In this slide contains diabetics, classification, symptoms, complication, invivo and invitro screening models of anti diabetics.
Presented by: GEETHANJALI ADAPALA (Department of pharmacology).
RIPER, anantapur
In this slide contains diabetics, classification, symptoms, complication, invivo and invitro screening models of anti diabetics.
Presented by: GEETHANJALI ADAPALA (Department of pharmacology).
RIPER, anantapur
Pharmacological screening of Anti-psychotic agentsAbin Joy
Presentation contents are:
Introduction, Definition of psychosis, Classification of anti-psychotics, MOA of anti-psychotic agents and screening models.
Preclinical screening of new substance for pharmacological activityShrutiGautam18
Preclinical study: A study to test a drug, a procedure, or another medical treatment in animals. The aim of a preclinical study is to collect data in support of the safety of the new treatment.
Pharmacological screening of Anti-psychotic agentsAbin Joy
Presentation contents are:
Introduction, Definition of psychosis, Classification of anti-psychotics, MOA of anti-psychotic agents and screening models.
Preclinical screening of new substance for pharmacological activityShrutiGautam18
Preclinical study: A study to test a drug, a procedure, or another medical treatment in animals. The aim of a preclinical study is to collect data in support of the safety of the new treatment.
Principles of cell viability assays by surendra.pptxSurendra Chowdary
1.DYE EXCLUSION ASSAYS:
Dye exclusion assays are the simplest methods that are based on utilization of different dyes such as trypan blue, eosin, congo red, and erythrosine B, which are excluded by the living cells, but not by dead cells.
For these assays, although staining procedure is quite straightforward, experimental procedure may be time-consuming in case of large sample sizes.
a. Trypan blue stain assay:
Trypan blue stain assay has initially been developed in 1975 to measure viable cell count and is still used as a confirmatory test for measuring changes in viable cell number caused by a drug or toxin.
Trypan blue stain, a large negatively charged molecule, is one of the simplest assays that are used to determine the number of viable cells in a cell suspension.
Principle:
The principle of this assay is that living cells have intact cell membranes that exclude the trypan blue stain, whereas dead cells do not.
Cell suspension is mixed with the trypan blue stain and examined visually under light microscopy to determine whether cells include or exclude the stain.
A viable cell will have a clear cytoplasm, whereas a nonviable cell will have a blue cytoplasm.
Reagent preparation:
To perform the trypan blue stain assay, 0.4% trypan blue stain and phosphate- buffered saline (PBS) or serum-free medium are obtained.
Trypan blue stain should be stored in dark and filtered after prolonged storage.
As trypan blue stain binds to serum proteins and causing misleading results, serum-free medium should be used to obtain reliable results.
Assay Protocol:
The cell suspension to be tested is centrifuged at 100 g for 5 min.
The supernatant is discarded and the pellet is resuspended in 1-ml PBS solution or serum-free medium.
Then, one portion of this cell suspension is mixed with one portion of trypan blue stain.
The mixture is allowed to stay at room temperature for 3 min. It is important to note that the cells should be counted within 3–5 min of mixing with trypan blue, as longer incubation periods will lead to cell death and hence reduced viability counts.
Following the incubation, a drop of the mixture is applied to a hemocytometer, which is placed on the stage of a binocular microscope.
Viable cells will remain unstained, and nonviable cells will stain, in the hemocytometer and these cells are counted separately.
.
Calculation:
After counting viable and nonviable cells, the total number of viable cells per milliliter of aliquot is determined by multiplying the total number of viable cells by 2, which is the dilution factor for trypan blue.
Similarly, total number of cells per milliliter of aliquot is determined by addition of number of viable and nonviable cells and multiplying it by 2.
Then, the percentage of viable cells is calculated using the following equation.
% Viable cells = Total number of viable cells per milliliter of aliquot × 100.
Total number of cells per milliliter of aliquot
2.COLORIMETRIC ASSAYS:
Colorimetric assays
In vitro methods for the assessment of general cellular toxicity,
End-points for the assessment of general cellular toxicity
Specialized cells commonly used in toxicology
Historically, genetic toxicology has been comprised of bacterial and cell based in vitro assays such as the Ames assay (a bacterial mutagenicity assay), Micronucleus and Chromosomal Aberration assays (mammalian cytogenetic assays), and Mouse Lymphoma Assay (in vitro mammalian cell gene mutation assay). These were routinely used for safety evaluation and are still part of the standard core battery. The emergence of new technologies has facilitated the development of in vitro methods for safe and effective drug and chemical testing.
This BioReliance® toxicology services webinar will explore alternative models, including 3D skin models that comply with the EC Scientific Committee on Consumer Safety (SCCS) recommendations. It will also discuss how the 3Rs (Replace, Reduce, Refine) Principle advocates the exploration of such alternative methods while achieving required goals.
In this webinar, you will learn:
• About in vitro alternatives to animal toxicity testing in pharma, chemical, tobacco, and personal care products.
• How the 3Rs (Replace, Reduce, Refine) Principle advocates exploring alternative methods without compromising the required goals.
• Alternatives to comply with the 7th Amendment to the EC Cosmetics Directive.
Historically, genetic toxicology has been comprised of bacterial and cell based in vitro assays such as the Ames assay (a bacterial mutagenicity assay), Micronucleus and Chromosomal Aberration assays (mammalian cytogenetic assays), and Mouse Lymphoma Assay (in vitro mammalian cell gene mutation assay). These were routinely used for safety evaluation and are still part of the standard core battery. The emergence of new technologies has facilitated the development of in vitro methods for safe and effective drug and chemical testing.
This BioReliance® toxicology services webinar will explore alternative models, including 3D skin models that comply with the EC Scientific Committee on Consumer Safety (SCCS) recommendations. It will also discuss how the 3Rs (Replace, Reduce, Refine) Principle advocates the exploration of such alternative methods while achieving required goals.
In this webinar, you will learn:
• About in vitro alternatives to animal toxicity testing in pharma, chemical, tobacco, and personal care products.
• How the 3Rs (Replace, Reduce, Refine) Principle advocates exploring alternative methods without compromising the required goals.
• Alternatives to comply with the 7th Amendment to the EC Cosmetics Directive.
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is an open access international journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
Evaluation of hepatoprotective agents - Hemant KanaseHemant Kanase
1. Introduction
2. Hepatotoxicity: Mechanism
3. Therapeutic strategies available – their limitations
4. In vivo models of liver damage
- Non-invasive model
a. Chemically induced hepatotoxicity
b. Drug-induced hepatotoxicity
c. Radiation-induced hepatotoxicity
d. Metal-induced hepatotoxicity
e. Diet-induced hepatotoxicity
Models of Acute Hepatitis
Models of chronic hepatitis
Models of fibrosis
Models of cholestasis
Models of steatosis
4. Problems faced with animal studies
5. In vitro models of liver damage
6. Advantages and disadvantages of in vitro models
7. Parameters of evaluation
8. Clinical Assessment
Acute scrotum is a general term referring to an emergency condition affecting the contents or the wall of the scrotum.
There are a number of conditions that present acutely, predominantly with pain and/or swelling
A careful and detailed history and examination, and in some cases, investigations allow differentiation between these diagnoses. A prompt diagnosis is essential as the patient may require urgent surgical intervention
Testicular torsion refers to twisting of the spermatic cord, causing ischaemia of the testicle.
Testicular torsion results from inadequate fixation of the testis to the tunica vaginalis producing ischemia from reduced arterial inflow and venous outflow obstruction.
The prevalence of testicular torsion in adult patients hospitalized with acute scrotal pain is approximately 25 to 50 percent
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
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2. “ Cancer is a disease which is characterized by
uncontrolled proliferation of cells that have
transformed from the normal cells of the body. “
CAUSE:
External Factors – chemicals, radiation, viruses, and lifestyle
Internal Factors – hormones, immune conditions, and
inherited mutations
Theories
› Cellular change/mutation theories
› Carcinogens
› Oncogenes/ protooncogenes
2
4. Although 92 approved anticancer drugs are available today for the
treatment of more than 200 different tumor entities, effective
therapies for most of these tumors are lacking.
Out of the 92 registered drugs, 17 are considered by oncologists to
be more broadly applicable and 12 additional agents are perceived
as having certain advantages in some clinical settings
They are mostly cytotoxic in nature and act by a very limited
number of molecular mechanisms.
Thus, the need for novel drugs to treat malignant disease requiring
systemic therapy is still pressing.
A preselection, called the screening process, is therefore required.
The aim of screening efforts is to identify products that will produce
antitumor effects matching the activity criteria used to define which
compounds can progress to the next stage in the preclinical
development program.
4
5. Development of multidrug resistance in patients.
Long-term treatment with cancer drugs is also
associated with severe side effects.
Cytotoxic drugs have the potential to be very harmful to
the body unless they are very specific to cancer cells.
New drugs that will be more selective for cancer cells
5
9. This assay is a sensitive, quantitative and reliable
colorimetric assay that measures viability, proliferation
and activation of cells.
The assay is based on the capacity of mitochondrial
dehydrogenase enzymes in living cells to convert the
yellow water-soluble substrate 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) into a dark
blue formazan product which is insoluble in water.
9
10. The amount of formazan produced is directly
proportional to the cell number in range of cell lines.
10
MTT Formazan
metabolically active Cell
Insoluble
11. It is performed to determine the Enzymatic properties.
Cells from particular cell lines in log phase of growth are
trypsinised,
It is counted in a hemocytometer and adjusted multiwell
plates (96 well plates)
The cells are treated with a various concentration of drug
for specified duration
11
METHOD:
12. After MTT dye is added in each well and plates are
incubated at 37° C for 4 hrs in a CO2 incubator.
The plates are taken out from the incubator and dark-
blue colored formazan crystal are thoroughly
dissolved in DMSO in room temperature.
The plates are then read on a ELISA reader at 570
nm
To calculate the percent cell viability with respect to
control is calculated .
12
13. Hemocytometer
The most common routine method for cell
counting which is efficient and accurate is with the use
of a hemocytometer.
13
14. 14
% cell viability =(OD of treated cells/ OD of
control cells) × 100
15. The Sulphorhodamine B assay measures whole-culture
protein content, which should be proportional to the cell
number.
Cell culture are stained with a protein staining dye,
Sulphorhodamine B.
SRB is a bright pink anionic dye that binds to basic amino
acid of cell.
Unbound dye is then removed by washing with acetic acid.
15
16. During the dead cell either lyse or are lost during
procedure, the amount of SRB binding is proportional
to the number of live cells left in a culture after drug
exposure.
16
17. Large-scale, morphological changes that occur at
the cell surface, or in the cytoskeleton, can be
followed and related to cell viability.
Damage can be identified by large decreases in
volume secondary to losses in protein and
intracellular ions due to altered permeability to
sodium or potassium.
Necrotic cells: nuclear swelling, chromatin
flocculation, loss of nuclear basophilia
Apoptotic cells: cell shrinkage, nuclear
condensation, nuclear fragmentation
17
18. Example: Morphological feature
(Human skin keratinocyte)
(A) (B)
Fig. Morphological feature of (A) normal human skin keratinocyte,
and differentiated human skin keratinocyte(B).
18
19. Example: Morphological feature
(Human skin fibroblasts)
(A) (B)
Fig. Morphological feature of (A) normal
human skin fibroblasts,
and aging human skin fibroblasts(B).
19
20. This assay is based on the structural integrity of the
cells.
Live cells possess intact cell membranes that exclude
certain dyes, such as tryphan blue, Eosin, or propidium,
whereas dead cells would have lost membrane integrity.
Hence they would take up the dyes while the live cells
exclude it.
METHOD:
1. Cell lines are counted, cultured and innoculated in 96
well plates as above.
20
21. 2. Cells were incubated with different concentrations of test
compounds for 4days.
3. Number of cultured cells in different wells were counted
using hemocytometer after staining with suitable dyes.
%cell viability = no.of viable cell
Total no.of cells (viable+dead)
×100
21
23. Advantages:
• Reduce the usage of animals.
• Less time consuming, cost effective & easy to manage
• Able to process a larger number of compounds quickly
with minimum quantity.
• Range of concentrations used are comparable to that
expected for in vivo studies.
Disadvantages:
• Difficulty in Maintaining of cultures.
• Show Negative results for the compounds which gets
activated after body metabolism and vice versa.
Impossible to ascertain the Pharmacokinetics
23
25. 1. Chemical carcinogen model
DMBA induced mouse skin papillomas
Two stage experimental carcinogenesis
› Initiator – DMBA (dimethylbenz[a]anthracene),
› Promotor – TPA (12-O-tetradecanoyl-phorbol-13-
acetate)
Mice : Single dose – 2.5 µg of DMBA , 5 to 10 μg of TPA
in 0.2 ml of acetone twice weekly.
Papilloma begins to appear after 8 to 10 wks - Tumor
incidence & multiplicity of treatment group is compared
with DMBA control group
25
26. Mice are topically applied a single dose of 2.5 µg DMBA in
acetone, followed by 5-10 µg of TPA in 0.2 ml acetone
twice weekly on the same site starting one week after
DMBA application.
Percent tumor incidence and multiplicity of treatment
groups is compared with DMBA control group.
Drug under test can be administered either topically or oral
route.
26
27. The tumor incidence in this model is usually about 100%
DMBA controls.
In repeated topical application of DMBA Alone has also
been shown to induced carcinogenesis.
27
Drug efficacy is measured as percent reduction in
carcinoma incidence, compared with that of carcinogen
control.
29. Mouse Mammary Tumor Virus (MMTV) was the first
mouse virus, isolated at Jackson labs as the “non-
chromosomal factor” that caused mammary tumors in
the C3H strain of mice.
Some viruses cause cancer via random integration in
certain cells
Some viruses carry cellular oncogenes
› Abelson murine leukemia virus – Abl
› Moloneymurine sarcoma virus – Raf
Engineered viruses now used routinely in the laboratory
to induce cancer.
29
30. Tumor cells or tissues (mouse or human) transplanted
into a host mouse.
Ectopic – Implanted into a different organ than the
original (typically subcutaneous or kidney capsule)
Orthotopic – Implanted into the analogous organ of the
original tumor.
Advantages :
› Typically cheap, fast & easy to use.
› Not covered by patents
30
31. In vivo screening tool implemented in 1995 by NCI.
12 human tumor cell lines (lung, breast, colon,
melanoma, ovary, and glioma.
Cells suspended into hollow polyvinylidene fluoride
fibers implanted IP or SC in lab mice
After in vivo drug treatment, fibers are removed and
analyzed in vitro
Antitumor (growth inhibitory) activity assessed
31
34. 2-D and 3-D Cell-based Assays in
Drug Screening
Currently, pharmaceutical firms spend a large amount of
money on the compound efficacy and cytotoxicity test.
There is still a 78% failure rate for all drugs, which may be
devastating to developing companies.
Effective compounds in vitro may be non-effective in vivo
for many reasons, including differences between in vitro
and in vivo target biology, interrelated biochemical
mechanism, metabolism, poor penetration into solid
tissues, etc.
34
35. Currently, almost all cell-based assays or biosensors are
developed in 2-D culture systems, although conventional 2-
D cultures usually suffer from contact inhibition and a loss
of native cell morphology and functionality.
In comparison with 2-D cultures, 3-D cell models create a
more realistic representation of real human tissues, which
is critical to many important cell functions, including
morphogenesis, cell metabolism, gene expression,
differentiation and cell-cell interactions.
35
36. 1.Ashish A., Sonia SY, Mark AH, and Minas TC., Pressure
Related apoptosis in Neuronal Cel Lines., Journal of
Neuroscience Research 2000 60: 495-503
2.Essentials of medical pharmacology ,by KD Tripathi, 7th
edition ,page no :857
3.https://www.slideshare.net/ashwinisomayaji7/screening-
of-anticancer-drugs
4.https://en.wikipedia.org/wiki/Chemotherapy.
5. http://www.embl-heidelberg.de/
ExternalInfo/karsenti/countingcells. html
36