Drug Discovery and Development Process of Anti-diabetic Plants

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Drug Discovery and Development Process of
Anti-diabetic Plants

• Dr. Basavaraj K. Nanjwade M. Pharm., Ph. D
• Professor of Pharmaceutics
• KLE University College of Pharmacy
• BELGAUM- 590010, Karnatka, India.
• Cell No 0091 974243100
• E-mail bknanjwade_at_yahoo.co.in

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Sources of drugs
Animal Insulin (Pig, cow)
Growth hormone (Man) (Creutzfeldt-Jakob) Plant
Metformin (Gallega officinalis )
Morphine (Papaver somniferum) Inorganic Arsenic,
Mercury, Lithium
Synthetic Chemical (Propranolol)
Biological (Penicillin)
Biotechnology (Human insulin)
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Drug Discovery
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Drug Discovery
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The Regulatory process

• Differs from country to country
• Demands safety and quality of product
• Encourages efficacy and need for product
• Grants clinical trials certificate if volunteer
  and animal data OK
• Approves protocols and examines data
• 50-400 volumes (30,000-150,000 pages)
• Original data available
• Two way process authority and company trying to
  produce a safe effective product
• Release for a specific purpose and use

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Marketing

• Getting the product right (packaging
  formulation)
• Right therapeutic slot
• Information on new drug
• Information for honest comparison
• Reporting problems
• Reporting new indications
• Therapeutic trends

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Schema diagram representing anti diabetic plants
data
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A screen shot of the database Database on
anti-diabetic plants home page with links and
dropdown search window.
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Annona squamosa (SugarApple)
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Nigella sativa
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Polyherbal formulation of Annona squamosa and
Nigella sativa

• This study was undertaken to investigate the
  effect of Polyherbal formulation of Annona
  squamosa and Nigella sativa on blood glucose,
  plasma insulin, tissue lipid profile, and
  lipidperoxidation in streptozotocin induced
  diabetic rats. Aqueous extract of Polyherbal
  formulation of Annona squamosa and Nigella sativa
  was administered orally (200 mg/kg body weight)
  for 30 days. The different doses of Polyherbal
  formulation on blood glucose and plasma insulin
  in diabetic rats were studied and the levels of
  lipid peroxides and tissue lipids were also
  estimated in streptozotocin induced diabetic
  rats. The effects were compared with tolbutamide.
  Treatment with Polyherbal formulation and
  tolbutamide resulted in a significant reduction
  of blood glucose and increase in plasma insulin.
  Polyherbal formulation also resulted in a
  significant decrease in tissue lipids and lipid
  peroxide formation. The decreased lipid peroxides
  and tissue lipids clearly showed the
  antihyperlipidemic and antiperoxidative effect of
  Polyherbal formulation apart from its
  antidiabetic effect.

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INTRODUCTION

• Diabetes mellitus is syndrome, initially
  characterized by a loss of glucose homeostasis
  resulting from defects in insulin secretion,
  insulin action both resulting impaired metabolism
  of glucose and other energy yields fuels such as
  lipids and protein. Experimental diabetes in
  animals has provided considerable insight into
  the physiologic and biochemical derangement of
  the diabetic state. Many of the derangement have
  been characterized in hyperglycemic animals.
  Significant changes in lipid metabolism and
  structure also occur in diabetes. In these cases
  the structural changes are clearly oxidative in
  nature and are associated with development of
  vascular disease in diabetes. In diabetic rats,
  increased lipidperoxidation was also associated
  with hyperlipidemia. Liver, an insulin dependent
  tissue that plays a pivotal role in glucose and
  lipid homeostasis and it is severely affected
  during diabetes.

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Animals

• Male Wistar albino rats (weighing 160200 g) were
  procured from Venkateshwara Enterprise, Bangalore
  and they kept in under standard environmental
  conditions (12 h light/dark cycles at 2528 0C,
  6080 relative humidity) in clean and dry cages
  and maintained in well-ventilated animal house.
  Animals were divided into 8 groups of five each
  and were fed with standard diet and water ad
  libitum. The study was approved by the
  Institutional Animal Ethics Committee.

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Preparation of drug

• The seeds of Nigella sativa obtained from Prgati
  Ayurvedic Drug store Belgaum and matured fruit of
  Annona squamosa from local market of Belgaum and
  they were authenticated from Botanical Survey of
  India, Pune (Maharastra).
• The extracts of the both antidiabetic plants were
  mixed and polyherbal formuation was prepared
  (Table 1). Five hundred grams of each plant
  (chopped into small pieces) was extracted
  individually and were soaked overnight in 1l of
  water. This suspension was filtered and the
  filtrates were pooled and the solvents were
  evaporated in a rotavapor at 4050 0C under
  reduced pressure and lyophilized.

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Chemicals

• Streptozotocin and other biochemicals used in
  this experiment were purchased from Sigma
  Chemical Company Inc., St. Louis, Mo, and USA.
  Enzyme linked immunosorbant assay (ELISA) kit for
  insulin assay was purchased from Boehringer
  Mannheim, Germany.
• Tolbutamide was a generous gift sample from Sun
  Pharmaceuticals Limited, Baroda, India. All other
  chemicals used were of analytical grade.

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Drug administration

• Polyherbal formulation of Annona squamosa and
  Nigella sativa was suspended in distilled water
  and administered orally through intragastric tube
  at the following doses of 50, 100 and 200 mg/kg
  body weight.

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Streptozotocin-induced diabetes

• Rats were made diabetic by single administration
  of streptozotocin (60 mg/kg/i.p) dissolved in 0.1
  M-citrate buffer, pH 4.5. Forty-eight hours
  later, blood samples were collected and glucose
  levels were determined to confirm the development
  of diabetes.
• Only those animals which showed hyperglycemia
  (blood glucose levels gt 250 mg/dl) were used in
  the experiment.

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Experimental design

• In the experiment, a total of 42 rats (30
  diabetic surviving rats, 12 normal rats) were
  used. The rats were divided into seven groups of
  six rats each after the induction of
  streptozotocin diabetes.

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Experimental design

• Group1 Normal treated rats.
• 2. Group2 Normal rats given aqueous solution of
  Polyherbal formulation (200 mg/kg body weight)
  daily using an intragastric tube for 30 days.
• 3. Group 3 Diabetic control rats.
• 4. Group 4 Diabetic rats given aqueous solution
  of Polyherbal formulation (50 mg/kg body weight)
  daily using an intragastric tube for 30 days.

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Experimental design

• 5. Group 5 Diabetic rats given aqueous solution
  of Polyherbal formulation (100 mg/kg body weight)
  daily using an intragastric tube for 30 days.
• 6. Group 6 Diabetic rats given aqueous solution
  of Polyherbal formulation (200 mg /kg body
  weight) daily using an intragastric tube for 30
  days.
• 7. Group 7 Diabetic rats given aqueous solution
  of Tolbutamide (250 µg/kg bodyweight) daily use
  an intragastric tube for 30 days.

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Experimental design

• At the end of 30 days, all the rats were killed
  by decapitation under pentobarbitone sodium (60
  mg/kg) anesthesia. Blood was collected in tubes
  containing potassium oxalate and sodium fluoride
  solution for the estimation of blood glucose and
  plasma was separated for the assay of insulin.

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Table 1 Polyherbal Formulation of Annona
Squamosa and Nigella Sativa (Composition and
Concentration).
Sl. No. Botanical Name Common Name Family Part used Conc. ()
1 Annona squamosa Sharifa Annonnaceae Matured fruits 50
2 Nigella sativa Kalonji Ranunculaceae Seeds 50
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Table 2 Changes in blood glucose and plasma
insulin levels of control and experimental animals
Group Fasting blood glucose (mg/dl) Plasma insulin (IU/ml)
Normal 81.04 2.29 11.26 0.96
Diabetic control 262.24 22.23 3.48 0.69
Diabetic Polyherbal formulation (50 mg/kg) 209.58 12.46 5.59 0.34
Diabetic Polyherbal formulation (100 mg/kg) 155.58 11.69 6.03 0.45
Diabetic Polyherbal formulation (200 mg/kg) 104.16 6.56 7.15 0.45
Diabetic Tolbutamide (250 mg/kg) 110.65 9.35 6.32 0.48
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Pharmaceutical Product Quality Cannot Be Tested
in - It Is Built in

• Pharmaceutical product quality is assured by
• comprehensive development program
• extensive manufacturing and environmental
  controls
• rigorous validation procedures and requirements
• The high quality thus built into the final
  product is ensured through in-process controls
  and verified in a series of confirmatory tests
  before each manufactured batch is released to the
  market

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Building-in of Quality Starts Early.Development
Builds-in Quality

• The chemistry, manufacturing and controls (CMC)
  aspect of drug development is focused on
  producing medicines suitable for human use with
  specified quality, safety and efficacy
  characteristics
• The drug development program is geared towards
• thorough understanding of the drug products
  performance
• identification of drug products critical
  characteristics (which would be monitored on a
  batch-by-batch basis)
• demonstration of drugs safety and efficacy
• ultimately leads to the review and approval of
  the drug

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Relationship between Safety, Efficacy and Quality

• Every drug product (with its specifications) has
  been thoroughly tested in clinical trials for
  safety and efficacy
• Specifications for release and stability testing
  may be equal to or tighter than the
  specifications for clinical trial batches
• Therapeutic indication and QC are considerations
  in establishing specifications

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Drug Development Process
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Drug Development Process
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Quality is Always Part of the Picture - Built-In
and Built-Up
Quality Control and Quality Assurance
Less established
Fully established
Commercial Manufacturing
Pre-IND
Phase I
Phase II
Phase III
Specification/Manufacturing Development for the
Product
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Drug Development Cycle
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Examples of QA QC Considerations During Drug
Development

• Evolution of documentation systems
• SOP
• change control
• trend analysis
• Evolution of QA and QC systems
• internal audits
• supplier audits
• document review (e.g., SOP, batch records,
  specifications, data)

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Chemistry Manufacturing Controls Evolve During
Drug Development

• The goal is to have process and product
  performance determined by the time of validation,
  although some level of validation occurs along
  the continuum and eventually leads to the
  full-scale validation.

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Examples of CMC Considerations During Drug
Development

• Selection of appropriate technology and raw
  materials
• Optimization
• of formulation and device
• of manufacturing process
• of specifications and analytical methods
• Careful selection and control of container
  closure systems
• Identification and control of critical
  manufacturing process parameters
• Process capability established
• Technical transfer to larger scale, i.e.,
  scale-up
• Process validation

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Process Registration Requirements

• Sponsor is required to describe how the product
  was developed
• Companies need to optimize, justify and register
  the entire recipe
• ranges
• temperatures
• mixing times
• hold times
• etc.
• quantities
• active ingredient
• excipients
• raw material specifications
• in-process limits
• in-process methods
• product specifications
• etc.

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Validation
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What is Validation?

• Documented evidence that the manufacturing
  process consistently produces product that meets
  predetermined specifications
• Defines product quality
• Developed and validated based on a thorough
  understanding of the critical process parameters
• Parameters are carefully controlled within the
  validated ranges to ensure a consistent
  manufacturing process.
• Manufacturing process validation consists of
  successfully manufacturing at least three
  full-scale batches in succession, which pass all
  in-process and product quality attributes

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Validation is Always Part of the Picture
Commercial Manufacturing
Pre-IND
Phase I
Phase II
Phase III
Final process validation
Specification Development
Re-validation
Ongoing Validation (DOE, IQ, OQ, PQ, PV)
DOE Design of Experiment IQ Installation
Qualification OQ Operational Qualification
PQ Performance Qualification PV Process
Validation

• The extent of IQ, OQ, PQ, validation, etc.
  depends on complexity of product

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Role of QC Tests

• Each batch of orally inhaled and nasal drug
  products (OINDP), manufactured by the validated
  process, is tested to the critical QC attributes
  as defined during development
• Confirms consistent performance
• The Delivered Dose Uniformity test for OINDP is
  one of several confirmatory QC tests of the
  finished product
• a result of a long and careful development and
  characterization process
• QC tests confirm the quality built-in through a
  well-understood and well-controlled manufacturing
  process

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Pre-Approval Inspection

• Confirms Facility is Ready
• Sponsor can do what they submit in the NDA
• Process is validated or validation protocols are
  in place
• Validation required prior to launch
• Thorough documentation review
• Quality systems are established and capable
• Confirms specifications are met
• Compliance versus Review Division
• Specifications may change based on NDA review

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Foreign Marketing Data

• Several Ayurvedic, Siddha and Unani drugs have
  been marketed for centuries in India. The drugs
  are also dispensed to the patients by recognized
  and qualified physicians of the complimentary
  systems.
• Most of these drugs have been found to be safe,
  It may be worthwhile for the, FDA to accord
  adequate weightage to the data already available
  in these countries in determining whether a drug
  has been used under particular conditions to a
  material extent and for a material time to
  qualify for inclusion in an OTC drug monograph.

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Foreign Market Data

• Since the FDA is proposing to make it available
  the facility for OTC marketing agencies in the
  United States, who are already marketing their
  products, the same exemptions can be made
  available to overseas marketing firms who have
  safely-marketed their products in their
  respective countries.

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Chemistry Manufacturing Controls (CMC)
information for the botanical drug products

• Botanical, drugs. are derived from vegetable
  matter and are usually complex mixtures. The
  chemical constituents of such a mixture are not,
  always defined and in most cases even the active
  constituent in a botanical drug is not defined
  nor its biological activity well characterized.

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Chemistry Manufacturing Controls (CMC)
information for the botanical drug products

• Therefore the CMC documentation would have to be
  substantially liberal in comparison to that of
  synthetic or highly purified drugs. Simple or
  combination tests like spectroscopic,
  chromatographic, fingerprints,, chemical and or
  biological assays can be the main reliability
  criteria to understand the product.
• While these tests may not generate the necessary
  specifics that are desirable, it can atleast
  bring about a rational approach to Quality
  Control.

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Applicability of Combination Drug Regulations

• It would be practical not to confine the
  botanical drug products that are derived from a
  single part of a plant such as leaves, stems,
  roots seeds under the fixed combination drug
  category.
• The current requirement of Botanical drugs
  composed of multiple parts of a single plant
  species under the combination requirement should
  also be revised and exemption accorded.

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Studies on bioavailability and drug interactions

• Bioavailability and Pharmacokinetic studies are
  cumbersome and extremely difficult to generate in
  complex formulations, where a number of herbs are
  involved. It would be desirable to have a very
  practical view in this area.
• Well-controlled clinical trials can substitute
  the bioavailability and Pharmacokinetic studies.

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Studies on bioavailability and drug interactions

• Ayurvedic and Siddha pharmacopoeia are full of
  formulations which have a. combination of several
  herbs and most of them can never be launched in
  the next decade or two if requirements are not
  simplified by the FDA.

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Natural Vs Cultivable source of Plants

• In a Country like India where the source of raw
  materials for Drug preparations is predominantly
  from the forests (80) a practical solution has
  to be found in connection with the source of the
  raw material for quality enforcements.
• When a medicinal plant is cultivated a lot of the
  Quality aspects are directly under the control of
  the grower.

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Natural Vs Cultivable source of Plants

• However, when the plant material is collected
  from the forests, it would be reasonable to
  presume that the origin of the source is, from
  nature and variations if any would be natural.
• The document should highlight separate guidelines
  for natural and cultivable sources for
  manufacturers using plants from the above two
  sources

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Conclusions

• Pharmaceutical quality is built-in through the
  entire drug development process
• validation is key element of ensuring quality
• in-process controls assure quality during
  manufacturing
• Specifications established based on thorough
  understanding of process

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BIRDEM ANRAP, Dhaka, Bangladesh
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• THANK YOU

Cell No 0091 974243100 E-mail
bknanjwade_at_yahoo.co.in
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