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  • In Vivo Study of Macrolide

    Antibiotics & Optimized

    Recrystallized Agglomerates

  • Chapter 10: In vivo study of macrolide antibiotics and optimized recrystallized

    agglomerates

    Direct tabletting and BA improvement of MA by spherical crystallization tech. 286

    10. In vivo study (Bioavailability) of MA and optimized recrystallized agglomerates:

    10.1. Introduction:

    During the past decade, formulation and delivery of Active Pharmaceutical Ingredients

    (APIs) have played a critical role in the development and commercialization of new

    pharmaceutical products. The major objective of formulation chemistry is to improve

    bioavailability, stability and convenience to the patient. Bioavailability means the rate

    and extent to which the active substance or therapeutic moiety is absorbed from a

    pharmaceutical form and becomes available at the site of action. The bioavailability of an

    orally administered drug depends on its solubility in aqueous media over the pH range of

    1.0–7.5 and the rate of mass transfer across biological membranes. A limiting factor in

    the oral bioavailability of poorly water soluble compounds is the inadequate dissolution

    rate. Development of solid dosage forms for water insoluble drugs has been a major

    challenge for pharmaceutical scientists for decades to improve BA. It is well known that

    drug efficacy can be severely limited by poor aqueous solubility because the driving

    force for absorption of most drugs across biological membranes is concentration of drug

    in solution. Dosage forms that enter the stomach and travel down the gastrointestinal tract

    must release the drug in solution to achieve good drug bioavailability. Consequences of

    poor solubility include low bioavailability, large inter and intra subject variation, and

    large variations in blood drug concentrations under fed versus fasted conditions.

    Poor bioavailability can be also due to poor solubility, degradation in GI lumen, poor

    membrane permeation and presystemic elimination (1, 2). By many estimates up to 40

    percent of new chemical entities (NCEs) discovered by the pharmaceutical industry today

    and many existing drugs are poorly soluble or lipophilic compounds which leads to poor

    oral bioavailability, high intra- and inter-subject variability and lack of dose

    proportionality (3). Thus, for such compounds, the absorption rate from the

    gastrointestinal (GI) lumen is controlled by dissolution (4). The ability to deliver poorly

    soluble drugs will grow in significance in the coming years as innovator companies rely

    upon NCEs for a larger share of the revenue within the pharmaceutical market. Many

    technological methods of enhancing the dissolution characteristics of slightly water-

    soluble drugs have been reported in various literatures (5). These include reducing

    particle size to increase surface area (6), solubilization in surfactant systems, formation of

    water-soluble complexes, use of pro-drug, drug derivatization and manipulation of solid

    state of drug substance to improve drug dissolution, i.e. by decreasing crystallinity of

    drug substance (7). Recently, natural polymers such as polysaccharides and proteins have

    received much attention in the pharmaceutical field owing to their good biocompatibility

    and biodegradability (8).

    The spherical crystallization technique has already been successfully applied to improve

    the micromeritic properties of several drugs such as acebutolol hydrochloride, celecoxib,

    and mefenamic acid etc [9-11]. Besides modifying the size and shape, flowability,

    packability and bulk density of the particles, this technique can also be exploited to

    increase solubility, dissolution rate and hence bioavailability of poorly soluble drugs (12).

    In the spherical crystallization technique the manipulation of solid state of drug substance

    to improve solubility and drug dissolution i.e. by decreasing crystallinity of drug

    substances by recrystallizing the drug substances in different solvents by using different

    pharmaceutical excipients. Hence the objective of present investigation is to evaluate the

  • Chapter 10: In vivo study of macrolide antibiotics and optimized recrystallized

    agglomerates

    Direct tabletting and BA improvement of MA by spherical crystallization tech. 287

    in vivo performance of optimized recrystallized agglomerates of MA with respect to

    pharmacokinetic parameters like Cmax, Tmas and AUC in animal models.

    10.2. Bio-analytical method development (13-20):

    HPLC equipment

    The HPLC system consisted of PU – 2080 plus intelligent system pump, a sampler with

    20μl loop, a UV detector UV– 2075 plus intelligent and an interface LC-Net II/ADC, all

    from Jasco (Tokyo, Japan). The reverse-phase column was a HiQ Sil C18-W,

    4.6mm×250mm column.

    Sample preparation:

    1 ml blood of rat was collected from Retro orbital vein and immediately transferred into

    anti-coagulant free 1.5 ml centrifugation tube and subjected to centrifugation at room

    temperature at 8000 rpm for 10 min. The serum obtained from plasma was mixed with 1

    ml of known concentration of drug in acetonitrile and centrifuged at 8000 rpm for 2 min.

    The resultant supernatant solution was further filtered through 0.2 µ filter and injected

    manually in 20 µl loop.

    10.2.1. Azithromycin:

    Chromatographic conditions:

    Mobile phase: phosphate buffer pH 6.5(0.02M): acetonitrile (25:75)

    Flow rate: 0.9 ml/min

    Retention time: 7.9 min

    Sample volume: 20 µl

    Detection wavelength: 215 nm

    Concentration range: 50-1000 ng/ml.

    HPLC analysis: Azithromycin:

    Validation data was collected from three analytical runs. All rat serum lots have used to

    prepare calibration standards. Retention time of approximately 7.9 min was consistently

    observed for Azithromycin throughout all analytical runs. The obtained peak for ATM in

    plasma was mentioned in figure 10.1. Calibration curve data (Figure 10.2) for

    Azithromycin in rabbit serum demonstrate that the calibration curve was linear in the

    concentration range from 50-1000 ng/ml. The correlation coefficient was found to be

    0.9993.

  • Chapter 10: In vivo study of macrolide antibiotics and optimized recrystallized

    agglomerates

    Direct tabletting and BA improvement of MA by spherical crystallization tech. 288

    Fig 10.1: HPLC chromatogram of Azithromycin.

    Figure: 10.2 Standard calibration curve of Azithromycin by HPLC method.

    Calibration Report

    Component: ATM

  • Chapter 10: In vivo study of macrolide antibiotics and optimized recrystallized

    agglomerates

    Direct tabletting and BA improvement of MA by spherical crystallization tech. 289

    User: Venkat, Group: MA, Model: Y = AX

    Nb of Points: 7, A = 149.4062, B = 0.0000

    Correlation = 0.99638

    Standard Error Vy = 1971.2201

    Mean %Error = 5.446

    Equation: Y=151.0x-1157

    Table: 10.1. Observation table for standard curve for Azithromycin by HPLC

    method.

    Sr. No. Concentration

    (ng/ml) Area

    1 0.0 0.000

    2 50 7520.1867

    3 100 14590.3537

    4 200 33250.7174

    5 400 52557.4249

    6 600 85875.1324

    7 800 116202.9881

    8 1000 156565.5573

    The linearity was performed with a 7 point calibration curve. The method was found to

    be linear over the examined concentration range 50-1000 ng/ml. The average calibration

    equation could be described by: Y=151.0x-1157, with a correlation coefficient of 0.9964.

    Where the y is the ratio of the peak area of ATM and internal standard and x is the

    concentration (ng/ml). The limit of detection (LOD) was 20 ng/ml with HQC

    (1000ng/mL) and MQC (400ng/mL). The retention time is 7.9 min.

    10.2.2. Roxithromycin:

    Chromatographic conditions:

    Mobile phase: phosphate buffer pH 6.5(0.02M): acetonitrile (24:76)

    Flow rate: 0.5 ml/min

    Retention time: 7.4 min

    Sample volume: 20 µl

    Detection wavelength: 205 nm

    Concentration range: 2 –12 µg/ml

    HPLC analysis of Roxithromycin:

    The chromatographic peak due to RTM was observed at 7.4 min, which indicated

    sensitivity and selectivity of the developed method. The calibration curve was plotted in

    the range between 2-12 µg/ml. The obtained peak for RTM in plasma was mentioned in

    figure 10.1. Correlation coefficient of 0.99996 indicated linearity of the method within

    the calibration range. Calibration curve and statistical analysis of RTM was reported in

    following figure: 10.4.

  • Chapter 10: In vivo study of macrolide antibiotics and optimized recrystallized

    agglomerates

    Direct tabletting and BA improvement of MA by spherical crystallization tech. 290

    Figure: 10.3. HPLC chromatogram of Roxithromycin.

    Figure: 10.4. Standard calibration curve of Roxithromycin by HPLC method.

    Calibration Report.

    Component: RTM