous form β, and then the formation of anhydrous form α. Heating of the monohydrate form resulted in a removal of the hydrated water to give anhydrous form α. Form γ and form α adsorbed water vapor rapidly under ordinary relative humidity conditions and transformed into the hemihydrate and monohydrate, respectively.

Method: Transformation Kinetics are checked by differential scanning calorimetry

Unit-2:

SOLUBILITY ANALYSIS

Solubility in water

:

The data demonstrate that from pH 0.6 to 5.8, the solubility of levofloxacin is essentially constant (approximately 100 mg/ mL). Levofloxacin is considered soluble to freely soluble in this pH range, as defined by USP nomenclature. Above pH 5.8, the solubility increases rapidly to its maximum at pH 6.7 (272 mg/ mL) and is considered freely soluble in this range. Above pH 6.7, the solubility decreases and reaches a minimum value (about 50 mg/ mL) at a pH of approximately 6.9.

Partion Coefficient

:

Method: Shake flask method is used to determine Pka Membrane Permeabiliy: High permeability drug

Permeability classification of representative fluoroquinolones by a cell culture method:

This study was undertaken to categorize representative fluoroquinolone drug substance permeability based on the methods outlined in the Food and Drug Administration's biopharmaceutic classification system (BCS) Guidance for Industry. The permeability of ciprofloxacin, levofloxacin, lomefloxacin, and ofloxacin was measured in an in vitro

Caco-2 assay with previously demonstrated method suitability. The permeability class and efflux potential were ascertained by comparing test drug results with standard compounds (metoprolol, atenolol, labetalol, and rhodamine-123). All 4 quinolones drugs demonstrated concentration-dependent permeability, indicating active drug transport. In comparing absorptive versus secretive in vitro transport, the tested fluoroquinolones were found to be subject to efflux in varying degrees (ciprofloxacin > lomefloxacin > rhodamine 123 > levofloxacin > ofloxacin). Based on comparison to

labetalol, the high permeability internal standard, ciprofloxacin was classified as a low permeability drug, whereas lomefloxacin, levofloxacin, and ofloxacin were classified as high permeability drugs. The in vitro permeability results matched human in vivo data based on absolute bioavailabilities. This laboratory exercise demonstrated the applicability of an in vitro permeability method for classifying drugs as outlined in the BCS Guidance.

Dissolution: Drug exhibit good dissolution properties.

Method and Equipment: Rotating Basket Apparatus is used.

Drug is placed in the Rotating Basket Apparatus and the dissolution of compound is determined

UNIT-3:

STABILITY ANALYSIS

Hydrolysis

:

Stability-indicating RP-HPLC method for levofloxacin in the presence of degradation products, its process related impurities and identification of oxidative degradant

The objective of current study was to develop a validated specific stability indicating reversed-phase liquid chromatographic method for the quantitative determination of levofloxacin as well as its related substances determination in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its process related impurities. Forced degradation studies were performed on bulk sample of levofloxacin as per ICH prescribed stress conditions using acid, base, oxidative, water hydrolysis, thermal stress and photolytic degradation to show the stability indicating power of the method.

RESULT:

Significant degradation was observed during oxidative stress and the degradation product formed was identified by LCMS/MS, slight degradation in acidic stress and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies and the impurity spiked solution. Good resolution between the peaks corresponds to process related impurities and degradation products from the analyte were achieved on ACE C18 column using the mobile phase consists a mixture of 0.5% (v/v) triethyl amine in sodium dihydrogen orthophosphate dihydrate (25 mM; pH 6.0) and methanol using a simple linear gradient. The detection was carried out at 294 nm. The limit of detection and the limit of quantitation for the levofloxacin and its process related impurities were established. The stressed test solutions were assayed against the qualified working standard of levofloxacin and the mass balance in each case was in between 99.4 and 99.8% indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per ICH requirements. The developed LC method was found to be suitable to check the quality of bulk samples

of levofloxacin at the time of batch release and also during its stability studies (long term and accelerated stability).

CHAPTER#06

INVESTIGATIONAL NEW DRUG

APPLICTION

Documents required by Ministry of Health for the approval of Clinical Trials in Pakistan

Documents

Investigator Brochure. Final protocol.

Informed Consent (English and Urdu )Form Fees 5000.

(Head of Account) C-Non Tax Revenue

C02- Receipts from Civil Administration and other Functions. C028-Social Services.

C02841-Health-Other Receipts List of participating countries. Phase of Trial

Quantity of drug to be imported on Form 4 of Drugs Import & Export Rules 1976 along with the sites where trial is to be conducted.

CV’s of Investigators.

Ethics committee approval of sites, with complete composition of committee i.e names and designation of members.

GMP Certificate along with CPP/Free Sale Certificate of Country of Origin. Pre- clinical/ Clinical data/ Safety studies.

Summary of the Protocol

Summary of the IB ( for quick review on drug). Adverse Event Reporting Form .

Number patients to be enrolled in each center Name of Monitors/ Clinical Research Associate Evidence of registration in country of origin

Copy of registration letter if drug is registered in Pakistan Sample of label of drug

CHAPTER#07

CLINICAL TRIALS

Our product is converted into a suitable dosage form. Than we submitted investigational to FDA. All preclinical studies mentioned and took permission for clinical trials.

PHASE-1 CLINICAL TRIALS: Aims:

To study the safety of the drug in healthy volunteers

No. of Patients:

We selected 20-100 healthy voluntaries for phase 1 trials.

Procedure:

We administered 1/10 of no effect dose of animals. This purloins gets stable so we increased the dose gradually and checked the response. In this phase, Parmacokinetics and Pharmacodynamic studies of a drug are undertaken to determine its toxicity, metabolism, absorption, distribution and elimination and pharmacological action preferred route of administration and safe dosage.

Duration:

Phase-1 survives usually for 1-2 years.

Results:

We conducted study under careful circumstances by personals trained in clinical pharmacology. The clinical research on ranitidine is preceded to phase-2 because phase 1 studies showed promise and no drug reaction, which became evident.

PHASE-2 CLINICAL TRIALS:

Phase II studies are sometimes divided into Phase IIA and Phase IIB.

¾ Phase IIA is specifically designed to assess dosing requirements (how much drug should be given).

¾ Phase IIB is specifically designed to study efficacy (how well the drug works at the prescribed dose(s)).

Aims:

¾ This trial aims to demonstrate conclusively efficacy of drug in relation to its safety.

¾ Pharmacokinetics of a drug should be investigated in patients because they may handle it differently from healthy people. Variation may occur due to the following reasons:

• Effect of disease

• Age as compared with that of the volunteers studies that in phase-1 trials.

No. of Patients:

We recruited hundred of patients for the conformation of phase-1 trials.

Duration:

It took 1-2 years for its completion.

A STUDY TO EVALUATE THE PHARMACOKINETICS AND SAFETY OF LEVOFLOXACIN IN PATIENTS WITH VARYING DEGREES OF RENAL

FUNCTION PURPOSE:

The primary objective was to evaluate the pharmacokinetics and safety of two dosing regimens of Levofloxacin in patients with varying degrees of renal function.

Condition Intervention Phase

Renal Diseases Drug: Levofloxacin Phase 2

Study Type Interventional

Study Design Treatment, Randomized, Open Label, Parallel Assignment,

Pharmacokinetics Study

Official Title An Open-Label Randomized Multiple-Dose Study to

Evaluate Levofloxacin Steady-State Pharmacokinetics and Safety in Subjects With Varying Degrees of Renal Function

FURTHER STUDY DETAILS AS PROVIDED

Primary Outcome Measures:

Evaluation of the pharmacokinetics of two dosing regimens of Levofloxacin in renally impaired and dialysis patients.

Secondary Outcome Measures:

Safety of two dosing regimens of Levofloxacin in renally impaired and dialysis patients.

Estimated Enrollment: 60 Study Start Date: October 2007 Study Completion Date: April 2009

Detailed Description:

In this multiple-dose study conducted at 4 centers, the pharmacokinetics of two dosing regimens of levofloxacin were assessed in medically stable men and women with varying degree of renal function. The study consisted of a 21 day pretreatment screening phase, a 7-day open label treatment phase, and a 7 day posttreatment phase (or a follow-up phase for subjects with early study withdrawal). Patients were randomized into 1 of 10 treatment groups, for a total of 6 patients per group, based on degree of renal function to ensure that creatinine clearance values within each group represented the full range of values defined in the Food and Drug Administration's (FDA) 1998 guideline for pharmacokinetic studies in patients with impaired renal function. Fifty-nine patients were enrolled in the study. All patients received a single 750-mg dose of levofloxacin on Day 1; subsequent doses of either 250, 500, or 750 mg of levofloxacin (q24h or q48h) were based on renal function. Blood samples were collected from each patient from Day 1 to Day 14 for pharmacokinetic evaluation. Urine was collected on Days 1 and 7 before dosing and over specific time intervals up to 24 or 48 hours postdosing depending on the patient's dosing regimen. Dialysate samples were collected on Day 7 from HD patients immediately before dosing (as

dialysis began) and at the end of the dialysis treatment. Patients were confined overnight at the study unit on Days 0, 1, 6, and 7, and remained confined until the 24 hour blood samples were collected on Days 2 and 8. Safety was based on the incidence, relationship to therapy, and severity of treatment-emergent adverse events and on changes in clinical laboratory values (hematology, chemistry, and urinalysis), vital sign measurements, electrocardiograms (ECGs), and physical examination findings.

Single 750-mg dose of levofloxacin on Day 1; subsequent doses of Levofloxacin 250 milligram (mg), 500 mg, and 750 mg tablets administered every 24 hours for 7 days or every 48 hours for 7 days

Eligibility Criteria:

Ages Eligible for Study: 18 Years to 65 Years Genders Eligible for Study: Both

Accepts Healthy Volunteers: No

Inclusion Criteria:

¾ BMI between 18 and 35 kg/m2

¾ No prescription or over-the-counter medications for previous 7 days ¾ Negative tests for drug and alcohol abuse, HIV, hepatitis B and hepatitis C ¾ Medically stable based on medical history, physical examination, 12-lead

electrocardiograms, toxicology, antigen, and antibody screens, and clinical laboratory evaluations

¾ Stable renal function based on calculated creatine clearance for non-dialysis patients and the same dialysis treatment for at least 6 months prior to screening for dialysis patients

¾ Patients with creatinine clearance ≤80 mL/min who require treatment for renal impairment or other chronic disease (e.g., well-controlled diabetes, hypertension) must be on a stable treatment plan (medicines, doses, and regimens) for at least 2 months prior to Day 1 and during the entire study ¾ Hematocrit (hct) within the normal range based on patients' renal function at

Exclusion Criteria:

¾ Allergic reaction to quinolones

¾ Known or suspected allergy to heparin

¾ Clinically significant ECG or clinical laboratory abnormalities

¾ Creatinine clearance <80 mL/min whose medical condition was unstable ¾ Creatinine clearance >= 80 mL/min who required concomitant medication

during the study

¾ Poorly controlled type 1 or type 2 diabetes

¾ Patients with creatinine clearance >= 50 mL/min with screening blood pressure outside the normal range (sitting systolic blood pressure <90 or >140 mm mercury [Hg] or diastolic blood pressure <60 or >90 mm Hg)

¾ Patients with CLCR <50 mL/min who had sitting systolic blood pressure <90 or >160 mm Hg, or diastolic blood pressure <60 or >90 mm Hg

¾ Required immunosuppressive medications for treatment of immune-mediated renal disease or kidney transplant

¾ Pregnant or breastfeeding

Results:

We conducted study under careful circumstances by personals trained in clinical pharmacology. The clinical research on ranitidine is preceded to phase-3 because phase-2 studies showed promise and no drug reaction, which became evident.

Phase-3 Clinical Trials:

The phase-3 studies are intended to assess the drug-s safety, effectiveness and most desirable dosage in treating a specific disease in a large group of subjects. Basically phase-3 involves the comparison between the existing therapies of a particular disease and the new drug.

A Study of the Safety and Effectiveness of Levofloxacin Compared With Ceftriaxone Sodium or Cefuroxime Axetil in the Treatment of Adults With

Pneumonia Purpose:

The purpose of this study is evaluation of the safety and effectiveness of levofloxacin, an antibiotic, compared with ceftriaxone sodium or cefuroxime axetil in the treatment of adults with pneumonia.

Condition Intervention Phase

Pneumonia Drug: levofloxacin Phase II

Phase III

Study Type: Interventional

Study Design: Treatment, Randomized, Open Label, Active Control, Parallel

Assignment, Safety/Efficacy Study

Official Title: A Multicenter, Active-Controlled, Randomized Study To Evaluate

The Safety And Efficacy Of Levofloxacin Versus Ceftriaxone Sodium Or Cefuroxime Axetil In The Treatment Of

Community-Acquired Pneumonia In Adults

Further study details as provided

Primary Outcome Measures:

Clinical response rate (reduction in signs and symptoms, improvement in x-ray findings) at post-therapy (5 - 7 days after the last dose of study drug).

Secondary Outcome Measures:

Rate of elimination of disease-causing bacteria, by patient, and by type of bacteria; incidence of adverse events; changes in physical examination and laboratory tests after treatment with study drug

Estimated Enrollment: 528 Study Start Date: September 2005 Estimated Study Completion Date: January 2007

Detailed Description:

This is a randomized, open-label, parallel group, multicenter study to determine the safety and effectiveness of levofloxacin (500 mg once daily by mouth) compared with ceftriaxone sodium (1 - 2 grams administered into a vein or muscle once daily or in divided doses twice daily for 7 - 14 days) or cefuroxime axetil (500 mg by mouth twice daily for 7 - 14 days) in adults with community-acquired pneumonia. The study consists of 4 visits: one visit for screening and enrollment, and 3 visits for assessment of safety and effectiveness (one visit on Day 2 - 4 [on-therapy], one visit [post-therapy] 5 - 7 days after the last dose of the study drug, and one visit [post-study] 21 - 28 days after the last dose of the study drug). The total duration of patient participation in the study is approximately 6 weeks. Levofloxacin is an antibacterial agent used for the treatment of many types of infections in adults. The purpose of this study is to compare the safety and effectiveness of levofloxacin with other frequently used antibiotics (ceftriaxone sodium or cefuroxime axetil) in the treatment of adults with pneumonia acquired in the community. The primary efficacy assessment is the clinical response 5 - 7 days after the last dose of study drug, (categorized as cured, improved, or failed) based upon changes in signs and symptoms, and changes in x-ray findings, Safety evaluations (incidence of adverse events, physical examination, and laboratory tests) are performed throughout the study. Cost-effectiveness is also assessed for the study drugs. The study hypothesis is that treatment with levofloxacin will be at least as effective as ceftriaxone sodium or cefuroxime axetil in treating patients with pneumonia acquired in the community, and that it will be well tolerated. Levofloxacin 500 mg by mouth once daily; ceftriaxone sodium (1 - 2 grams administered into a vein or muscle once daily or in divided doses twice daily); or cefuroxime axetil (500 mg by mouth twice daily). Treatment duration is 7 - 14 days.

Eligibility Criteria:

Ages Eligible for Study: 18 Years and older Genders Eligible for Study: Both Accepts Healthy Volunteers: No

Inclusion Criteria:

¾ Diagnosis of pneumonia based upon clinical signs and symptoms of a lower respiratory tract infection including at least 2 of the following: fever, cough, greenish-yellow mucus produced on coughing, chest pain, shortness of breath, or evidence of decreased lung function during the physical examination ¾ Has chest x-ray findings consistent with acute pneumonia

¾ Previously received antibiotics for pneumonia if the duration of therapy was <= 24 hours, or if greater than 24 hours, but without improvement or stabilization with that therapy

Exclusion Criteria:

¾ Previous allergic or serious adverse reaction to any antibiotic similar to those used in this study or to penicillin

¾ Collection of pus in the cavity between the lung and the membrane that surrounds it

¾ Has cystic fibrosis

¾ Has a lung infection due to fungus, bacteria, or virus known prior to the start of the study to be resistant to any of the study drugs

¾ Has severe kidney failure, decrease in white blood cell count, seizure disorder, or an unstable psychiatric condition.

Results:

Levofloxacin 500 mg by mouth once daily is more efficacious as compared to ceftriaxone sodium (1 - 2 grams administered into a vein or muscle once daily or in divided doses twice daily); or cefuroxime axetil (500 mg by mouth twice daily)

A Study to Compare the Safety and Effectiveness of 2 Doses of Levofloxacin Given for Different Time Periods in Patients With Pneumonia

Purpose:

The purpose of this study is to evaluate the effectiveness and safety of two antibiotic regimens in the treatment of community-acquired pneumonia in non-hospitalized adult patients. A 5-day course of 750 milligrams of levofloxacin given once daily will be compared to a 10-day course 500 milligrams of levofloxacin given once daily.

Condition Intervention Phase

Pneumonia Drug: levofloxacin Phase III

Study Type: Interventional

Study Design: Treatment, Randomized, Double-Blind, Parallel Assignment,

Safety/Efficacy Study

Official Title: Multicenter, Double-Blind Randomized Study to Compare the

Safety and Efficacy of Levofloxacin 750 mg Once Daily for Five Days vs. Levofloxacin 500 mg Once Daily for 10 Days in the Treatment of Mild to Severe Community-Acquired Pneumonia in

Adults

Further study details as provided

Primary Outcome Measures:

Clinical response rates based on signs and symptoms at posttherapy visit.

Secondary Outcome Measures:

Microbiologic eradication rates at posttherapy visit; Clinical response rates (chest x-ray findings and signs/symptoms) and microbiologic eradication rates at poststudy; Incidence of adverse events.

Enrollment: 530 Study Start Date: March 2007

Study Completion Date: June 2009

Detailed Description:

Levofloxacin is an antibiotic that is approved by the FDA for the treatment of sinusitis, chronic bronchitis, skin infections, urinary tract infections, and community-acquired pneumonia. This multicenter, double-blind (neither the patient nor the study doctor will know the dose of levofloxacin being administered) study evaluates the effectiveness and safety of two antibiotic regimens in the treatment of community-acquired pneumonia in adult patients. A 5-day course of 750 milligrams of levofloxacin given once daily will be compared to a 10-day course 500 milligrams of levofloxacin given once daily. Patients receive levofloxacin by mouth or through a vein depending on the severity of their pneumonia. Patients are assessed after 3 days of treatment; treatment is discontinued if no significant improvement is noted. Patients showing signs of improvement continue in the study, with assessments on study days 12-16, and 17-21 (posttherapy visits), and 31-38 (poststudy visit). Effectiveness is assessed by measuring the ability of the study drug to eliminate bacteria causing pneumonia and to reduce the signs and symptoms of pneumonia. Chest x-rays and laboratory tests for presence of bacteria are performed during the study. Safety evaluations (incidence of adverse events, physical examinations, laboratory tests) are performed throughout the study. The study hypothesis is that levofloxacin administered at a higher dose for a shorter duration is at least as effective as levofloxacin administered at a lower dose for a longer duration in the treatment of community-acquired pneumonia and is generally well-tolerated.

Levofloxacin, 500 milligrams (mg) by mouth or through vein daily for 10 days or 750 mg by mouth or slowly through a vein daily for 5 days

Eligibility Criteria:

Ages Eligible for Study: 18 Years and older Genders Eligible for Study: Both Accepts Healthy Volunteers: No

Inclusion Criteria:

¾ Diagnosis of community-acquired pneumonia as follows: clinical signs and symptoms of a lower respiratory tract infection and chest-x-ray findings consistent with pneumonia within 24 hours before entry into the study

¾ At least one of the following: abnormal temperature (high or low) or abnormal white blood cell count

¾ Previous antibiotic treatment <= 24 hours or, if the duration of treatment was >= 72 hours and that therapy failed based on at least 2 of the following: fever within 12 hours of entry into the study, chest x-ray findings have worsened compared to the initial chest-x-ray, white blood cell count is significantly increased, respiratory rate higher than at the start of treatment and >= 20 breaths per minute or need for supplemental oxygen if not previously needed ¾ Patients whose infection is acquired in the community or, if in a nursing home,

who had been living there < 14 days

¾ Fine Class (rating scale used to assess patients' overall condition which includes information such as age, gender, other diseases, physical examination and laboratory findings) score <= 130 upon admission (patients with Fine Class scores > 70 but < = 130 must initially be hospitalized

¾ Patients with scores of <= 70 may be treated as outpatients or hospitalized at the discretion of the investigator)

Exclusion Criteria:

¾ Pneumonia known or suspected to be due to a bacteria resistant to levofloxacin ¾ Previous allergic or serious reaction to or failed therapy with levofloxacin or

similar drugs

¾ Hospitalized within 2 weeks before entry in the study or within 1 month before entry in the study if treated with antibiotics

¾ Pneumonia acquired in a hospital ¾ Cystic fibrosis or other lung disorders ¾ Receiving chronic steroid treatment

¾ Received assistance from a machine to breathe within the previous month

Results:

Levofloxacin, 500 milligrams (mg) by mouth or through vein daily for 10 days show better results than 750 mg by mouth or slowly through a vein daily for 5 days.

WE GIVE CONSTANT REPORTS ON PROGRESS OF EACH

PHASE TO THE AUTHORITY.

CHAPTER#08

LONG TERM ANIMAL TOXICITY

STUDIES

LONG TERM ANIMAL TOXICITY STUDIES

Levofloxacin, when administered orally to rats at a dose of 20 mg/kg, was absorbed primarily from the small intestine, and the maximum serum concentration (2.5 ug/ml)

was reached 0.5 hours after administration, except for the level, in the central nervous system and fat, levofloxacin concentration in almost all tissues of the body were higher than the serum level, demonstrating the good transference to tissues. Drug concentrations in the main organs were high in the kidneys and liver and lowest in the brain

Long term toxicity studies show following results;

Repeated dose toxicity:

Studies of one and six month’s duration by gavage have been carried out in the rat and monkey. Doses were 50, 200, 800 mg/kg/day and 20, 80, 320 mg/kg/day for 1 and 6 months in the rat and 10, 30, 100 mg/kg/day and 10, 25, 62.5 mg/kg/day for 1 and 6 months in the monkey.

Signs of reactions to treatment were minor in the rat with slight effects principally at 200 mg/kg/day and above in reducing food consumption and slightly altering haematological and biochemical parameters. The “No Observed Adverse Effect Levels” (NOEL) in these studies were concluded to be 200 and 20 mg/kg/day after one-and six months, respectively.

Toxicity after oral dosing in the monkey was minimal with reduced body weight at 100 mg/kg/day together with salivation, diarrhoea and decreased urinary pH in some animals at this dose. No toxicity was seen in the 6-months study. The NOELs were concluded to be 30 and 62.5 mg/kg/day after 1 and 6 months respectively.

The “ No Observed Adverse Effect Levels” (NOEL) in the six-month studies were concluded to be 20 and 62.5 mg/kg/day in the rat and monkey respectively.

Subacute Toxicity:

Following 4-weeks oral administration to rats, no toxicological changes in clinical signs, hematology, blood chemistry urinalysis and histopathology were observed in the 50 mg/kg and 200 mg/kg administered groups. At a dose of 800 mg/kg, however, increased M/E ratio of bone marrow cells, decreased neutrophil count and slight degeneration of the articular cartilage of limb joint were observed. Following 4 weeks oral administration to cynomolgus monkeys, no toxicological changes were observed at doses of 10 and 30 mg/kg, but salivation, diarrhea, slight inhibition of body weight gain and decrease in urine pH were observed at 100 mg/kg.

Chronic Toxicity:

Following 26 weeks oral administration to rats, no toxicological changes were observed at a dose of 20 mg/kg, but salivation and high urinary pH were observed at doses of 80 and 320 mg/kg. In addition, at a dose of 320 mg/kg, increased feces and enlargement of goblet cells in cecal mucosa were seen. Following 26-weeks oral administration to cynomolgus monkeys, no toxicological changes were observed at doses of 10, 25 and 62.5 mg/kg

Genotoxicity:

Levofloxacin did not induce gene mutations in bacterial or mammalian cells but did induce chromosome aberrations in Chinese hamster lung (CHL) cells in vitro at or above 100 μg/ml, in the absence of metabolic activation.

In-vivo tests (micronucleus, sister chromatid exchange, unscheduled DNA synthesis, dominant lethal tests) did not show any genotoxic potential.

Reproductive studies:

¾ Fertility study : No effects were observed on fertility in either sex or on fetuses after oral administration to rats at upto 360 mg/kg.

¾ Teratogenic study: No effects were observed on fetuses or neonates after oral administration to rats upto 90 mg/kg. Moreover lethal effect to embryos and fetuses, growth retardation in fetuses and neonates or teratogenesis were not observed in rabbits after oral administration at 50 mg/kg.

¾ Prenatal & postnatal study: No effects Were observed on maternal parturition and nursing or on neonates in rats after oral administration of upto 360 mg/kg.

Antigenicity:

No specific antibody to levofloxacin was produced in mice, guinea pigs, and rabbits concurrently treated with adjuvants. In PCA test using serum of experimentally sensitized animals, mice showed positive reaction, but

guinea pigs and rabbits were negative, and systemic anaphylactic reactions were not observed in guinea pigs.

Mutagenicity:

Chromosomal aberration test and sister chromatid exchange test using cultured

Chinese hamster cell showed positive results. However, in vivo studies for the same items, mouse bone marrow micronucleus test

and sister chromatid exchange test results were negative. Moreover, the reverse mutation test, induced mutation frequency test, HGPRT test, in vivo unscheduled DNA synthesis test, and dominant lethal test were negative.

Effect on kidneys:

Following oral administration of upto 120 mg/kg to rabbits for 10 days, no abnormalities were observed in renal function and morphology.

Effect on eyes:

Eye toxicity tests in pigmented rats orally administered 100 mg/kg/day for 14 days showed no changes in electroretiongram, ophthalmological examination and histopathology.

Effect on articular cartilage:

When levofloxacin was orally administered to juvenile rats (3 to 4 weeks of age) and beagle dogs (4 months) for 7 days, lesions were seen in the articular cartilage in rats at 300 mg/kg or more and in dogs at 10 mg/kg or more. Juvenile dogs were more susceptible to the chondrotoxicity. When levofloxacin was orally administered to young adult dogs (13 months of age) for 7 days, very mild toxicity was observed at 40 mg/kg. However, in adult dogs aged 18 months in which the drug was administered for 14 days, no toxicity was observed at a high dose of 30 mg/kg.

Albino mice were orally given levofloxacin and subsequently irradiated with UVA (wave length 320-400nm), and auricular thickness was measured. Phototoxicity (increase in thickness) was not shown at 200 mg/kg.

CHAPTER#09

PRODUCT FORMULATION

Levomax 500mg tablets:

Each film-coated tablet of Levomax contains 500mg of levofloxacin as active ingredient corresponding to 512.46mg of levofloxacin hemihydrate.

List of excipients:

Levomax 500mg film-coated tablets contain the following excipients for a weight of 630mg respectively.

Tablet core

Crospovidone

Methylhydroxypropylcellulose Microcristalline cellulose

Sodium stearyl fumarate

Tablet coating

Methylhydroxypropylcellulose Titanium dioxide

Talc

Polyethylene glycol (E 171) Yellow ferric oxide (E 172)

Machinery and Equipments:

¾ Weighing Balance ¾ Glen Mixer ¾ Fitzpatrick Mill ¾ Stainless Steel Spatula ¾ Multiple Punching Machine ¾ Fluidized Bed Dryer

CHAPTER#10

MANUFACTURING PROCESS

QUALITY CONTROLS

MANUFACTURING PROCESS

Levomax tablets of the same dosage amount are manufactured in batches. After careful weighing, the necessary ingredients are mixed and compressed into units of granular mixture called slugs. The slugs are than filtered to remove air and lumps, and are compressed again into numerous individual tablets. Documentation on each batch is kept throughout the manufacturing process and finished tablets undergo several tests before they are bottled and packaged for distribution.

The procedure for manufacturing levomax tablets, known as dry granulation or slugging, is as follows:

Weighing:

The active ingredient-levofloxacin, the lubricant-sodium steryl fumerate and other excepients are weighed separately in sterile canisters to determine if the ingredients meet pre-determined specifications for the batch size and dosage amount.

Mixing:

Sodium steryl fumerate is dispensed into cold purified water, than heated and stirred until a translucent paste forms. The active ingredient, the microcrystalline cellulose, a part of binder-methyl hydroxypropyl cellulose and a part of lubricant -sodium steryl fumerate are next poured into one sterile canister, and the canister is wheeled to a mixing machine called Glen Mixer. Mixing blends the ingredients as well as expels air from the mixture.

Slugging:

The mixture is than mechanically separated into units, which are generally from 7/8 to 1 inches (2.22 to 2.54 cm) in size. These units are called slugs.

Dry Screening:

Next, small batches of slugs are forced through a mesh screen by a handle-held stainless steel spatula. Large batches in sizable manufacturing outlets are filtered

through machine called Fitzpatrick mill. The remaining lubricant is added to the mixture, which is blended gently in a rotary granulator and sifter. The lubricant keeps the mixture from sticking to the tablet machine during the compression process.

Compression:

The mixture is compressed into tablets either by a single punch machine (for small batches) or by a rotary tablet machine (for large scale production).

On single punch machines, the mixture is fed into one tablet mold (called a die cavity) by a feed shoe. After this, the powder is compressed into tablet with the help of a punch. This punch descends into the die compressing the mixture into a tablet.

On a rotary tablet machine, the mixture runs through a feed line into a number of die cavities that are situated on a large steel plate. The plate revolves as the mixture is dispensed through the feed line, rapidly filling each die cavity. Punches, both above and below the die cavities, rotate in sequence with the rotation of the die cavities. Rollers on top of upper punches press the punches down onto the die cavities, compressing the mixture into tablets, while roller-activated punches beneath the die cavities lift up and eject the tablets from the die platform.

Coating: Film coating;

The optimization of film coating may be necessary to improve adhesion of the coating to the core, to decrease bridging of intagliations, to increase coating hardness or to improve any other property that the formulator deems deficient. The development scientist has to consider three major factor which affect the film quality –tensile strength of the film coating formulation, elasticity of the resultant film and the film tablet surface interaction. Due to these considerations, it becomes very important to use the most optimized coating formulation in order to get the best results.

Film coating involves the deposition, usually by a spray method, of a thin film of polymer formulation around each tablet core. It is possible to use conventional panning equipment but usually specialized equipment is employed to take advantage of the fast coating times and higher degree of automation possible.

The coating liquid contains a polymer in suitable liquid medium together with other ingredient such as pigments and plastesizers. This solution is sprayed on a rotating,

mixed tablet bed. The drying condition results in the removal of the solvent leaving a thin deposit of a coating material around each tablet core.

Film Coating Parameters:

Inlet Air Temperature 50°C

Exhaust Air Temperature 60°C

Air Flow 245 CMH

Spray Rate 06 g/min

Atomization Air 2.0 bar

Pan Speed 20 RPM