This is the presentation on Role of advancement in instrumentation in pharmacodynamic evaluation of drugs
in clinical trials.
CONTENTS
Concept of medical instrument and instrumentation
Centrifuge
PCR
HPLC
Flow cytometry
Mass SPECTROMETRY
Minimally invasive technologies in PD
Conclusion
5. DRUG DEVELOPMENT
Understanding disease
Identifiying and validating gene/protein
responsible for disease (Target
discovery)
Lead compound is a substance with
greatest potential for successful
iteraction with the target (lead
identification)
6. Concept of medical instrument and instrumentation
According to WHO Medical device or Instrument means any machine,
implement, appliance, apparatus, implant, calibrator or in vitro reagent,
material, software made by its manufacturer to be used for the human
beings for the purposes of:
Diagnosing, preventing, monitoring, treating a disease
Diagnosing, monitoring, treating, alleviating an injury
Investigating, replacing, modifying or supporting the anatomy/physio
Sustaining or supporting life
Control of conception
Disinfecting medical devices
Dispensing information for any medical purpose
7. • In Clinical Pharmacology, it further indicates the use of instruments to
screen the activity of the drug and to assess its effects.
• The instruments are also essential to measure the adverse effects of
the drug.
• Clinical Pharmacology is a well-established branch which attempts to
connect the gap between medical practice and laboratory science.
Hence, instrumentation with all its components is an integral part of
clinical pharmacology.
• Thus, instrumentation can even be considered as a subdivision of
clinical pharmacology.
8. Need of Instrumentation in Clinical Pharmacology
• Like medicines, medical instruments have become an indispensible
part of the patient health care – at the bedside, at rural health clinics
and also at large, specialized hospitals.
• They include a vast range of equipments, from simple tongue
depressor to haemodialysis machines.
• In other words instruments have become important in the health care
and research and especially in clinical pharmacology where they are
required for the various purposes such as standardization of
data/results/observation, accuracy, reliability, reproducibility,
minimization of human error and therapeutic drug monitoring.
• Any technology comes at a cost and instrumentation is no exception as
these devices cost governments a substantial amount of money.
9. Current regulatory scenario of medical instruments in
India
• The Central Drugs Standard Control Organization (CDSCO) is the main
regulatory organization in India.
• It regulates import, commerce and manufacturing of medical devices.
The medical devices are mentioned as drugs under the Drugs and
Cosmetics Act.
• The function of CDSCO is to prepare standards for drugs, cosmetics,
diagnostics and medical devices. It also grants licenses to drug
manufacturing companies and importers.
• It also regulates new drug trials in India.
10. Commonly Used Instruments in Pharmacology Laboratory
Kymograph (Sherrington-Starling Kymograph)
Student’s Physiograph
Analgesiometer
Convulsiometer
BP Apparatus
Electrolyte Analyzer
Body Plethysmograph
12. 1. Centrifuge Machine
The most commonly used instrument in the biomedical research to prepare or
separate a sample liquid/semiliquid for the further analysis.
Centrifuge is the instrument which separates solid or large particles from liquid on
the basis of density, shape or size.
Application of Centrifuge
Low-speed centrifuge is used for separating red blood cells or platelets from
whole blood samples
Other like protein precipitation, tissue culture, subcellular
Isolation (i.e., ribosomes, Golgi bodies, etc.), plasmid preparations
and DNA/RNA separations are important.
Ultracentrifuge is used in the separation of virus particles,
DNA, protein, or RNA fractionations, or lipoprotein flotation
13. 2. Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) is a technique used to produce multiple
copies of a specific DNA sequence in short duration from a small initial
sample.
The PCR works on the principle of amplification of a targeted single
strand DNA exponentially.
Further, total process is classified into three cycles namely,
(1) Denaturation of DNA template and primer,
(2) Annealing of single stranded DNA to primer, and
(3) Extension and Elongation of DNA strand
14. A real-time polymerase chain reaction (real-time PCR), also
known as quantitative polymerase chain reaction (qPCR),
is a laboratory technique of molecular biology based on
the polymerase chain reaction (PCR).
It monitors the amplification of a targeted DNA molecule during the
PCR (i.e., in real time), not at its end, as in conventional PCR.
Real-time PCR can be used quantitatively (quantitative real-time
PCR) and semi-quantitatively (i.e., above/below a certain amount
of DNA molecules) (semi-quantitative real-time PCR).
15. Application of PCR and Real-Time PCR
Real-Time PCR can be applied to traditional PCR applications as well as
new applications that would have been less effective with traditional
PCR.
Primary application is in vitro amplification of specific target DNA
sequences. But, the limitation of the process is, it requires highly
skilled person, extremely liable to contamination and it may not easy
to set up a quantitative assay.
Evaluating the Pharmacodynamic Effect of Antimalarial Drugs in
Clinical Trials by Quantitative PCR.
The ongoing development of new antimalarial drugs and the
increasing use of controlled human malaria infection (CHMI) studies
to investigate their activity in early-stage clinical trials require the
development of methods to analyze their pharmacodynamic effect.
16. This is especially so for studies where quantitative PCR (qPCR) is
becoming the preferred method for assessing parasite clearance as
the study endpoint.
Viral Quantitation
Quantitation of Gene Expression
Array Verification
Drug Therapy Efficacy
DNA Damage measurement
Quality Control and Assay Validation
Pathogen detection
Genotyping
17. 3. HPLC
High-performance liquid chromatography (HPLC), is a chromatographic
technique used to separate, to identify and to quantify each component in a
mixture.
HPLC is considered an instrumental technique of analytical chemistry.
In biomedical research, HPLC is a qualitative and quantitative process.
HPLC is based on the principle of partition differentiation between the moving
solvent (mobile phase), and the column packed particles (stationary phase) to
separate a test substance.
18. Mainly, two principal factors that determine the overall separation
power are mechanical separation power (produced by the column
length, particle size, and packed-bed uniformity) and chemical
separation power (produced by the packing material and the mobile
phase).
Hence, degree of separation of two compounds is known as
chromatographic resolution which depends on high flow pressure
through packed particle size and length of the column.
HPLC has the ability to separate, identify, and quantitate the compounds
that are present in any sample that can be dissolved in a liquid.
20. Application OF HPLC
HPLC is widely used in biotechnological, biomedical, and biochemical
research but very commonly used to determine concentration of drug
in plasma/serum in the therapeutic drug monitoring (TDM)
It is also used along with mass spectrometry
21. 4.Liquid Chromatography and Mass Spectrometry(LCMS)
Mass spectrometry (MS) is an analytical technique that measures
the mass-to-charge ratio of ions.
In general, coupling of a mass spectrometer to an HPLC system is called
LC/MS.
So, it is combination of powerful analytical separation technique with a
powerful analysis and detection of mass technique.
Salient feature of LCMS is that it can detect the ions ranging from at
least 2 to 3,000 mass-to-charge ratio (m/e).
22. Application of LCMS
qualitative and quantitative analysis,
impurities identification,
metabolite studies,
pharmacokinetics,
pharmaceutical and biomedical applications in several industries and
research institutes.
Recent application is extended in determination of drugs and its
metabolites concentration in phase 0 or microdosing study during the
drug development process.
23. 5. Flow cytometry
It is the instrument which simultaneously measures and analyses the
observations of multiple physical characteristics of single particles i.e.
cells, such as particles relative size, relative granularity or internal
complexity and relative fluorescence intensity and the process by which
these observaions and result obtained is k/a flow cytometry.
Applications
Cncer diagnosis and prognosis
Immunofluorescence
Cell therapy and transplantation
Immunologic diseases
25. 6. PET SCAN
The use of PET to examine the behavioral, therapeutic and toxic
properties of drugs and substances of abuse is emerging as a
powerful new scientific tool.
PET provides a new perspective on drug research by virtue of its
ability to directly assess both pharmacokinetic and pharmacodynamic
events in humans and in animals.
These parameters can be assessed directly in the human body both in
healthy volunteers and in patients.
Moreover, the new generation of high-resolution, small-animal
cameras hold the promise of introducing imaging in the early stages
of drug development and make it possible to carry out longitudinal
studies in animals and to study genetically altered animals.
26. This places PET in a unique position to contribute significantly to the
process of drug development through understanding the molecular
mechanisms underlying drug action while addressing some very
practical questions such as determining effective drug doses for
clinical trials for new drugs, determining the duration of drug action
and examining potential drug interactions.
In the perspective of DDR, PET may extend its range of use from
Phase 0 evaluation of pharmacokinetics and biodistribution/
bioavailability, to the measurement of Proof-of-Concept, in particular
wherever a direct connection between PoC and in vivo performance
(e.g. receptor affinity/occupancy and target treatment endpoints) can
be sought.
27. Positron emission tomography (PET)
Technology and applications (possible tracer/marker)
1. General characteristics for imaging
Strengths Sensitivity; specificity; kinetic resolution; low dose administered
Weaknesses Lack of chemical resolution; methodology developments required
Opportunities Molecular imaging possible; proof of principle and new pharmacokinetic/
pharmacodynamic studies possible
28. 1. Imaging glucose utilization ( 18 F-FDG)
Strengths For grading and response assessment
Weaknesses Inflammation can give false-positives
Opportunities Utility increasing; availability of standards for large trials
2. PK of labeled drugs/molecules (5- 18 F-fluorouracil)
Strengths PK obtained for tumor and normal tissues
Weaknesses Lack of chemical resolution
Opportunities New labeling strategies; improved data analysis
29. Strengths Direct and rapid assessment
Weaknesses Thymidine metabolism can complicate data analysis
Opportunities Nonmetabolized thymidine analogs
3. Imaging cell proliferation ( 11 C-thymidine)
4. Blood flow and blood volume imaging ( 15O H2O/CO)
Strengths Endpoint for antivascular therapy; index for drug uptake and clearance
Weaknesses Poor signal-to-noise ratio in some tumors
Opportunities Improved sensitivity of scanners
30. 5. Measurement of tissue pH ( 11 C-bicarbonate)
6. Drug mechanism of action studies ( 11 C-temozolomide)
7. Protein synthesis measurements ( 11 C-methionine)
Strengths Tissue pH measured
Weaknesses Does not differentiate pHi from pHe
Opportunities Understanding drug effects
Strengths Provides proof of principle in patients
Weaknesses Not applicable to all drugs
Opportunities Evidence of activity in vivo
Strengths Endpoint for certain drugs
Weaknesses Tracer metabolism can complicate data analysis
Opportunities Improved data analysis
31. 8. Thymidylate synthase inhibition ( 11 C-thymidine)
9. Imaging multidrug resistance phenotype ( 11 C-daunorubicin)
10. Hypoxia imaging ( 18 F-FMISO)
Strengths Endpoint for certain drugs
Weaknesses Application is limited to initial drug effect
Opportunities Several compounds can be evaluated
Strengths Patient selection for reversal of phenotype
Weaknesses Methodology developments required
Opportunities Improved synthesis; new tracers
Strengths Patient selection for several therapeutic agents
Weaknesses Current tracers have high nonspecific signal
Opportunities New tracers
32. 11. Cell surface and nuclear receptor imaging ( 18 F-FES)
12. Imaging gene expression ( 124 I-FIAU)
13. Angiogenesis imaging ( 124 I-VEGF)
Strengths For grading and response assessment
Weaknesses Requires extensive validation
Opportunities Direct clinical assessment
Strengths Monitoring gene therapy; studying transcription
Weaknesses Methodology developments required
Opportunities More sensitive animal scanners; molecular imaging of tumor
bearing/transgenic mice
Strengths Response assessment
Weaknesses Not verified in humans
Opportunities Understanding tumor biology
33. 14. Apoptosis imaging ( 124 I-annexin)
15. Imaging protein – protein interactions
Strengths Response assessment
Weaknesses Difficulty to differentiate necrosis
Opportunities Evaluating several therapies; dose optimization in radiotherapy
Strengths Endpoint for certain drugs
Weaknesses Methodology developments required
Opportunities Patient selection
34.
35. 7. MAGNETIC RESONANCE TECHNIQUES
The Food and Drug Administration (FDA) has targeted imaging as a
tool that can help overcome stagnation in drug development.
MRI and, to a lesser extent, MRS are beginning to have important
roles in anticancer drug trials.
MRI is routinely used in the initial evaluation of tumor size, shape and
anatomic appearance and changes in these parameters during
therapy can be used to assess and quantify the PD effects of a drug.
In addition, dynamic contrast-enhanced MRI (DCE-MRI) is proving
increasingly valuable for assessing PD endpoints.
Other MRI approaches, as well as MRS, also have PD applications, and
in some cases MRS can even be used for minimally invasive
monitoring of anticancer drug uptake and metabolism.
36. Magnetic resonance imaging (MRI)/Magnetic resonance spectroscopy
(MRS)
1. Volumetric imaging
2. Contrast enhanced imaging
Strengths High soft-tissue contrast and resolution;
rapid
Weaknesses Limited utility in bone and lung;
CT also has improved cost and availability
Opportunities Recent advances in lung cancer imaging; availability increasing
Strengths Reflects permeable vasculature,
increased blood volume and perfusion
Weaknesses Not applicable to all tissues; timing crucial;
limited standardization; expensive;
other techniques also reflect tumor activity though with reduced resolution
Opportunities New (larger) contrast agents; improved computing power and processing
37. 3. Permeability imaging
4. Perfusion/blood volume imaging
Strengths Based on contrast enhanced imaging, but images can be quantitative;
obtained simultaneously with imaging
Weaknesses Requires specialized sequences;
other radiolabeled methods available though with poorer resolution
Opportunities Functional information related to vasculature
Strengths Based on contrast enhanced imaging or uses intrinsic contrast mechanism,
still developing; simultaneous with imaging
Weaknesses Requires specialized sequences;
usually only provides relative changes
Opportunities Functional information on drug delivery
38. 11. Measurement of oxygenation (PFCs)
12. Measurement of lactate
Strengths T1 provides sensitive and quantitative measure of pO 2
Weaknesses Low solubility of PFCs;
artefacts, e.g., macrophage uptake of PFCs
Opportunities Intratumoral injection for regional pO2
Strengths Tumor grade and metastatic marker;
glycolysis endpoint
Weaknesses Lipid suppression or complex measurement techniques required;
currently only applicable for brain in clinic
Opportunities Developments in data acquisition
39. 13. Measurement of pH
14. Glucose utilization
Strengths pHi and pHe measured simultaneously
Weaknesses Exogenous probe required for pHe;
not relevant for all drugs
Opportunities Cell uptake of weak electrolytes is pH-dependent
Strengths Changes in rates of glycolysis using 19 F- or 13 C-labeling
Weaknesses Low sensitivity;
high doses may alter tumor physiology/biochemistry
Opportunities Improved modeling of glycolytic enzyme activity
40. 15. Assessment of fructose-1,6 bisphosphate using 31 P
Strengths Changes in fructose-1,6 bisphosphate as a marker for apoptosis
Weaknesses Poor resolution in vivo,
not verified in animals in vivo;
low sensitivity compared to other in vitro methods
Opportunities Identification of chronology of apoptosis events
41. 8. COMPUTED TOMOGRAPHIC SCANNING
In PD studies, CT is used mainly to measure changes in the volume of
disease with treatment.
CT can also be used with X-ray – dense contrast agents, such as
iodinated materials, to assess some of the functional properties of
tumors.
CT can also be helpful in determining the boundaries of the tumor
and the extent of its invasion into adjacent tissues and can identify
involvement of lymph nodes, particularly when such involvement has
caused an increase in lymph node size or an abnormal lymph node
appearance.
In clinical trials of innovative therapies, CT is most commonly used to
assess the size of the primary tumor and the extent of metastatic
disease.
42. Computed tomography (CT)
1.Volumetric imaging
Strengths Excellent cross-sectional anatomy;
good soft-tissue contrast;
good visualization of bone
Weaknesses Soft-tissue contrast often less good than MRI; limited to direct transaxial
views; other views can be reconstructed but may suffer resolution loss
Opportunities Spiral CT aids volume interpretation
2. Contrast enhanced imaging
Strengths Uptake depends on perfusion and permeability;
standard technique provides good contrast
Weaknesses Limited range of agents; some side effects, high radiation dose limits repeat
studies and duration, including pediatric application
Opportunities Enables wider application than magnetic resonance
43. 3. Permeability imaging
Strengths Based on Contrast enhanced imaging
images can be quantitative
Weaknesses Requires special software; not widely used.
Limited range of agents; some side effects,
high radiation dose limits repeat studies and duration, including pediatric
application
Opportunities Can be included in wider range of studies
44. 9. ULTRASOUND SCANNING
Current developments in ultrasound technologies include three-
dimensional ultrasound imaging techniques and the use of ultrasound
contrast agents, which show promise in assessing vascular delivery of
agents.
Although ultrasound is of considerable assistance in diagnosis, its use
for morphologic assessment in serial studies is limited because the
technique is operator dependent, which makes it difficult to
reproduce imaging planes.
Color Doppler ultrasound measurements have been used to assess
tumor response to conventional therapies; as with other modalities,
contrast agents may also prove helpful.
45. The major application of ultrasound in therapeutic trials is to assess
changes in tumor size.
Ultrasound scanning using microbubble contrast agents has potential
value in the measurement of perfusion in response to antivascular
and antiangiogenic agents.
46. Ultrasound
1. Sectional imaging
Strengths High resolution cross-sectional images;
new developments leading to 3-dimensional acquisitions;
real-time imaging method;
can characterize tissues based on a range of properties, such as scatter and
speed of sound
Weaknesses Soft-tissue contrast can be limited by noise;
images dependent on optical window into region;
images are operator dependent;
normally does not provide contiguous volume coverage;
no documented record of entire volume, meaning audit trail more difficult
than that for CT or MRI
Opportunities Useful for size measurements
47. 2. Flow measurements
3. Contrast agents
Strengths Can measure flow directly by Doppler shift;
good for large vessels;
good time resolution;
can measure tissue perfusion
Weaknesses Hard to make truly quantitative
Opportunities Can assess potential for drug delivery
Strengths New ultrasound contrast agents provide potential for functional
measurements; microbubbles can be used for perfusion measurements;
microbubbles are intravascular; can use with Doppler perfusion methods
Weaknesses Cannot measure vascular permeability
Opportunities Can measure vascular input functions;
potential for agents targeted at vascular endothelial surface receptors
51. Conclusions
Drug development is a complex and time consuming process which a
lead compound has to undergo
Medical device or instrument means any machine, implement,
appliance, apparatus, implant, calibrator or in vitro reagent, material,
software made by its manufacturer to be used for the human beings
for multiple purposes.
The identification and implementation of PK/PD endpoints provide a
powerful mechanism for improving the quality of drug development,
allowing prioritization of the drugs that are most likely to succeed,
accelerating their regulatory approval, and reducing the overall risks
and costs of pharmaceutical development
52. Funding bodies and industry are required to invest more in the
development, validation and standardization of minimally invasive
methodologies as well as in the development of instrumentation.
Notas del editor
Drug development is a complex and time consuming process which lead compound has to undergo. It is designed to ensure that only those pharmaceutical products that are both safe and effective are brought to market for the benefit of the patient.
Phase1 – human , efficacy/tolerability – MTD
Phase2- dose searching for efficacy
Phase3- multicentric, safety efficacy,
15 yrs
Drug development is a complex and time consuming process which lead compound has to undergo.
It is designed to ensure that only those pharmaceutical products that are both safe and effective are brought to market for the benefit of the patient.
Pharmacodynamic evaluation of drugs comes in phase 1/2/3
Instrumentation in Clinical Pharmacology
The concept of instrumentation not only the science of installation or manufacture of an instrument but also encompasses the science of measurement, calibration, validation and regulation related to instruments within a production or manufacturing area.
Before coming to actual instrument its important to know some basic concepts in instrument and instrumentation
Central Drugs Standard Control Organization (CDSCO)
A plethysmograph is an instrument to measure changes in volume within an organ or whole body.
Maximal Electroshock Seizure (MES) Convulsiometer
Kymographh- graphical amplified measurable response of a muscle or tissue (contraction/relaxation) against a given concentration of drug or stimuli, hence the resolution of the tissue response is increased.
Physiograph - electronic stimulator
Elevated Plus Maze
Morris Water Maze
Passive Avoidance Test
Type of centrifugation depend on speed and temperature
The amplification depends on the reaction cycle and the cycle is nothing but the different heating cycle.
Polymerase chain reaction (PCR) tests are used to detect HIV's genetic material, called RNA. These tests can be used to screen the donated blood supply and to detect very early infections before antibodies have been developed. This test may be performed just days or weeks after exposure to HIV.
Anneal - recombine (DNA) in the double-stranded form.
https://youtu.be/iQsu3Kz9NYo
and being highly sensitivity
HPLC is a dynamic adsorption, analytical process with multiple applications characterised by a high resolution, rapid analysis and high accuracy and precision.
https://youtu.be/ZN7euA1fS4Y
Basis of Column Selection
Column selection is an important part in the HPLC, which has whole influence on the process.
So, the selection of column depends on the several factors which are molecular weight of the sample (Exclusion limit molecular weight),
Ability to dissolve the sample (Applicable to packing materials) and
molecular weight distribution (Range of calibration curve).
Efficiency of HPLC is a measure of mechanical separation
power whereas selectivity of HPLC is a measure of
chemical separation power.
Adsorption is defined as the deposition of molecular species onto the surface. (activated charcoal)
There are two common Atmospheric Pressure Ionization (API) LC/MS processes; Electrospray Ionization (ESI) and Atmospheric Pressure Chemical Ionization (APCI). Both are soft ionization technique and are compatible with most chromatographic separations.
APCI is regarded as a soft ionization process which results in a mass spectrum typically dominated by a single ion (either + or –) that corresponds directly to the molecular weight of the compound, which is protonated in the positive ion mode (M+H)+ or deprotonated in the negative ion mode (M-H)-
BYF - Bupi Yishen Formula
To identify chemical constituents in any herbal preparation.
Ultra-high performance liquid chromatography with linear ion trap mass spectrometry and triple-quadruple tandem mass spectrometry methods were developed for qualitative chemical profiling and multi-components quantitative analysis in Chinese medicine preparation BYF (Bupi Yishen Formula).
Summary of strengths, weaknesses, and opportunities for using minimally invasive technologies in pharmacokinetic and pharmacodynamic studies
10. FACS – FLUORESCENCE ACTIVATED CELL SORTING
Drug Development Research (DDR)
The process of separation of pure enantiomers from their racemic modification is called resolution.
Technology and applications (possible tracer/marker)
Technology and applications (possible tracer/marker)
pHi = intracellular pH;
pHe = extracellular pH.
Temozolomide is an oral chemotherapy drug. It is an alkylating agent used as a treatment of some brain cancers; as a second-line treatment for astrocytoma and a first-line treatment for glioblastoma multiforme
Examples of Thymidylate synthase inhibitors
Raltitrexed, used for colorectal cancer since 1998
Fluorouracil, used for colorectal cancer[4]
Daunorubicin, also known as daunomycin, is a chemotherapy medication used to treat cancer.[1] Specifically it is used for acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), and Kaposi's sarcoma.[1] It is used by injection into a vein.[1] A liposomal formulation known as liposomal daunorubicin also exists.[1]
Vascular endothelial growth factor (VEGF), originally known as vascular permeability factor (VPF), is a signal protein produced by cells that stimulates the formation of blood vessels.
iodine I 124 FIAU A radioconjugate containing the nucleoside analog 2'-fluoro-2'-deoxy-iodo-1beta-D-arabinofuranosyl-5-iodouracil (FIAU) labeled with the radioisotope iodine I 124 with positron-emitting activity.
Drug-drug interaction (DDI) is a significant cause of adverse drug reactions (ADRs), especially in patient populations on multiple medications.
The FDA is interested in using functional and/or molecular imaging—especially MRI and positron emission tomography (PET)—to better delineate the details of responses to therapy.
PME = phosphomonoester;
PDE = phosphodiester;
PFC = perfluorocarbon;
UICC
CT measurement of changes in disease volume is usually performed to support bidimensional assessments of response as recommended by the Union Internationale Contre le Cancer (UICC) or unidimensional assessment of response, as recommended by Response Evaluation Criteria in Solid Tumors (RECIST), although its multislice capability also allows accurate assessment of tumor volume.
Rendition - interpretation
conventional imaging not registered to an orthogonal coordinate grid, limiting reproducibility;