Cdpceut00001 PDF
Cdpceut00001 PDF
Cdpceut00001 PDF
N. SREE HARSHA
Doctor of Philosophy
In
Pharmacy
DEPARTMENT OF PHARMACEUTICS
AL-AMEEN COLLEGE OF PHARMACY
HOSUR ROAD, BANGALORE - 560 027
2005
This is to certify that the dissertation entitled “Drug targeting
to lungs by way of microspheres” is a bonafide research work
done by N.Sree Harsha in fulfillment of the requirement for the
degree of Doctor of Philosophy.
Date:
Place: Bangalore N.Sree Harsha
This is to certify that the dissertation entitled “Drug targeting
to lungs by way of microspheres” is a bonafide research work
done by N.Sree Harsha under the guidance of Dr. Shobha Rani.
R.H. Professor & Head, Department of Pharmacy Practice, Al
Ameen college of pharmacy, Bangalore.
Date: Date:
Place: Bangalore Place: Bangalore
I hereby declare that the Rajiv Gandhi University of Health
Sciences, Karnataka shall have the rights to preserve, use and
disseminate this disseratation in print or electronic format for
academic / research purpose.
Date:
N. Sree Harsha
Place: Bangalore
With deep sense of respect, I thank Rajeev D. Hiremath . for timely suggestion
and encouragement throughout my work.
All the non-teaching staff of Al-Ameen college of Pharmacy have always helped
in a long way, thanks to Mrs Sabiha Banu, Mrs.Sujatha, Ms.Syeda, Mrs
Jayamma, Mr. Shekar, Mr. Gowda, Mr. Irshad (Jr and Sr.), Mr.
Siddaraju, Mr.Shivanna, Mr.Ravi, Mr.Shareef and Mr. Javed.
Its great pleasure to express my thanks to Chairman Aizaz Khan, Secretary Mansur Ali Khan, Director
Tappan Naik and Principal Mr. M. N. Narasimha Rao for all the support.
Thanks to Dr.Mohan , Principal, PES College of Pharmacy and all the staff of
PES for their encouragement and concern.
With great appreciation, I thank Principal Dr. Krishnamurthy rao and staff
of Milind College of Pharmacy for their support and motivation and also I thank
Mr. Ravi for helping me in lab.
I wish to thank a crew of people for all the technical support provided at all
stages of my research work.
Thanks to Mrs. Sheela Verma of Microlabs, Hosur for the gift sample of
ofloxacin and for determination of drug content by HPLC method.
Thanks to Mr. Santosh SRF for helping me in animal studies.
Thanks to Dr. Suguna, Prof and Head, Veternary College for Histopathological
studies.
Thanks to Mr.Jairali , IISc for carrying out DSC studies at SSCU unit.
Thanks to Mr. Rajesha B.C . Formulator at Apotex Research Pvt. Ltd for all his
intellectual support in optimization studies.
Thanks to Mrs. Vanaja. K for her inspiring words, support and for proof
reading of my thesis.
Thanks to Mr. Narendra , Health scribe for proof reading of my thesis.
All in all, my parents are my life’s greatest treasure. Thank you mom and dad.
LIST OF ABBREVIATIONS USED
10. Beta Macro rate constant associated with the elimination phase
11. Beta-HL The half-life associated with the macro constant Alpha,
25. IV Intravenous
27. K10-HL the half-life associated with rate constant K10, sometimes
Denoted as K10-HL
36. SC Subcutaneous
The efficacy of drug candidates is frequently limited by their inability to reach the target
site of action, especially when they are administered through conventional dosage forms
or drug delivery systems. Targeted drug delivery systems has increased the quantum of
drug reaching the site and simultaneously decrease the amount being distributed to other
parts of the body. Microspheres are solid colloidal particles ranging in size from 1 to 1000
µm, particles with diameter 7 – 15 µm when given through IV route are trapped by the
first capillary system encountered. The capillary sizes of the lungs are of dimensions
lesser than that of the injected microspheres. This phenomenon offers a greater scope of
targeting the drug to the lungs in a more efficient manner, which can act as good targeted
drug delivery platforms especially for pneumonia infections. The research work is aimed
at developing microparticulate targeted drug delivery system of ofloxacin employing
albumin and gelatin as macromolecular carriers for passive targeting to the lungs resulting
in the improved therapeutic efficacy of the drug in case of pneumonia. Pre-formulation
studies were carried out to confirm the suitability of the polymers. Ofloxacin
microspheres were prepared by emulsion polymerization method and mathematically
optimized for the desired particle size range (8-12 µm). Optimized formulae were
GLOME -03 and ALOME -03ofloxacin gelatin and albumin microsphere respectively.
The drug and polymer(s) possessed acceptable suitability and compatibility as evidenced
by the pre-formulation studies. Optimization formulations were in the size range of 8 –
12µm. The surface morphology of the particle were spherical. In-vitro release studies
showed 99.3% and 91.62 release for gelatin and albumin microspheres, release kinetics
showed that the drug release followed Peppas model for both gelatin and albumin
microspheres. Differential scanning colorimetry and X-ray diffraction analysis proved
that the drug is entrapped in gelatin and albumin microspheres. Residual solvent was well
below the permissible ICH limits and provides its safety for formulations. The
formulations were found stable. In-vivo tissue distribution studies of GLOME –03 and
ALOME –03 showed the largest value of AUC for lung when compared with other
organs. The drug distribution was 90.98% and 91.7% for GLOME –03 and ALOME –03.
Pharmacokinetics of the formulations after I.V administration followed two-compartment
model.
Keywords : Microspheres, targeting, lungs, ofloxacin, gelatin, albumin, optimization, in-vivo
TABLE OF CONTENTS
Title Page
1. Introduction 1 - 29
1.1. Targeted Drug Delivery 1
1.2. Drug – carrier delivery systems 4
1.3. Colloidal drug delivery systems 9
1.4. Microsphere 11
2. Objectives 30-31
4. Methodology 62-113
microspheres . 79
6. Summary 204-213
7. Conclusion 214-215
8. Bibliography 216-235
9. Annexures 236-237
9.1. List of papers presentation 236
9.2. Ph.D. registration letter 237
are more or less evenly distributed within the body. Moreover, to reach the
target area, the drug has to cross-different biological barriers – organs, cells,
targeting.
It has been almost 200 years since Paul Ehrlich first formulated the idea of
drug targeting. It was after visiting the opera ‘Der Freischtz’ in which the
Freikugeln plays a major role; Ehrlich came upon the idea of Zauberkuglen –
magic bullets. The Freikugeln in the opera could be fired in any direction, yet
still reach their goal. On similar lines Ehrlich imagined that targeted tiny
drug-loaded magic bullets would target to the required site of action, while
interaction with cell and cell membrane related structure and functions
subsequent control of drug input rate. 3 The efficacy of many drugs is often
cases, only a small amount of the administered dose of the drug reaches this
site, while the major drug amount is distributed to the rest of the body
forms freely travel throughout the body, leading to uptake by cells, tissues or
organs other than where their pharmacological receptors are located. This
The lack of target - specificity can be attributed to the formidable barriers that
the body presents to a drug. A drug taken orally must withstand large
produce therapeutic effect, the drug must selectively access and interact with
its pharmacological receptors, and the concentration of the drug at the active
the drug and dose related toxicity is frequently observed. Further, it cannot be
assured that the drug will reach its desired destination in adequate
drug reaching the site but also simultaneously decrease the amount being
distributed to other parts of the body. Thus, a target oriented drug delivery
system supplies the drug selectively to its site(s) of action(s) in a manner that
the target sites and protects the body from adverse reactions because of
concentration 5, 6,7
and simple.
Different Approaches of Drug Targeting
There are three approaches for drug targeting. The first approach involves the
use of biologically active agents that are both potent and selective to a
forms of active drugs, that when they reach the active sites become activated
approaches are very successful in some cases, most of the times it is not
formidable cost and the long time it takes for drug discovery.
For this reason, the third approach, i.e., the delivery of the original drug by
specially designed drug delivery systems is the better and the only feasible
system depends on the timely availability of the drug in active form at the
application takes for granted that the drugs included in the system will be
expected to “seek out” the preferred site and consequently the drug could be
directed to the intended site of action. For this to happen, the particulate
carrier must have an access to the intended site of drug action, and further it
8
must be able to avoid interactions with other sites within the body.
drug.
• Protect the drug from, metabolism and immune system recognition until it
• Interact selectively with the cells of the target site, if equipped with
• Retain the drug within the particle while ‘in transit’, and release the drug
(polymers) to direct a drug to its target site in the body. Depending on the
carrier system, the drug can be either molecularly entrapped within the carrier
drug – carrier delivery systems is that the distribution of drugs in the body
depends on the physico- chemical properties of the carrier and not those of
carrier.
The various methods of vectoring the drug to the target site by the drug –
• Passive targeting
• Inverse targeting
• Active targeting
• Double targeting
some colloids to be taken up by the RES especially in liver and spleen has
made them as ideal vectors for passive hepatic targeting of drugs to these
drug carriers to redefine its biofate. The natural distribution pattern of the
facilitation of the binding of the drug-carrier to target cells through the use of
active targeting.
Active targeting can further be classified into Ligand mediated targeting and
physical targeting.
Ligand mediated active targeting: Targeting components, which have
selectively deliver the drug to the cell or group of cells generally referred
apoprotein coat serves as a ligand for the LDL receptors expressed in the
body.
programmed and monitored at the external level (ex vivo) with the help of
bed.
Double Targeting: For a new future trend, drug targeting may be combined
with a methodology, other than the passive and active targeting for drug
temporal control of drug delivery. The temporal control of drug delivery has
for a drug, such as zero order release. When these two methodologies are
A large number of drug - carrier delivery systems have been conceived and
developed for the purpose of drug targeting. Among these systems, colloidal
promise for reaching the goal of drug targeting and because of their small size
(<1 micron), colloidal drug carriers come close to Ehrlich’s idea of magic
bullets.
1.3. COLLOIDAL DRUG DELIVERY SYSTEMS
fine particles are larger than most molecules but so small that they cannot be
Due to the small size of the particles, colloidal drug delivery systems can be
In simple colloids, clear distinction can be made between the dispersed phase
Multiple colloids involve the co-existence of three phases of which two are
oil-in-water-in-oil (o/w/o).
matrix of drug and the core material, e.g.: Nanospheres) or capsular (in which
the liquid drug core is surrounded by the carrier material, e.g.: Nanocapsules)
The biodistribution of colloidal particles is dependent on the route of administration
and physicochemical properties such as particle size and surface characteristics (e.g.:
surface charge and surface affinity). If the route of administration is via intravenous
injection, large particles (>7 micron) are retained in the pulmonary region whereas
overcoming the main physiological barriers to achieve drug targeting, namely to the
The following colloidal dosage forms are commonly used in drug targeting:
• Nanoparticles10
• Liposomes
• Polymeric microspheres
• Macromolecular microspheres
Among the entire colloidal dosage forms microspheres are popular as they posses
polymers and natural products like starches, gums, proteins, fats and waxes. The natural
polymers of choice are albumin and gelatin, the synthetic ones being polylactic acid and
polyglycolic acid. The polymers used in their manufacture are chosen according to their
back to the 1930s and the work of Bungenberg de Jong and coworkers on the
encapsulation was by the National Cash Register Company for the manufacture of
carbonless copying paper. The technology and applications have advanced over the last
several decades. The agricultural, food, household products, medical, graphics, and
cosmetics industries use this technology. The potential use of microspheres in the
pharmaceutical industry has been considered since the 1960s. Since then this concept
mundane and inherent challenges associated with the present-day formulation problems.
The current drug delivery systems in use, in many cases, fails to meet the need of the
efficient drug delivery at the target site / organ and thereby eliciting a less efficacious
a certain extent. Microspheres have emerged as one of the desired methods of drug
administration since they offer a sophisticated grasp over the control aspects of drug
include: 14
• Taste and odor masking
• Delay of volatilization
• Freedom from incompatibilities from drugs & excipients especially the buffers
targeted medications.
The microsphere drug delivery method thus facilitates accurate delivery of small
quantities of potent drugs, reduced concentration of drug at sites other than the target
organ or tissue, protection of instable drug before and after administration prior to the
availability at the site of its action. The other major advantage offered by the
pharmacokinetic profile, tissue distribution, and cellular interactions of the drug can
The efficient targeting of drug with microspheres is due to its particle size. The size
plays a significant role in controlling the drug delivery to the target organ and the
The capillary sizes of the lungs are of dimensions lesser than that of the injected
microspheres. This phenomenon offers a greater scope of targeting the drug to the
indicated that the numbers of administered particles as well as their size are
crucial. Particle size greater than 10µm are trapped in the lungs with almost 99%
within 1-2 minutes where subsequent pharmacological response takes place. The
blood-activity profile will depend on the nature of the microspheres and the size
has no role to play. The blood activity- time profile closely follows bi-exponential
pattern of clearance. The initial part is rapid due to the “burst effect” and the
second phase has a much longer half-life and significantly longer release times.
Though the primary seat of activity is observed in the lungs, some activity is also
observed in the liver, spleen, heart and kidneys. The mechanisms of release of
burst mechanism)
polymer)
The combinations of one or more mechanisms are seen in many cases. The release
profile of the drug from the microsphere depends on the nature of the polymer
used and the nature of the drug, micro-morphology of carrier and the nature of the
drug.
Figure 1.3: Factors influencing the microspheric drug release
The osmatically driven burst mechanism water/moisture diffuses into the core
factors namely,
In pore diffusion method, the waterfront diffuses towards the core, the
dispersed drug gets dissolved thereby creating a water filled pore network
through which the active principle diffuses out in a controlled manner. In case
the accumulation of the monomer in the release medium. The erosion of the
polymer begins with the changes with the microstructure of the carrier,
leading to the rapid release of the active ingredients. The rate of extent of
water intake determines the release profile of the microspheres, which in turn
depends on the polymer used, porosity of the polymer matrix and protein drug
loading.
Microsphere Preparation
microspheres since they are designed to elicit a targeted release in vivo. The
include: 14
manufacture include:
3. Coacervation
4. Spray drying
5. Solvent evaporation
6. Precipitation
7. Freeze drying
collected. For highly aqueous soluble drugs, a non-aqueous phase can be used
to prevent loss of drug to the external phase. Wax-coated microcapsules are
inexpensive and often used, release the drug more rapidly than polymeric
Spray coating and pan coating employ heat-jacketed coating pans in which the
solid drug cores particles are rotated and into which the coating material is
sprayed. The core particles are in the size range of micrometers up to a few
millimeters. The coating material is usually sprayed at an angle from the side
into the pan. The process is continued until an even coating is completed.
Coating a large number of small particles may provide a safer and more
adaptation of the fluid-bed granulator. The solid core particles are fluidized
by air pressure and a spray of dissolved wall material is applied from the
perforated bottom of the fluidization chamber parallel to the air stream and
onto the solid core particles. The fluidized-bed technique produces a more
coacervate phase. The coacervate forms around any core material that may be
present, such as drug particles. Agitation of the coacervate system can prevent
Spray Drying
practice and the production of sterile materials. The drug and the polymer
the feed rate of the polymer drug solution, the nozzle size, the temperature in
the drying and collecting chambers, and the size of these chambers.
containing the polymer and the drug may be dispersed in an aqueous phase
employed to evaporate the more volatile organic solvent and leave the solid
to evaporate the more volatile organic solvent and leave the solid polymer–
Precipitation
removed from the droplets by the use of a cosolvent. The resulting increase
suspension of microspheres.
than its critical micelle concentration, are present in the aqueous phase.
radicals diffuse into these swollen micelles and begin the polymerization
the monomer and the drug are contained within micelles composed of
Crystallography
9 X-Ray Diffraction analysis (XRD)
characterization
therapy. However in almost one-third of cases the specific microbial etiology is non-
specific.
Initial antimicrobial therapy is often empirical and is based on the setting in which
chest radiography, results of staining of sputum or other infected body fluids, and
After the etiologic agent is identified, specific antimicrobial therapy can be chosen.
Pathology
upon the causative microorganism rather than upon these anatomic characteristics.
Epidemiology
animals, and contacts with other ill individuals as well as the clinician’s knowledge
about the microbial etiology of a given case of pneumonia, since the outbreaks are
The relative frequency of various pulmonary pathogens varies with the setting in
which the infection was acquired e.g., place of dwelling or hospital. In patients
Pseudomonas aeruginosa, are estimated to account for more than 50% of cases of
pneumonia, while Staphylococcus aureus is responsible for more than 10%. Enteric
aerobic gram-negative bacilli and P. aeruginosa are more common among who
CLINICAL MANIFESTATIONS
Pneumonia is found to have several clinical presentations; the decision on the line
presented in two syndromes; (i) the typical presentation (ii) atypical presentation.
(i) The "typical" pneumonia syndrome is characterized by the sudden onset of fever,
cough productive of purulent sputum, and in some cases pleuritic chest pain; signs
breath sounds, and rales) anomalies can be found in radiographical diagnosis. The
other bacterial pathogens, such as H. influenzae and mixed anaerobic and aerobic
fatigue, sore throat, nausea, vomiting, and diarrhea), and abnormalities on chest
conditions like deterioration in mental status, renal and hepatic abnormalities, and
bacterial infection may either follow the viral infection without interruption or be
separated from the viral infection by several days of transient relief of symptoms.
condition, with persisting or renewed chills, fever, and cough productive of purulent
Patients with hematogenous S. aureus pneumonia may present with fever and
dyspnea only. In these cases the inflammatory response is initially confined to the
consolidation develop only after the infection extends into the bronchi. These
patients are usually gravely ill, with intravascular infection as well as pneumonia,
endemic and epidemic proportions currently making this one of the difficult
disease to manage and is rated as the sixth highest disease in terms of contraction
and fatality17. The current therapeutic practices to contain and cure pneumonia
since pneumonia is caused by a variety of bacterial flora and hence a low success
ofloxacin by oral or parenteral routes does not provide the most efficacious mode
1000 µm, particles with diameter 7 – 15 µm when given through IV route are
act locally on the organ of infection will improve the therapeutic efficacy, reduce
the body and protects from adverse reactions due to repeated or high
dosing.
To prepare microspheres using natural, biodegradable and
biocompatible polymers.
To obtain the desired particle size range of the microspheres for lung
formulation.
histopathological studies.
REVIEW OF LITERATURE
the drug of choice in the current therapy of pneumonia and hence was chosen
Description
fluoro-2-3-dihydro-3-methyl-10-(-4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-
Clinical Pharmacology
achieved after I.V administered may be upto 50% higher, but distribution and
administration. The peak serum concentration after a 400 mg once daily I.V
dose of ofloxacin are 8.1±2.1µg per ml. Steady state is generally reached
within 24 – 36 hours.
metabolites.
Inactivation in body: Serum protein binding of ofloxacin is about 30%.
penetrates well into bronchial secretions, lung tissue and pleural fluid.
ofloxacin is present in saliva, lung tissue, pleural fluid, blister fluid, bone,
results from interference with the enzyme DNA gyrase, which is needed for
infections.
reaction and rashes were also noted in patients treated with the most common
doses of ofloxacin.
maintained.
respiratory tract infections, skin and skin structure infections and bone &
structure and bone and joint, the recommended adult dosage is 400 mg IV
every 8 hours. The recommended adult dosage for mild, moderate and severe
3.2.1 GELATIN
firm gel. The isoelectric point of type A gelatin is between pH 6.3 and 9.2 and
obtained from the partial hydrolysis of collagen derived from the skin,
connective tissue and connective tissue bone and sinews. It obtained either
250 is a widely used choice because they offer good particle size range, drug
loading and the entrapment rate, the drug release kinetics is controllable by
the reaction type and concentration of the linking agent usually formaldehyde
µm. The size and the surface characterizes of the gelatin microspheres are
crucial, since they exert a great influence on the macrophage uptake; and are
non-toxic and readily excreted. Apart from this, it is a soluble polymer it can
be modified to prepare drug delivery systems. 14, 20, 21, 22, 23, 24
3.2.2 ALBUMIN
they are used to target tumor cells due to their selective uptake of protein
The widely used and the method of choice is emulsion polymerization using
absorption and drug release profile which are desirable properties for
hydrochloride, was chosen as drug model and loaded into the microspheres.
period decreased both the swelling and the in vitro drug release processes of the
linked with GAL are well tolerated in vivo. These results suggested the potential
technique: the microspheres had a median particle size of 16.2 +/- 4.2 micro and
were prepared using a stirring speed of 600 rpm for 5 min at 80 degrees C. L-
dopa was incorporated into the microspheres with an efficiency of 65 +/- 6.7%.
L-dopa was released from the microspheres, showing an initial fast release rate,
design was used to optimize the technology of preparation, the appearance and
size distribution were examined by scanning electron microscope, and the
might recognize the target organ (Lewis lung carcinoma). These findings
more than in control (BSA-NS coated with mouse IgG) at 24 hr after the
elimination from the body and their degradability suggested that side
avoided. 78
systems for haematoporphyrin and/or its zinc derivative was probed at the
the slow-releasing pool was higher, the high-affinity pool being the fast-
is one of those drug carriers. The progress has been made during the last
intra-arterial use against human primary metastatic liver tumor was also
discussed. 81
microspheres showed a biphasic profile and the release rates were reduced
cross-linking time. 82
slower release (about 36% in 4 h) profile was obtained for the highly
were carried out with MCF-7 breast cancer cell line. Free 5-fluorouracil
indicated that the diameter of PYM-GMS was more appropriate for the
which significantly reduces the circulating drug level and the dosage,
and intestinal emptying time of the beads was observed, which was also
phase was created as follows. First, the aqueous disperse phase was fed
prepared by the suspension method using toluene as the oil phase. Rat
cells retained its spherical shape, which is similar in vivo, and showed no
full factorial design study. The influence of the concentration of PVA and
the polymers tested on particle size and zeta potential value was evaluated
before and after freeze-drying of the prepared particles. The original 2(2)
design for poloxamer and carbopol, in order to fit the measured data to a
macromolecules. 89
copolymer (PHBV) (Mw = 630kD, 21% mol HV) were loaded with
18. A fluid mechanics-based correlation for the average size of Bovine Serum
well suited for the controlled release of MTZ and were promising for anti-
cancer chemotherapy. 92
collecting the particles in a liquid bath was evaluated. The protein bovine
studies indicated that the degree of uptake by the lungs was higher than
that of the other organs. All these results demonstrated that terbutaline
free and in the size range 1-5 micron. The drug release from the
The type of surfactant affected the microsphere shape and BSA release.
the first time in the absence of any surface-active agent, using paraffin oil
particle size distribution in the range 50-400 microns. This method, being
simple and cost effective, could be a promising technique for the large-
and aqueous volume. These factors significantly affected the sphere size,
polydispersity and yield. Of all the factors investigated, only the albumin
high yields (> 80%). The study concluded that this new preparation
method for albumin nanoparticles was suitable for future drug targeting
studies. 102
30. Human serum albumin (HSA) microspheres were produced in the size
heating times (> 30 min) for stabilization. This paper describes the
systemic side effects. The percentage accumulated was higher in the lungs
than in the other organs for both albumin and gelatin microspheres,
albino mice. After the microscopic determination of the lungs and liver,
the spherical microspheres were observed after 10,30 and 60 min, and 6
hydroxy urea in the prepared microspheres was recorded when t 10% drug
phase. 106
liver at a higher concentration and that use of the two formulations might
nanoparticles was developed. After the first desolvation step, the low
for cell uptake studies and fluorescent nanoparticles showed a high uptake
that oxidized dextran can form a cross-linked gelatin network which can
these formulations. HPLC data revealed that the drug was stable in these
formulations for atleast 6 months. The FTIR studies indicated the absence
possibly modify gelatin dissolution and drug release. The effect of this
emulsion process, using 2:1 as the weight ratio of LTH to gelatin was
particle size and size distribution, LTH content, in vitro release, stability,
microns. The release t 1/2 of LTH when given LTH-GMS was about 6 times
and blood, and was about 6 times as much as that of the LTH solution
coacervation method. In vitro studies were carried out and the results
into the gelatin microspheres and the spheres were characterized for the in
showed that 51% of the entrapped drug was released during the first day
and the release followed approximate zero order kinetics from day 2
onwards. The total release of FITC-BSA lasted for about 8 days. SDS-
PAGE analysis revealed that BSA was not degraded by this preparation of
microspheres. 116
were acquired through orthogonal test. The paddle method was used to
45. Microspheres are drug carrier system which ensures controlled release
serum albumin and bovine serum albumin, which were frequently used
different stirring rate. In the first part of for, drug content, payload,
microspheres containing the active substance were injected into the tail
were studied: (1) type of albumin; (2) albumin concentration; (3) speed
surfactant (Span 85); (7) type of oil; and (8) mixing-cell with or
with natural biodegradable gelatin as the load material and castor oil as
the oil phase. The experimental conditions were optimized; the mean
volume diameter obtained being 9.7 microns and the mean rate of
prepared. Release of the drug from the DHAQ-GMS in vitro was much
slower and its t1/2 was 4 times longer than that of pure DHAQ. The
prepared and injected into the tail vein of mice. The results showed
was regular, the mean size was 13.13 +/- 3.55 microns, embedding
ratio was 21.62% and the release characteristics in vitro were in accord
mice. The total amount in the lung was about 97% of the injected dose
53. Factorial concept was utilized to optimize the size and drug loading of
stirring rate at low and high levels. For two levels of the four variables
concentration, low water-oil phase ratio and low stirring rate. 127
4.1.1 Estimation Procedures of the drug
^ Accuracy
^ Precision
• Repeatability
• Intermediate Precision
• Reproducibility
^ Specificity
^ Detection Limit
^ Quantitation Limit
^ Linearity
^ Range
^ Robustness
Accuracy
accepted reference value and the value found. This is sometimes termed
trueness.
Precision
and reproducibility.
Repeatability
Repeatability expresses the precision under the same operating conditions over a
Intermediate precision
Reproducibility
Quantitation Limit
products.
Linearity
Range
The range of an analytical procedure is the interval between the upper and
regression analysis. The general purpose of linear regression (the term was
first used by Pearson, 1908) is to learn more about the relationship between
mX+ c’. Thus, the Y variable can be expressed in terms of a constant (c)
and a slope (m) times the X variable. The constant is also referred to as the
The regression line expresses the best prediction of the dependent variable
line. The deviation of a particular point from the regression line (its
predicted value) is called the residual value. The smaller the variability of
the residual values around the regression line relative to the overall
most cases, the ratio would fall somewhere between these extremes, that is,
between 0.0 and 1.0. 1.0 minus this ratio is referred to as R-square or the
coefficient of determination. The R-square value is an indicator of how
well the model fits the data (e.g., an R-square close to 1.0 indicates that we
have accounted for almost all of the variability with the variables specified
in the model).
Procedure
Ofloxacin
Estimation by spectrophotometry
The working stock (3) solution was scanned in the range of 200nm to
400nm after correcting the base line with a reagent blank (0.1N HCL). The
λ max was found to be 297 nm. Hence all further investigations were carried
From the working stock (3) solution different aliquots of 1.0, 2.0, 3.0…
with 0.1 N HCl. The absorbance was measured against a reagent blank at
297 nm and the standard graph was plotted, which is shown in figure 4.1.1.
Recovery studies
stock solution was again added in a 10 ml volumetric flask and the volume
made up with 0.1 N HCl. The absorbance of the resulting solution was
in table 4.1.1
0.7
0.6
ABSORBANCE (nm)
0.5
0.4
y = 0.0319x
0.3 2
R = 0.9986
0.2
0.1
0
0 5 10 15 20 25
CONCENTRATION (mcg/ml)
R2 Recovery ( % ) Linearity
(mcg/ml)
Spectrophoto- 0.9986 95.08 0 – 20
metric estimation of Standard calibration curve – Absorbance = 0.0319 x
ofloxacin 0.1 N HCl concentration
The coefficient of determination ‘R 2 ’ are within acceptable limits for the
was reproducible and hence reliable for routine analysis of drug from
formulations.
albumin and gelatin were found suitable polymers for the preparation of
microspheres.
Procedure
prepared by the water in oil emulsion and spray drying method. 28 The
Both the polymers were found to be suitable for the formulation of microspheres. The
which were prepared by water in oil emulsion method and shrivel (surface folding)
albumin-0.5g; emulsifier-0.5ml) section 4.1.3 which was almost near to the specified
range and microspheres prepared by spray drying technique had an average size of
12µm and 10µm respectively, which was within the specified range.
Many different techniques have been proposed for the production of microspheres and
it was suggested that more than 200 methods could be identified in the patent
literature.29
method. We have selected one chemical and one mechanical process for the
preparation of microspheres.
It has been reported that many scientists have prepared microspheres by water in oil
emulsion technique and spray drying technique for lung targeting.12, 30, 31, 32
Procedure
microspheres preparation from natural polymer, viz. water in oil emulsion and
spray drying method. The blank microspheres formed were characterized for
their surface morphology and Particle size. Thus, two methods of preparation
solution was prepared in 10 ml of water and the dissolution process was aided
by warming the solution. The solution was stirred for 20 min at 25 0 C prior to
the same temperature the emulsifying agent tween 85 was added into the two
phase system and was stirred continuously for 5 min then the emulsion was
filtration process with a sintered glass filter. This process however was
prepared by taking 2 gms of albumin. This was solubilized; then the contents
were slowly added to a beaker containing 650 ml of sesame oil and stirred for
1h at 1000 rpm, to this 0.5 ml of tween 85 was added and was stirred
The microspheres were isolated by filtration and washed with n-hexane and
working formula.
44
Preparation of Gelatin/Albumin Microspheres Spray Drying Method
microspheres. The solutions of the polymers viz. gelatin and albumin were
with an average particle size of 58µm with gelatin and 42µm with albumin.
microspheres with size of 12µm (gelatin) and 10µm (albumin). The shriveled
and bored-out appearance of the spray-dried microsphere was seen during the
SEM surface morphology studies figure 5.1.4 & 5.1.5. Hence sensing upon in
this inelegance of the product and potential failure in case of drug loading,
the spray drying process was discontinued in favor of emulsion
polymerization. 33
found unsuitable for the intended purpose. The ideal particle size range for
in the range of 5-15µm. Hence it was decided to optimize the size range and
34
4.1.4. Sterilization Studies
tissues.
attacks these same amine groups. 35 Any residual gas can induce a toxic
polymeric biomaterials.
In dry heat sterilization, the biomaterial is exposed to 160 – 190 0 C, but the
Aseptic filtration though a popular method for parenterals again could not be
residue.
size (laser diffraction) studies were carried out on these formulations prior to
irradiation. After sterilization, the particle morphology and particle size was
Results
importantly the particles did not break and were intact. A slight increase in
the particle size (1.2µm – 3 µm) was observed after sterilization with γ -
albumin (1:1) and physical mixture of ofloxacin: gelatin (1:1) using. Samples
press at a force of 5.2 τ cm -2 for 3 minutes. The scanning range was 400 –
reference with which the spectrums of the physical mixtures were compared.
prominent difference in the spectrums was seen. Thus, it was concluded that
The formulation studies constitute the phase of the work involving the
phase. This phase of work helps in deciding the right working formulae for
the microspheres, which during this phase of work were finalized upon
warming and the drug was added to the gelatin solution. The concentration of
drug was varied according to the varying concentration of Gelatin (1:1 Drug:
Gelatin). The solution was stirred for 20min at 25 0 C and the solution was pre
same temperature. This two-system, plus Tween 85 was added to obtain a w/o
as shown in table - 4.2.2. A solution of albumin was prepared and the drug
was added to the albumin solution. The contents were slowly added to a
beaker containing 650 ml of sesame oil and stirred for 1h. This two systems
plus Tween 85 was stirred. The temperature was raised to 40 0 C for hardening
hexane and dried at room temperature. The preparation was carried out
Concentration of
0.5g 2g
Gelatin (gms)
Concentration of
0.5ml 2 ml
Emulsifying agent (ml)
Table 4.2.2: Variables operating range for albumin microspheres
Concentration of
0.5g 2g
Albumin (gms)
Concentration of
0.5ml 2 ml
Emulsifying agent (ml)
simply by trial and error while varying one factor at a time. This approach is
To over come this, the approach is to optimize - that is, to determine the
region in the factors that leads to the best possible response. In addition to the
experience of the formulator, techniques are available that can aid the
formulator’s choice of formulation components which will optimize one or
for formulation. If all the variables are included in the experimental design,
The formulator must first identify the potentially dominant parameters that
established between what is worth and what is not worth investigating. The
which purposeful changes are made to input variables or factors, and data are
collected at each run. This is used to identify the process conditions that
influence best output and then determine the input variable (factor) settings
that maximize results and to draw logical and purposeful conclusions from the
identify a "vital few" controllable factors and to correlate the factor settings
that optimize the response. Response surface methods are employed to:
response
suitable technique for the modeling and analysis of problems in which the
Experimental Plan
formulations were prepared as per table - 4.2.4 to elucidate the effect of the
and the dependent response variable measured was the particle size
The obtained results were first fitted into different regression models like
modified, mean, linear, quadratic and cubic. The model with significantly
(ANOVA) was performed [Probability F value less than 0.05 (95% level of
The contour, response surfaces and interaction graphs were plotted to obtain
have a maximum desirability (of 1.000) are considered always. 30, 41, 42
formulations as per the probable solutions and subsequent correlation with the
predicted values. That probable solution which gave the desired response and
which was viable was taken as the final optimized formula for the preparation
of microspheres.
Experimental Plan
A central composite design was created to study the main effects and
formulations were prepared as per table 4.2.12 to elucidate the effect of the
and the dependent response variable measured was the particle size
The obtained results were first fitted into different regression models like
modified, mean, linear, quadratic and cubic. The model with significantly
(ANOVA) was performed [Probability F value less than 0.05 (95% level of
The contour, response surface and interaction graphs were plotted to obtain
equation) required to obtain the desired response. Only those solutions that
have a maximum desirability (of 1.000) are considered always. 30, 41, 42
formulations as per the probable solutions and subsequent correlation with the
predicted values. That probable solution which gave the desired response and
which was viable was taken as the final optimized formula for the preparation
of microspheres.
4.2.4. FINAL OPTIMIZED FORMULAE AND FORMULATION CODES
OF OFLOXACIN GELATIN AND ALBUMIN MICROSPHERES
The final optimized formulae of all the formulations and their respective
formulation codes are given in table 4.2.9 and 4.2.16 for gelatin and albumin
microspheres respectively.
All the further studies were carried out using Glome–03 and Alome–03.
the three optimized formulas had the required particle size range 5 – 15 µm
(SAL) is required for parenteral products which come in direct contact with the
tissues.
high temperature (greater than 121oC) and steam at high-pressure. These conditions
membrane proteins and lipids. Steam sterilization is rapid compared to other methods,
however this requires about 30 minutes of exposure this degrades most polymeric
biomaterials under the harsh process conditions of steam sterilization, which often
exceed the glass transition (Tg) and melting temperatures (Tm).37 As a result,
Ethylene oxide (EtO) kills microorganisms by alkylating the amine groups on nucleic
acids. However, EtO is also toxic to humans, since it attacks these same amine
groups.35 Any residual gas can induce a toxic response in vivo. Therefore, gas
In Dry heat sterilization the polymers are subjected to a temperature 160 – 1900C, but
due to the problem of melting and softening of the polymers, this method is was not
Although aseptic filtration is a popular method for parenteral sterilization, again this
could not be employed for these microspheres formulations because they are colloidal
Gamma (γ) radiation is a type of radiation that sterilizes microspheres by ionizing the
nucleic acids of the contaminating microorganisms. The use of γ radiation has been
sterilization, an only gamma irradiation method was found to the best-suited method
optimized formulae (formulation codes – ALOME -03 & GLOME -03 – since
they are optimized formulae and were chosen in a random manner to prevent
bias over the particle size) by using the emulsion polymerization method.
sterilization, the particle morphology and particle size was again carried out.
since this study accounts for the amount of microsphere obtained at the end of
the preparation; and the polymer and the drug which went into its
Procedure
The equal quantities of polymer and drug (1 gram each) were taken for
(theoretical yield) and the actual yield got as the results of this formulation
amount of drug that is entrapped in the microsphere and the drug, which is
adsorbed in the surface of the polymer. This study carries vital importance in
surface studies since this is indicative of the measure of drug that goes in
polymer or as free drug and as a faction which is found entrapped within the
polymeric matrix.
microsphere should take into account the free adsorbed and entrapped
that, the total drug content of the microspheres should quantify all the three
portions of the drug – free, adsorbed and entrapped and they should be
Procedure
a) Free drug
b) Adsorbed drug
c) Entrapped drug
mixture was shaken well in order to liberate the free drug present in the
polymeric matrix. This was quantified by measuring the absorbance on
min, and then centrifuged at 3000rpm for 2 min. The supernatant was
The leftover residue from the extraction of free and adsorbed drug was
mixed with 5ml of glacial acetic acid 0.1M the samples were
The free drug in the microsphere was determined using the formula
Qm
L= X 100
Wm
Qm
L= X 100
Wm
The amount of drug entrapped in the microsphere was determined using the
formula
Qp
E= X 100
Qt
Examining the surface of a polymeric drug deliver system can provide vital
distribution and morphology of the surface and the encapsulated matrix can
The most common technique used for characterizing the surface morphology
loading the samples for taking the photomicrograph, samples are coated (20 –
providing heavy metal atoms with incident beam of electron and, to conduct
the accumulated sample charge and heat to the sample holder. The coating
evaporation.
Procedure
codes GLOME -03 & ALOME-03 respectively) was observed by SEM – SEM
(Joel, Japan). The microspheres were placed on graphite surface and coated
with gold using an ion sputter (Joel, Japan) and were observed at 12KV.
ensure that particle size of the formulation lies in the optimal range. This is
the crucial factor since the target activity as intended in this study exclusively
depending upon the size itself. These methods use different physical
chief attraction of this technique is the quick and easy measurement of size
distribution over a broad range of 0.1 – 1000 µm. The dry powders is sampled
and sized by this method. Helos is an example of the instrument utilizing this
other manufacturers, especially in the lower size range. This is because all the
particles are not spherical always and because each manufacturer uses own
The particle size distribution studies of the microspheres were carried out by
Laser Light Diffraction and Ultrasonic techniques; HELOS laser system for
Procedure
The free flowing microspheres were added into sample holder of the HELOS
The in vitro release studies were carried out as per the following procedure to
study the drug release pattern to note the extent and pattern of the drug
The compartment were stirred and the drug which diffuses out of the
assayed. 48, 49
In this study, the in vitro release patterns were studied using conventional
(PBS), pH 7.4 at 37±1 o c. Sink conditions were maintained and throughout the
Aliquots were withdrawn at specific time intervals and the same volume of
dissolution medium was added to the flask to maintain a constant volume. The
nm, and the data were used to calculate a cumulative drug release profile from
The in vitro release studies were carried out for albumin and gelatin
terms of drug release, and therefore its efficacy. The initial release is
adsorption and do not involve in any chemical bonding with the polymer.
When such microspheres are briefly washed in a sink condition, these drug
particles would be washed, the drug bound to the surface of the microspheres
are rapidly washed, and would lead way to a rapid release of the drug in the
early stages. However, this phenomenon holds good for only a brief period
with maximum drug release. After this time window, the release pattern is
Since the microspheres are heterogeneous systems, this suggests that the drug
matrix are phase separated into two distinct domains a bi-phasic system, the
microspheres.
The entrapped drug, which separated into a bi-phasic system, upon the
liberation from the polymeric matrix, and through diffusion process the drug,
into account all the variants mentioned above. The loading of drug into the
polymer matrix also plays a significant role in the release profile of the drug
(in this case 1:1); a point of domain separation is reached and the drug
particles come in contact with one another, and the drug diffuses out of the
matrix. Leaving the solvent filled channels open. Through this void channel,
the drug is preferentially released paving way for a controlled drug delivery
state, coupled with the size of the microsphere, the targeting can be achieved
organ (in this case –lungs) can be attained satisfactorily with the aforesaid
conditions.
However to quantify the drug release pattern, the drug depletion from
• Predict the value of the response variable for any value of the predictor
Since the response and the predicator responses in this case may not follow a
thereby predicting the release rate (as in the ideal case). Regression equation
have adopted different approaches to curve fit; this study has used the
prominent regression models to study the drug release and their mode of
2. Higuchi Model
4. Peppas Model
The best model of the fit is determined by the R 2 and adjusted R-values. They
response values that is explained by the predictor. Higher the R 2 value, best is
the fit offered by the equation / model. The fitted line in the plot is a
The method used to draw the line using a 95% confidence interval and around
3σ values were used to attain precision in the replicate runs. This was
The DSC technique can provide qualitative and quantitative information about
the drug inside the polymer matrix can be assessed, which may emerge in
display relevant properties during in vitro release and hence can be utilized as
drug’s melting point when present in this mixture disappears in the drug
unloaded microsphere (the ones without drugs). This is indicative of the fact
inside the polymer matrix formation, even if the drug loading is of higher
drug delivery system depends on the careful selection of the excipients, which
are added to the formulation. Ideally, these excipients should not interact with
the drug leading to its degradation. This fact is indicated by the appearance of
thermal signature with a broadened peak base, at the end of the thermal
spectra this might not happen in all cases since the thermal activation energy
Interpretation of DSC
An interaction on DSC will show changes in melting point (peak shift) and/or
transition temperature and peak shape and area by virtue of mixing two
no new thermal events occur or are lost by mixing the two components, no
melts. 60
methods like TLC is that neither long-term storage of the mixture is required
the interactions.
efficacy.
PW 170 system (Philips USA) with Cu-K 2α radiation (40KV, 30 mA; scan
ofloxacin albumin and ofloxacin gelatin microspheres are given in the figures
1to5.
4.2.15. RESIDUAL SOLVENT ANALYSIS
Residual solvents in pharmaceuticals are volatile organic chemicals that are used in
Presently in the pharmaceutical industry special importance is given for the residual
solvent testing as they will affect the physicochemical properties of the drug
potential risk to human health due of their carcinogenicity and ototoxicity 20, 25, 33, 61, 62
polymerization method were tested for acetone. The acetone levels were
Procedure
Instrument : Shimadzu Gas Chromatograph GC 17A
Mobile Phase : Nitrogen.
Column : SGE BP-5 capillary column (0.25mm x 30m, 0.25mm).
Column pressure : 30 kpa.
Temperature : Column - 140 0 , Injection port – 220 0 & Detector 250 0 .
Injection volume : 1 µl.
Flow rate : 20 ml/min.
Detector : Flame ionization detector.
Ret. Time : Methanol – 2.5 min, Dichloromethane – 5 min &
Acetone – 7 min.
10 mg of microsphere formulation was extracted with 10-ml portion of
0.45 µm filters (Millipore, India) into 10-ml volumetric flasks and the volume
made up with DMSO. From this filtrate, 1.0 µl was injected into the gas
that the product intended to be used is of adequate quality, and has safety
specifications. 51, 63
gelatin microspheres in this case) varies with time under the influence of a
to establish a re-test period for the drug substance or a shelf life for the drug
procedure.
This guideline provides generic directive to study the stability of drug product on a
real time basis – since they provide a more realistic data. The storage conditions
described in the guideline are based on worldwide storage temperature (mean kinetic
temperature- MKT) and percent relative humidity (RH) conditions. The countries of
the world are segregated into four zones depending on their climatic conditions,
namely, Zone I, II, III and IV. India is designated zone III / IV for its tropical
presence and high levels of ambient temperature and humidity levels.64 Minimum
time period to be covered by the stability analysis is at least 12 months for long-term
• Testing has to be conducted in the final package, i.e., in the containers and
• ICH guidelines suggest a sampling frequency of every 3 months during the first
year, every 6 months during the second year and then annually for drug substances
• Although the guideline does not propose a testing frequency for samples stored
under accelerated conditions, the FDA guideline has set a frequency of 0, 2, 4 and
study.
The parameter used for evaluation of the drawn samples generally varies with the type
of dosage form. As per the ICH guideline “Specifications: Test procedures and
acceptance criteria for new drug substances and new drug products – Q6A”, one
quality characteristics of the product, which are susceptible to change during storage,
in addition to drug assay and the analysis of degradation products, thus covering the
Procedure
The stability protocol was designed based on the ICH ‘Q1AR’ and ‘Q6A’ guidelines.
polymers chosen. Since these polymers were of protein origin and have relatively low
thermal stability profiles – gelatin and albumin degenerate 500C and above 250C
respectively.
The stored samples were tested for their drug content, particle size distribution, and
for any physical change. The drug content was determined through a UV
The testing was carried out at 0, 2, 4 & 6 months (as per FDA guideline) for
accelerated storage condition; and at 3-month intervals for a period of 12 months for
The in vivo studies are a key component in this study since they provide
Experimental Procedure
adult albino mice weighing between 20±2 gms were selected at random and
divided into 6 groups to test the drug release at the following time periods –
10 min, 30 min, 1 hr, 3 hrs, 6 hrs, and 12 hrs. Prior to the administration, the
study groups were randomly divided into 6 groups, with 6 animals in each
group. All the animals were kept on starvation 12 hours before injection, with
intervals as stated earlier the animals were injected with the microspheres (for
both gelatin & albumin) via the tail route vein; and were sacrificed by
cervical dislocation. The organs, which were studied, for target action – viz.
lungs, liver and spleen were extracted. The tissue samples were stored for 24
described below.
Sample preparation
0.1mg/ml. The protein in the mixture was precipitated (to prevent interference
mixture was then vortexed for 5 seconds to aid the mixing process and was
The method was chosen for its capability to determine ofloxacin in body fluids –
serum and blood as well as tissues. The samples were prepared as follows; the organs
extracted, weighed and were subjected to cutting to tissues with scalpel, homogenized
with 1-3 ml buffer, centrifuged at 9600 g for 5 min, three cycles, injected a 20µl
aliquot of the preparation. HPLC variables were as follows; column used was 200X
45 µm Nucleosil C18. The mobile phase used was a mixture of methanol, methyl
cyanide buffered with pH 3.0 with phosphoric acid in the ratio 13:7:80 the pH
adjusted to 3.0 with phosphoric acid (buffered with was 15mM phosphoric acid
96
adjusted to pH with 3.0 with tetrabutylammonium hydroxide). The HPLC
Parameter Specifications
column C18 type 200 X 45µ m Nucleosil
MeOH:MeCN:buffer 13:7:80
Mobile
adjusted to pH 3.0 with
Phase
phosphoric acid
Flow rate 1 ml/min
injection
20-100µl
volume
Detector F ex 278 em 446
Limit of
10 ng/ml
detection
formulations to the lungs and compared with the conventional dosage of the
drug.
delivery system can be made available for routine use, it is important that the
of drug, in all major tissues of the body, following its administration via the
administered via test targeted drug delivery system, the denominator refers to
that the tissue ‘i ’ is exposed to drug to a greater extent following the use of
efficacy of two delivery systems in reference to one tissue, it does not provide
non-target tissue. Here values of te > 1 indicate greater selectivity of the delivery
system for the target tissue, as compared to the non-target tissue against which this
parameter is estimated. Implicitly, the higher the value of te the greater is this
selectivity.
The drug targeting efficiency of a delivery system against a given non-target tissue, a
Where the denominator refers to the sum total of drug exposure to all the tissues,
Or
Pharmacokinetics in simplest terms is how drugs move in the body and how
It is concerned with the study and characterization of the time course of drug
of the drug and the physiological properties of the body membranes and
fluids.
The drug then interacts with receptors and causes therapeutic or / and toxic
responses.
Drugs are in a dynamic state within the body. The biological system is very
complex and the kinetics of the drug is very complicated. One can describe
the drug kinetic processes or interpret the data using simple mathematical
models.
hypothesis.
Types of PK models
1. Compartmental
2. Non-compartmental
3. Physiological
Among all the three, compartmental analysis is the traditional and most
Compartmental model
compartments that are connected reversibly with each other. The kinetics of
disposition.
compartment.
3. One can estimate the amount of drug in any compartment of the system after the
There are two types of compartment models, namely, mammillary and caternary
model. Among two compartment models mammillary model are the most common
compartments are connected to central compartments like satellites with the central
into the tissue changes that might have occurred due to the pre-existing disease conditions
Histopathological studies are a method of determining the tissue compatibility with the
portion of the drug and excipient are accumulated in specific tissues (such as lung, liver &
spleen) and therefore, determining the compatibility between these tissues and the
formulations.
Procedure
were sacrificed by excess anesthesia and the lungs, liver and spleen were dissected and
washed with cold saline. The organs were pressed between filter pads and weighed.
Liver and spleen tissues were fixed in 10% neutral formalin using standard techniques
and stained with hematoxylin and eosin (H&E) stain for histopathological examination.
All tissue samples were examined and graded under light microscopy with 500x
magnification71
Table 25 1
5. RESULTS & DISCUSSION
The particle size optimization data is shown in the table 4.2.4. The results
values of R 2 and model sum of squares. ANOVA was performed and the
ANOVA proved that the model was significant (with a probability F value
graph was plotted figure 4.2.1, 4.2.2 & 4.2.3, followed by numerical
affects the particle size. From the numerical optimization results, solution
Design Summary
Study Type : Response Surface
Experiments: 15
Initial Design: Central Composite
Blocks: Nil
Design Model: Quadratic
points) was adopted since it is employed to analyze the relationships between multiple
variables with a reduced number of experimental runs. The particle size of the Gelatin
Ofloxacin microsphere was selected as the response to be measured, since this offers a
showed the particle size of microspheres to lie in a range of 0.32 – 22µm, and is not
affected by transformations and this followed a linear setup which indicated that the
response measured holds good over a range of other factors viz. stirring, polymer
concentration and the amount of emulsifier used. This would in turn help in fixing the
optimal particle size range, which has the maximum efficacy and the effect of other
DESIGN-EXPERT Plot
Particle Size
X = A: Stirring RPM
Y = C: Amt. of EA
Actual Factor
B: Gelatin Conc. = 1.89
42.48
31.22
19.96
8.71
Particle Size
-2.55
0.50
0.88
5000
1.25
4000
C: Amt. of EA
3000 1.63
2000
A: Stirring RPM 1000 2.00
upon the particle size, the gelatin concentration increases with that of the
particle size the gelatin microspheres. This can be attributed to the increasing
lowers the subdivision of the gelatin solution into smaller droplets during
emulsification and finally increases the particle size. The increases in drug
concentration therefore had no effect on the particle size and in turn depend
upon the variabilities like stirring rate, concentration of the gelatin and
emulsifier.
Figure 4.2.2: 3D RSM Graph of ofloxacin gelatin microsphere - factor: amount
of emulsifying agent
DESIGN-EXPERT Plot
Particle Size
X = A: Stirring RPM
Y = B: Gelatin Conc.
Actual Factor
C: Amt. of EA = 1.00
42.48
31.22
19.96
8.71
Particle Size
-2.55
0.50
0.88
5000
4000 1.25
In this 3D RSM the increase in the amount of the emulsifying agent decreases
the viscosity present in the system and thereby lowering the particle size, the
decrease in the RPM in interaction with the gelatin concentration increase its
particle size and vice versa. This figure is indicative that optimal particle size
formation depends upon the correct RPM and the concentration of the
polymer.
Figure 4.2.3: 3D RSM graph of ofloxacin gelatin microsphere - factor: stirring
rate
DESIGN-EXPERT
Pl t
Particle
X = B: Gelatin
Si
Y = C: Amt. of
C
EA
Actual
A:
F Stirring
t RPM 42.48
=
4840
31.22
19.96
Pa
8.71
rtic
le
-2.55
Siz
e
0.50
0.88
2.00
1.63 1.25
1.25 C: Amt. of EA
1.63
0.88
B: Gelatin Conc. 0.50 2.00
In the 3D RSM the stir factor is found to have the influence on the
RPM’s the figure is indicative that the particle size would be lower range due
to the higher dispersion of the emulsifying agent influencing particle size and
vice versa and it also suggests that the optimal particle size formation can be
Gelatin - Gl -
Polymer
Albumin - Al -
Drug Ofloxacin -O-
Emulsion Polymerization -E-
Method of Preparation
Spray Drying -S-
Microspheres -M-
The Anova for response Surface design showed a quadratic setup, which indicates the
employment of CCD. The higher sum of square values for the factor concentration of
gelatin indicates that this factor is influenced by the other factors – stirring rate and amount
of emulsifier used. The influence exerted by these two values almost similar. The sum of
squares for the gelatin factors as well, is predicted by this model in comparable terms. And
output of interacting factors shows a higher value for factors, which involves gelatin. This
shows that the system is influenced by the inclusion of gelatin – supported by the other two
EA = Emulsifying Agent
The numerical optimization study was conducted to study the constraints on
the design space and the vulnerability of the experimental model, this is
important since it suggest factors, responses and the goal for each variable
solutions can be arrived at in quantitative terms, i.e. the likely behavior of the
the design space. The influences of stirring rate, polymer concentration and
data table 4.2.9 suggested the closeness of the predicted results with that of
1.00
8.00 12.00
1.00
8.00 12.00
1.00
8.00 12.00
and further evaluated for the response i.e., particle size. It was seen that the
response was almost similar to the response predicted by the design expert
software.
All the further studies were carried out using GLOME-03. Since all the three
optimized formulae had the required particle size range of 5 – 15 µm for lung
studies.
5.1.2. Optimization of ofloxacin albumin microspheres
The particle size optimization data is shown in the table 4.2.12. The results
of R 2 and model sum of squares. ANOVA was performed and the results are
ANOVA proved that the model was significant (with a probability F value of
The three-dimensional response surface graph along with the contour graph
concentration most significantly affects the particle size. From the numerical
optimization results, solution 2,1 & 7 was selected randomly as the optimized
Design Summary
Study Type : Response Surface
Experiments: 15
Initial Design: Central Composite
Blocks: Nil
Design Model: Quadratic
Response Transformatio
Units Observations Minimum Maximum Model
n
Particle Size Microns 15 3 35 None Quadratic
transformations and this followed a quadratic setup which indicated that the
response measured holds good over a range of other factors viz. stirring,
polymer concentration and the amount of emulsifier used. This would in turn
help in fixing the optimal particle size range, which has the maximum
Sum of Mean
Source DF F Value Prob. > F
Squares Square
Model 1356.29 9.00 150.70 15.30 0.0039
A 273.78 1.00 273.78 27.80 0.0033
B 264.50 1.00 264.50 26.86 0.0035
C 86.99 1.00 86.99 8.83 0.0311
A2 27.97 1.00 27.97 2.84 0.1528
B2 47.69 1.00 47.69 4.84 0.0791
C2 73.88 1.00 73.88 7.50 0.0408
AB 92.85 1.00 92.85 9.43 0.0278
AC 0.08 1.00 0.08 0.01 0.9303
BC 360.80 1.00 360.80 36.63 0.0018
Residual 49.24 5.00 9.85
Lack of Fit 49.22 1.00 49.22 7837.34 < 0.0001
Pure Error 0.03 4.00 0.01
Total 1405.533 14
2
R = 0.9650
The polynomial equation giving the mathematical relationship between each
Particle
Size =
( RPM) X -0.004009167
(Albumin Concentration) X 49.60111111
(Amount of Emulsifying Agent) X 50.88444444
(RPM) 2 X 8.175E-07
(Albumin Concentration) 2 X 7.591111111
(Amount of Emulsifying Agent) 2 X -9.448888889
(RPM) X Albumin Conc. X -0.005563333
(RPM) X (Amount of Emulsifying
Agent) X 0.000166667
(Albumin Concentration)
X (Amount of Emulsifying
Agent) X -29.24444444
-34.67555556
Figure 4.2.7: RSM graph of ofloxacin albumin microsphere - factor :
amount of emulsifying agent
DESIGN-EXPERT Plot
Particle Size
X = A: Stirring RPM
Y = B: Albumin Conc.
Actual Factor
56.51
C: Amt. of EA = 1.25
44.49
32.46
20.44
Particle Size
8.42
2.00
5000.00
1.63
4000.00
1.25
3000.00
B: Albumin Conc.
0.88 2000.00
A: Stirring RPM
0.50 1000.00
upon the particle size, the albumin concentration increases with that of the
the viscosity lowers the subdivision of the albumin solution into smaller
droplets during emulsification and finally increases the particle size. The
increases in drug concentration therefore had no effect on the particle size and
in turn depend upon the variabilities like stirring rate, concentration of the
DESIGN-EXPERT Plot
Particle Size
X = A: Stirring RPM
Y = C: Amt. of EA
Actual Factor
B: Albumin Conc. = 1.25
34.59
25.28
15.96
6.65
Particle Size
-2.66
2.00
5000.00
1.63
4000.00
1.25
3000.00
C: Amt. of EA0.88 2000.00
A: Stirring RPM
0.50 1000.00
In this 3D RSM the increase in the amount of the emulsifying agent decreases
the viscosity present in the system and thereby lowering the particle size, the
decrease in the RPM in interaction with the albumin concentration increase its
particle size and vice versa. This figure is indicative that optimal particle size
formation depends upon the correct RPM and the concentration of the
polymer.
Figure 4.2.9: RSM graph of ofloxacin albumin microsphere factor -
stirring rate
DESIGN-EXPERT Plot
Particle Size
X = B: Albumin Conc.
Y = C: Amt. of EA
Actual Factor
A: Stirring RPM = 3000.00
50.93
36.95
22.98
9.00
Particle Size
-4.97
2.00
2.00
1.63
1.63
1.25
1.25
C: Amt. of EA0.88 0.88
B: Albumin Conc.
0.50 0.50
In the 3D RSM the stirring factor is found to have the influence on the
RPM’s the figure is indicative that the particle size would be lower range due
to the higher dispersion of the emulsifying agent influencing particle size and
vice versa and it also suggests that the optimal particle size formation can be
The Anova for response Surface design showed a quadratic setup, which
vindicates the employment of CCD. The higher sum of squares values for the
exerted by these two values is almost similar. The sum of squares for the
output of interacting factors shows a higher value for factors, which involves
albumin. This shows that the system is influenced by the inclusion of albumin
the design space and the vulnerability of the experimental model, this is
important since it suggest factors, responses and the goal for each variable
solutions can be arrived at in quantitative terms, i.e. the likely behavior of the
data, table 4.2.16, which suggested the closeness of the predicted results with
Rounded
Emulsifie Parti
Polymer & its Rate of -up
Formulation Drug (g) r & its cle Formulatio
Concentratio stirring RPM(to
Method Ofloxacin concentra size n code
n (g) (RPM) nearest -
tion (ml) µm
500s)
Emulsion 0.93 0.93 1 4121.38 4000 8.96 ALOME-01
Polymerizati 0.83 0.83 1 3089.31 3000 10.49 ALOME-02
on 0.77 0.77 1 2616.28 2500 11.29 ALOME-03
RPM = Rotation Per Minute, EA = Emulsifying Agent,
gms = grams, ml = milliliter, µm=micrometer
Figure 4.2.10:Parameters suggestions for ALOME-01
1.00
8.00 12.00
Desirability = 1.000
1.00
8.00 12.00
Desirability = 1.000
Figure 4.2.12: Parameters suggestions for ALOME-03
1.00
8.00 12.00
Desirability = 1.000
and further evaluated for the response i.e., particle size. It was seen that the
response was almost similar to the response predicted by the design expert
software.
All the further studies were carried out using Alome-03. Since all the three
optimized formulas had the required particle size range 5 – 15 µm for lung
studies.
5.2. FORMULATION CHARACTERISIZATION STUDIES
• GLOME-03
• ALOME-03
The recovery / yield studies of microspheres are shown in table 5.1.1. The
higher than 91%. The maximum yield is observed in case of ALOME 03,
prepared by same method, factors like nature of polymers, and the extent of
cross – linking, which decides the drug polymer binding, would have
Amount of Amount of
Sl. Formulation
adsorbed drug Adsorbed drug
No. code
(mg ± SD) (%)
Amount of drug
Sl. Formulation Amount of drug
entrapped (mg ±
No. code entrapped (%)
SD)
surface and were spherical to near spherical for all the formulations analyzed.
the spray drying process (in both polymers) showed a drug dissociated and
higher temperature and pressure experienced by the sprayed droplet inside the
cyclone chamber of the spray drying apparatus and shear caused by the spray
nozzle of the spray dryer setup since the formulation was prepared by
dispersing the drug into a polymer solution prior to spraying, this might have
also caused a phase separation between drug and the polymer solution which
could have resulted in drug dissociation from its polymer substrate in the
in the temperature - pressure gradient across the height of the cyclone, this
might have caused by relatively quick drying of the outer core of the
microsphere while its inner core is still wet – as the inner core continues to
across the core of the microsphere, this in turn causes a shriveled appearance
as the spherical shape is not perfectly formed due to this boring –out effect in
the latter case Fig 5.1.4 & 5.1.5. This however does not happen in the case of
The particle size of ofloxacin gelatin and ofloxacin albumin microspheres ranged
from 0.32 to 22 µm and 3 to 35 µm respectively table 5.1.5. It was observed that the
size of the microspheres was increased with an increase in the concentration of the
polymer, the size of the microspheres was decreased with increasing with stirring rate
and increase in the concentration of emulsifying agent decreased the particle size.
All these results indicated that the particle size of the microspheres could be modified
Furthermore, considering that microspheres lie within the size range of 5 - 15 µm, this
are required for deposition in the lungs all these microspheres can be used for lung
targeting.
Release of Ofloxacin
Time (hrs.) from Albumin
microspheres (%)
0.5 26.2
1 42.1
1.5 53.2
2 64.4
3 72.1
4 87
5 92.3
6 99.3
Table 5.1.7: In vitro curve fits for various release systems for albumin
microspheres
Adjusted
Sl# Equation R R2 K P n
R2
Higuchi, (Square
2. 0.9929 0.9858 0.9858 41.9723 <0.0001 -
Root Time)
Baker and
5. 0.9733 0.9473 0.9473 0.0504 <0.0001 -
Lonsdale
Figure 5.1.12: In vitro release profile of ofloxacin albumin microsphere - First
order release pattern
120
80
Drug Release (%)
60
40
20
0
0 2 4 6
Time in hours
160
140
% Drug release Vs Time in hrs
Higuchi, Square Root Time
120 Time (hrs) vs Release (%)
Drug Release (%)
100
80
60
40
20
0
0 2 4 6
Time in hours
Figure 5.1.14: In vitro release profile of ofloxacin albumin microsphere - Hixon -
crowell pattern
120
80
60
40
20
0
0 2 4 6
Time in hours
120
80
60
40
20
0
0 2 4 6
Time in hours
Figure 5.1.16: In vitro release profile of ofloxacin albumin microsphere - Baker -
Lonsdale plot
120
80
Drug Releae (%)
60
40
20
0
0 2 4 6
Time in hours
The co-relation Coefficient value R 2 is taken into account to decide upon the
relevance of the model /curve fit which will best describe the extent of fit.
The anova (P) values is lesser than that of the 95% confidence interval (0.05)
the fit data are derived form the R 2 values for noting the extent of fit.
According to the R 2 values given by different data fits, the Peppas model
seems to be an ideal fit – 0.9868. This proves that Peppas fit to the ideal
According to Peppas fit, the release of the drug is decided upon by the
variation of Fick’s law of diffusion. The factors, which control this, are
vitro. The drug release is also affected by the morphological and structural
crystallites, degree of swelling of the matrix, the size of the mesh formed by
polymer plays a role in the release of the drug. In light of these facts stated
which releases the drug from the polymeric matrix based upon the extent
drug due to diffusion might also be a possibility. Due to this reason the
release profile shows an initial surge in the release pattern due to swelling of
the matrix (above 50% in the first 1.5 hours in this case) - releasing the
diffusion and there after a more controlled release pattern controlled by the
earlier stated facts. Due to this, the release pattern can be sustained for a
longer time line producing a control over the release profile of the
microsphere in vitro.
Table 5.1.8: Drug release profile of ofloxacin from gelatin microspheres
as a function of time
Release of Ofloxacin
Time (hrs.) from Gelatin
microspheres (%)
0.5 32.27
1 41.95
1.5 48.71
2 53.34
3 58.56
4 67.25
5 71.02
6 74.84
7 78.23
8 83.03
10 88.02
12 91.62
Table 5.1.9: In vitro curve fits for various release systems for
gelatin microspheres
Sl Adjusted
Equation R R2 K P n
# R2
Higuchi, (Square
2. 0.8863 0.7855 0.7855 30.2577 <0.0001 -
Root Time)
Baker and
5. 0.9861 0.9723 0.9723 0.0278 <0.0001 -
Lonsdale
Figure 5.1.17: In vitro release profile of ofloxacin gelatin microsphere –Baker
and Lonsdale
120
80
60
40
20
0
0 2 4 6 8 10 12 14
Time in hours
120
80
Drug release (%)
60
40
20
0
0 2 4 6 8 10 12 14
Time in hours
Figure 5.1.19: In vitro release profile of ofloxacin gelatin microsphere - Hixon
and Crowell
120
80
Drug Release (%)
60
40
20
0
0 2 4 6 8 10 12 14
Time in hours
120
80
60
40
20
0
0 2 4 6 8 10 12 14
Time in hours
Figure 5.1.21: In vitro release profile of Ofloxacin Gelatin microsphere - First
Order
120
80
Drug Release (%)
60
40
20
0
0 2 4 6 8 10 12 14
Time in hours
The co-relation Coefficient value R 2 is taken into account to decide upon the
relevance of the curve fit which will best describe the extent of fit undergone
by the release pattern of the drug from the formulation. Since the anova (P)
values is lesser than that of the 95% confidence interval (0.05) the fit data
here too were derived form the R 2 values for deciding on the degree of the
curve fit. According the R 2 values given by different data fits, the Peppas
model figure 5.1.18 seems to be an ideal fit in this case too – 0.9970. This
proves that Peppas fit to the ideal candidate regression pattern as undergone
In Peppas fit model as described earlier, the release of the drug is decided
upon by the diffusion of the polymeric matrix and the drug release is
released drug in vitro. The drug release is also affected by the morphological
and structural characteristics of the polymers, thus it can be surmised that the
vitro runs) and the solute concentration. It can be decided from these facts,
releases the drug from the polymeric matrix based upon the extent diffusion,
diffusion. This results in the manifest the release profile shows an initial
surge – “burst release” (53.34% in the first 2 hours) the release pattern due to
microsphere surface and there after by diffusion and there after a more
controlled release pattern. Due to this the release pattern is be sustain for a
longer time line producing a control over the release profile of the
In the present study, the DSC analysis were performed to find out the physical
nature of ofloxacin entrapped in the gelatin and albumin microsphere and also
the pure drug, raw polymer and drug loaded microspheres (1:1 drug/polymer
gas.
Procedure
analysis. Along with blank, drug loaded, and a physical mixture of Ofloxacin
and blank gelatin/ albumin microspheres in the ratio of 1:1 was analyzed in
Curves of DSC are shown in figure 5.1.22 to 5.1.28 from which one can
conclude that ofloxacin microsphere was not a physical mixture, but exists as
ofloxacin and 386.87K for the blank microsphere, respectively; both almost
372.34K appeared. The DSC curve of the physical mixture also differed from
that of ofloxacin microsphere. In the present study, the DSC analysis was
shown in figure 5.1.25 & 5.1.28 respectively. The drug, ofloxacin showed an
endothermic peak at 545K, while the polymers albumin and gelatin showed an
545K figure 5.1.22. This phenomenon was observed for a plain sample of
of the polymer, which produces this effect. At the end of this effect a
denaturation of the polymer and is also indicative of the fact that the drug is
present in a physical mixture and devoid of any chemical interaction with the
The same Glass transition effect is observed in the thermogram of the plain
higher levels of moisture can lead to significantly shorter T g levels and the
Throughout the XRD runs conducted for ofloxacin bearing albumin and
was not visible in the spectra. However, the XRD patterns of drug ofloxacin
ingredient) form. It was expected the crystalline drug ofloxacin would show
this did not occur. These results indicates that drug present in the polymeric
from XRD runs concurs with reported findings of several other workers, and
also supports the DSC studies in terms of parallel results in form of absence
out that the drug is indeed present in a entrapped form. 52, 58, 59, 61, 75, 74
Figure 5.1.29: The characteristic XRD pattern obtained for ofloxacin
OFLOXACIN
3000
2500
2000
Relative Intensity
1500 Intensity
1000
500
0
0 5 10 15 20 25 30 35 40 45
2θ
The XRD graph shows the presence of the typical XRD pattern observed for
the crystalline drug ofloxacin figure 5.1.29. Due this nature of Ofloxacin, the
this graph elucidates the purity of the drug by means of its characteristic
peaks which pertains to the crystal lattice of the drug and can act as a
reference spectrum for drug for the microsphere formulated forms of the drug.
forms of the drug with two polymers helps in understanding the modifications
ALBUMIN
1600
1400
1200
1000
Relative Intensity
800 Intensity
600
400
200
0
0 10 20 30 40 50
2θ
The XRD pattern of the polymer albumin shows figure 5.1.30 a prominent
peak in the spectra, which may due to the crystalline nature of the polymer.
The prominence of the peak of albumin suggests that the polymer by itself
crystalline drug ofloxacin. This also suggests that the crystalline nature of
ofloxacin be decided by the inclusion of the polymer, which shall decide upon
GELATIN
1600
1400
1200
Relative Intensity
1000
800 Intensity
600
400
200
0
0 5 10 15 20 25 30 35 40 45
2θ
The XRD pattern of the polymer gelatin shows Figure 5.1.31 the presence of
junctions of the drug, which forms the structural basis of the polymer.
However, this property of the polymer is not fully exhibited in the spectra;
Gelatin Microspheres
1600
1400
1200
1000
Relative Intensity
800 Intensity
600
400
200
0
0 5 10 15 20 25 30 35 40 45
2θ
The XRD pattern of the gelatin microsphere shows Figure 5.1.32 the characteristic
crystalline signature of the partially crystalline polymer of gelatin. The XRD pattern
which appears in the spectrum is due to the partial crystallanity of the drug – existing
spectrum is the absence of the characteristic XRD pattern of ofloxacin. This may be
attributed to the following factors like the denaturation of the crystalline nature of the
drug is embedded in the polymeric matrix as a physical mixture which would lead to
Albumin Microspheres
1600
1400
1200
Relative Intensity
1000
800 Intensity
600
400
200
0
0 5 10 15 20 25 30 35 40 45
2θ
The albumin ofloxacin microsphere XRD spectrum also shows figure 5.1.33 an
physical nature of the drug due to the changes in the crystalline nature of the drug in
albumin polymeric matrix, which leads to the altered crystal signatures of the
crystalline drug ofloxacin. This suggests that the drug is either molecularly entrapped
the crystals of the drug which accounts for the absence of the drug peaks in the
spectrum. This infact acts as a pointer for the efficiency of the formulation adopted,
since it acts as a qualitative measure of either the encapsulation efficiency of the drug
or the entrapment capability of the drug in the polymer matrix, which would in turn
prominent difference in the spectrums was seen. Thus, it was concluded that
prescribed by the ICH guideline ‘Q3C’ 26 for the residual solvents. It was
importantly the particles did not break and were intact and withstood the
irradiation process. A slight increase in the particle size (1.2µm – 3 µm) was
The observations of long-term storage condition and accelerated storage condition are
shown in tables 5.2.1 and 5.2.2. The drug content values of the formulations in long-term
storage condition and accelerated storage condition along with their 95% confidence limits
Depending on the stability of the microspheres formulations with respect to the tested
parameters, a re-test date was arrived upon. After re-test if the formulations are found to
The formulations were stable for the 6-month period in long-term storage temperatures of 5
± 30C. The drug content of the microspheres formulations did not vary to a large extent. A
maximum decrease of 2.47% from the initial concentration observed in GLOME - 03, and
2.14 % in the case of ALOME -03. Minor changes in particle size were noticed during the
storage time while the stability studies were conducted, however the changes were found to
The formulations were stable for the 12-month period in long-term storage temperatures of
25 ± 20C and 60 ± 5% RH. The drug content of the microspheres formulations did not vary
to a large extent. A maximum decrease of 3.5% from the initial concentration observed in
both GLOME - 03 and ALOME -03. Minor changes in particle size were noticed during
the storage time while the stability studies were conducted, however the changes were
ALOME -03 99.02 98.22 97.37 96.88 11.4 11.3 11.3 11.4 - - - -
GLOME -03 99.20 98.12 97.68 96.73 9.5 9.4 9.4 9.4 - - - -
-: No physical
change
The average particle size also did not vary appreciably. More or less it was
around the initial particle size value. No physical changes, like liquefaction or
drug –ofloxacin.
An action plan was devised and the following considerations were addressed
¾ The both GLOME -03 and ALOME -03 microspheres were fixed with a re-
test date of 2 years from the date of their manufacture, as they were stable
¾ But in the case of ALOME -03 microspheres, the storage conditions were
¾ The re-test date of ALOME -03, were fixed at 1 year (provided, the
of manufacture as the long-term studies were conducted for this period and
the formulations were found to be stable during this period. 64, 26
1987)
The drug activity data shown as solid circles has a linear regression line
superimposed. The lower 95% confidence line was also graphed. The
accepted definition of the shelf life time is the x axis coordinate for the
intersection of the lower 95% confidence line with 90% drug activity
and Biologics," Food and Drug Administration, DHHS, February, 1987). This
104
ALOME -03
102 90% reg. line
Concentration of ofloxacin (%)
98
96
94
92
90
88
86
0 3 6 9 12 15 18 21 24 27 30
Time (months)
104
96
94
92
90
88
86
0 3 6 9 12 15 18 21 24 27 30
Time (months)
Figure: 5.2.3 Drug content plot of ALOME - 03 in real time storage
conditions
104
ALOME -03
102 90% reg. line
Concentration of ofloxacin (%)
98
96
94
92
90
88
86
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time (months)
104
GLOME -03
102 90% reg. line
Concentration of ofloxacin (%)
98
96
94
92
90
88
86
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (months)
5.4. IN-VIVO TISSUE DISTRIBUTION STUDIES
Results
The drug content (µg/ml) with time was determined in blood, liver, spleen and
lung. The results were calculated to express the percentage amount, as shown
The data of drug content change with time were treated to calculate the
The ofloxacin gelatin microspheres showed the largest value of AUC and r e
(compared with spleen) ~16.9 (compared with liver) ~33.7 (compared with
was 90.98%.
The ofloxacin albumin microspheres showed the largest value of AUC and re for
with spleen) ~18.5 (compared with liver) ~25 (compared with ofloxacin), table
AUC 1 2 3 4 5
Sample
GLOME Ofloxacin re te Te Te* T e * X 100
Blood 26.74 119.52 0.32 39.06 38.30
Liver 53.21 147.58 0.55 18.32 81.65
Spleen 18.79 9.80 1.92 52.80 28.33 0.90982 90.98
Lung Target
903.25 77.35 12.86 1495.98
tissue
∑ Te* = 1644.26
Table 5.3.5: Lung-targeting parameters of ALOME-03: Ofloxacin after
I.V. administration in mice (n=3)
AUC 1 2 3 4 5
Sample
ALOME Ofloxacin re te Te Te* T e * X 100
Blood 37.34 133.70 0.28 41.51 37.34
Liver 50.05 144.35 0.53 20.42 75.90
Spleen 26.54 9.73 1.84 58.42 26.90 0.91709 91.70
Lung 934.01 114.65 13.52 - 1550.18
∑ Te* = 1690.32
5.4.2. Compartment modeling
The data of GLOME –03 and ALOME –03 was subjected to compartment modeling
using with ‘WINNONLIN Version 1.5 Pharmacokinetic software’. The actual plot
exactly superimposed on the predicted line when two-compartment model was fitted
to the data. The plasma concentration and pharmacokinetical parameters are reported
in table 5.3.6 and 5.3.7 for gelatin and albumin microspheres respectively, 32, 48, 76
.
3.5
3.0
2.5
2.0
Observed
1.5 Predicted
1.0
0.5
0.0
0 2 4 6 8 10 12
Time in
h
Table 5.3.7: Pharmacokinetics parameters of ALOME-03
5.0
4.5
4.0
3.5
3.0
2.5
Observed
2.0 Predicted
1.5
1.0
0.5
0.0
0 2 4 6 8 10 12
Time in hours
5.5. HISTOPATHOLOGICAL STUDIES
Staining showed blue colored nucleus and cytoplasm with various shades of pink with
organs of control and microsphere formulations are shown in figures 5.4.1 to 5.4.9
below.
their inability to reach the target site of action, especially when they are
improved the scope of application of the drugs and has increased the quantum
of drug reaching the site and simultaneously decreasing the amount of drug
being distributed to other parts of the body. Thus, the pharmacotherapy of the
degradation or inactivation during transit to the target sites and protects the
Microspheres are solid colloidal particles ranging in size from 1 to 1000 µm,
which can act as good targeted drug delivery platforms if the particle size can
pulmonary region whereas small particles (<7 micron) are distributed in the
RES (liver & spleen). The entrapment of the microspheres in the lungs and
release of drug thereby results in improved therapeutic efficacy of the drug
and their compatibility with the drug. The method of estimation of the drug
The polymers chosen were albumin and gelatin, as they are natural,
Ofloxacin had a λ max of 297 nm and obeyed Beer’s law in the range of 0 – 20
mcg/ml when 0.1 N HCl was used as solvent. The recovery was found to be
degenerative changes existed as shown by the lack of extra peaks during the
compared to their individual thermograms of the drug and the excipient. This
was employed as it showed better output in terms size and drug entrapment
and far apart. However, the size range produced by this process was found
The ideal particle size range for lung targeting and subsequent mechanical
influences the particle size. Hence, it was decided to optimize and obtain the
& products, which had desired size 9.10 µm for GLOME-03 (ofloxacin gelatin
microspheres).
The optimized formulations were sterilized using γ – irradiation. After
importantly the particles did not break, were intact and withstood the
irradiation process. A slight increase in the particle size (1.2µm – 3 µm) but
within the required range was observed after sterilization with γ - irradiation.
solvent analysis
The microsphere yield / recovery for GLOME-03 was above 91.5% and
ALOME-03 above 94.48%. Free drug studies revealed that the drug added
during the preparation was not completely incorporated into the microsphere
matrix, there was some amount of un-entrapped drug which varied between
ALOME-03 respectively.
Drug entrapment studies showed that the drug entrapment efficiency of the
03) respectively.
surface and was spherical to near spherical, discreet for the microspheres
concentration of the polymer, the size of the microspheres was decreased with
All these results indicated that the particle size of the microspheres could be
µm, which are required for deposition in the lungs all these microspheres can
DSC prove that ofloxacin gelatin and albumin microspheres, which were
prepared, were not just a physical mixture, but existed as microsphere. There
were no new peaks and this confirmed that there was no chemical interaction.
tools. The analysis was performed in order to characterize the physical state
of the polymer and drug in the microspheres. Throughout the XRD runs
characteristic peak of the crystalline drug ofloxacin was not visible in the
form. It was expected the crystalline drug ofloxacin would show its specific
occur. These results indicated that drug present in the polymeric microsphere
in an amorphous form in the polymer matrix. The data from XRD runs also
of acetone was 1900 PPM and 2000 PPM for GLOME -03 and ALOME -03
respectively. The organic solvents were well below the permissible ICH limits
for residual solvents (5000 ppm for acetone), and the formulation are safe.
stability study protocol. The drug content in long-term storage conditions did
variation of 2.47% from the initial concentration was seen in case of gelatin
the maximum variation of 2.14% was observed, a re-test date of 2 years from
the date of manufacture was fixed, while a re-test date of 1 year was fixed for
The formulations were stable for the 12-month period in Accelerated storage
from the initial concentration was observed in both GLOME - 03 and ALOME
-03. Minor changes in particle size were noticed during the storage time while
the stability studies were conducted, however the changes were found
the first one hour, which reflected the significant amount of ofloxacin
practice this would lead to ‘burst effect’, which enables the preparation to
show fast effect to the patients. Although the ofloxacin release from gelatin
ALOME- 03. During the same period, the released amount was 99.3% and
91.62% respectively. The results indicated that the GLOME-03 and ALOME-
The data obtained form in-vitro release studies were fitted to various kinetic
equations to determine the mechanism of drug release and release rate using a
computer program sigma plot version 9.01. The co-relation Coefficient value
R 2 is taken into account to decide upon the relevance of the model / curve fit
which will best describe the extent of fit. According to the R 2 values given by
different data fits for ALOME - 03, the Peppas model was to be an ideal fit
having R 2 = 0.9868. According to Peppas fit, the release of the drug is decided
upon by the diffusion of the polymeric matrix and the drug release is
diffusion process which releases the drug from the polymeric matrix based
Due to this reason the release profile shows an initial surge in the release
pattern due to swelling of the matrix (above 50% in the first 1.5 hours in this
case) - releasing the adsorbed drug particles on the microsphere surface and
there after by diffusion and there after a more controlled release pattern
In case of ofloxacin gelatin microsphere (GLOME- 03) the ideal fit seems to
surge – “burst release” (53 % in the first 2 hours) the release pattern due to
microsphere surface and there after by diffusion and there after a more
controlled release pattern. Due to this the release pattern is be sustain for a
longer time line producing a control over the release profile of the
microsphere in vitro.
formulations to the lungs and compared with the conventional dosage of the
drug.
The organ targetability ofloxacin gelatin microspheres showed the largest
value of AUC and r e (time-averaged relative drug exposure) for the lung; the
90.98%.
value of AUC and r e (time-averaged relative drug exposure) for the lung; the
91.7%.
injecting into the animal. The cytoarchitecture of the tissues (lungs, liver &
show any degenerative changes when compared to that of the control animals.
7. CONCLUSION
properties and the particle size range was ensured by optimization for both
targeted drug delivery systems, which holds a very promising alternative over
the conventional means with which the most despondent and perplexive to
in intracellular therapy which otherwise was not favored due to the drawbacks
higher dose, which are known to cause ADRs and the unnecessary exposure of
The findings of the work can also be applied for developing effective blue-
print for targeted organ specific drug delivery; and the corollary of this work
means of passive targeting of drugs to the target organ and can be employed
Dekker; 1994
3. Vyas SP, Khar RK. Molecular basis of targeted drug delivery. In:
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9.1. LIST OF PAPERS PRESENTATION
International
1. Drug targeting to lungs by way of microspheres. International congress of
National
1. Drug targeting to lungs. 54th I.P.C at PUNE from 13th –15th December
2002.
47. 5.1.29 The characteristic XRD pattern obtained for ofloxacin 173