Advanced Pharmacology-I (PHR5001) Lecture 8: Analgesics (Centrally Acting)

1. Advanced Pharmacology-I(PHR5001)Lecture 8:Analgesics (Centrally Acting) Dr. M G Azam Asstt. Professor Dept. of Pharmacy, NSU

2. What is Pain? □ Pain can be defined as a somatic sensation of acute discomfort, a symptom of some physical hurt or disorder, or even emotional distress. • □ Pain is a part of a rapid warning relay instruction the motor neurons of the central nervous system to minimize detected physical harm. • □ Pain can be classified into two types: □ Chronic pain is pain that last much longer than pain normally would with a particular injury. • Acute pain is short-term pain or pain with an easily identifiable cause. Acute pain “is the body's warning of present damage to tissue or disease. Opioid Analgesics can be used to treat both types of pain

3. What Causes Pain? • Pain is caused by the stimulation of pain receptors which are free nerve endings. • “Nocireceptorsare pain receptors that are located outside the spinal column in the dorsal root ganglion and are named based upon their appearance at their sensory ends. These sensory endings look like the branches of small bushes. • There are two types of nocireceptorsthat mediate fast or slow pain signals • The perception of pain is when these receptors are stimulated and they transmit signal to the central nervous system via sensory neurons in the spinal cord.

4. Pain Signaling These neurons release excitatory neurotransmitters which relay signals from one neuron to another. “The signals are sent to the thalamus, in which pain perception occurs. From the thalamus, the signal travels to the somatosensory cortex in the cerebrum, at which point the individual becomes fully aware of the pain .

5. Terminology “Opium” is a Greek word meaning “juice,” or the exudate from the poppy seeds (Paper somniforum) “Opiate” is a drug extracted from the exudate of the poppy. □ Analgesia simply means the absence of pain without loosing consciousness. • □ “Opioid” is a natural or synthetic drug that binds to opioid receptors producing agonist effects: Used for thousands of years to produce: Euphoria, Analgesia, Sedation, Relief from diarrhea, Cough suppression □ Opium contains over 20 distinct alkaloids (morphine was the first alkaloid of opium to be isolated in 1803). By the late 19th century use of these “pure” opium derivatives spread throughout the medical world.

6. Endogenous Opioid Peptides • □ “Endogenous opioid peptides are the naturally occurring ligands for opioid receptors. The term endorphin is used synonymously with endogenous opioid peptides. These peptides are produced by the pituitary gland and by the hypothalamus. are found in the central nervous system mainly in limbic and brainstem areas associated with pain reception, and the certain areas of the spinal cord. • □ These natural peptides work as ligands that interact with their specific receptors causing structural changes that result in other changes in the effected neuron such as the opening or closing of ion gated channels or the activation or deactivation of certain enzymes. □ Opioid peptides work by modulating the release and uptake of specific neurotrasmitters in the neurons they are found. This alteration of neurochemicalbalance creates a vast amount of possible physiological effects, one of which is analgesia.

7. General Consideration of Narcotic Analgesics 【action mechanism】 ligands opioids receptor Gi inhibiting adenylatecyclase increasing potassium ion efflux or reducing calcium ion influx impeding neuronal firing and transmitter release

8. Classification of drug • Origin • natural opiates: morphine, heroin, codeine, thebaine, paraverine • synthetic analgesics: meperidine, methadone, fentanyl, anadol, etorphine, pentazocine. 2. potency • strong agonist: morphine, heroin (diacetylmorphine ), meperidine, methadone,fentanyl • moderate agonist: codeine, propoxyphene. • mixed agonist-antagonist: buprenorphine, pentazocine • antagonist: naloxone, naltrexone Fentanyl When codeine is administered ~10% is converted to morphine by O-demethylation (in the liver) cytochrome p450 Codeine

9. Opioid Antagonists Naloxone • Opioid Antagonists are used to treat opioid overdose cases. • Most are derived from Thebaine (an alkaloid of Opium) • The have strong binding affinity for the mu receptors • They work by competitive inhibition at the binding site (It binds but does not change the receptor while at the same time blocking the agonist). • Naloxone is administered intravenously. • It can rapidly produce the withdrawal symptoms associated with opioid addiction. • Naltrexoneis another example of an opioid antagonist. It is more potent than Naloxone and is used in the treatment of alcohol addiction but its mechanism in this treatment is unknown. Naltrexone

10. Mechanism of action • Activation of peripheral nociceptive fibers causes release of substance P and other pain-signaling neurotransmitters from nerve terminals in the dorsal horn of the spinal cord • Release of pain-signaling neurotransmitters is regulated by endogenous endorphins or by exogenous opioid agonists by acting presynaptically to inhibit substance P release, causing analgesia Primary Effect of Opioid Receptor Activation • Reduction or inhibition of neurotransmission, due largely to opioid-induced presynaptic inhibition of neurotransmitter release • Involves changes in transmembrane ion conductance • Increase potassium conductance (hyperpolarization) • Inactivation of calcium channels

11. The Opioid Receptors • There are 3 main opioid receptors, the mu receptor, the delta receptor, the kappa receptor. • The receptors are found on cell membranes of cells in the nervous system (neurons) and have different effects. μ-receptor • Morphine and its analogues (most opioids) bind to the mu-receptor it produces the effects of analgesia (μ1-receptors). The mu-receptor is also associated with other effects such as “sedation, reduced blood pressure, nausea, euphoria, decreased respiration, miosis (constricted pupils) and decreased bowel motility often leading to constipation (μ2-receptors). • When an opioid binds to the mu-receptor it induces a change in shape which in turn induces a change in the ion channels of the associated cell membrane.

12. μ-receptor • The mu-receptor opens up the ion channel allowing K+ • to flow out of the cell causing hyperpolarizationof the membrane potential. This hyperpolarization causes it to become extremely difficult for an action potential to be reached and therefore the firing of the neuron become far less frequent and the neurons excitability decreases . • The release of K+ also causes less calcium ions to enter the terminal end of the neuron. This is where neurotransmitters are stored and as a result this significantly reduces neurotransmitter release. • These effects of a ligand binding to a mu-receptor essentially turn off the neuron and in doing so block the relaying of pain signals from pain receptors. • Respiratory depression is considered the deadly side effect of opioid analgesic drugs. It is the cause of death in all overdose cases.

13. δ-receptor • The delta receptor is the strongest binding site of the body’s natural pain killer, the class of opioid peptides called the enkephalins. • Morphine and other commonly used opioid analgesics also bind to this receptor strongly and act as an agonist much like they do with the mu receptor. • The delta receptor is a G-protein linked receptor. When an agonist binds to the delta receptor is induces a conformational change that causes the activation of a specific G-protein. • This G-protein “inhibits the membrane bound enzyme adenylatecyclaseand prevents the synthesis of cAMP. The transmission of the pain signal requires cAMP to act as a secondary messenger, and so inhibition of this enzyme blocks the signal.

14. κ-receptor • The kappa receptor is associated directly with analgesia and sedation but with none of the undesired side effects associated with the mu receptor. • Because of this, it is an area of focus in current research and shows promise in the development of a safer analgesic. • When and agonist or ligand binds to the kappa receptor it induces a conformational change that results directly in the closing of the calcium ion channels in the terminal of the neuron and the neuron can not relay pain messages. • There are not many significant agonist of the kappa receptor known. K-receptors only effect nerves that relay “pain produced by non-thermal stimuli and mu receptors inhibit all pain signals.

15. In vivo method for evaluating Analgesics (centrally acting) 1. Haffner’s tail clip method in mice 2. Tail flick or other radiant heat methods 3. Hot Plate Method 4. Tail immersion tests 5. Formalin test in rats

16. 1. Haffner’s Tail Clip Method • Purpose and Rationale: • The method was described by Haffner who • observed that raise of tail in mice treated • with morphine or similar opioid like drugs is less sensitive to noxious stimuli. • Procedure: • The test compounds & the drug are usually administer S.C or orally to fasted male mice (weight 18-25 g) 15, 30, 60 min prior testing. • An artery clip is applied to the root of the tail of mice to induce pain (The animal quickly responds to this noxious stimuli by biting the clip). • The time between the stimulation onset and response is measured by a stopwatch in 1/10 sec increments.

17. Haffner’s Tail Clip Method • Evaluation • A cut-off time is determined by taking the average reaction time + 3 x STDEV of combined latencies of the control mice. • Any reaction time of the test animals which is greater than cut-off time is called a positive response indicative of analgesic activity. • Critical Assessment of this test • The test does not need any sophisticated equipment but a skilled observer is required. • Peripheral analgesic ,such as Salicylate type are not detected by this test.

18. 2. Radiant Heat method • Purpose and rationale • This test was developed by Wolff et al for quantitative measurement of pain threshold against thermal radiation and evaluation of analgesic activity of opiates. • This method is very useful for discriminating between centrally acting morphine like analgesics and non-opiate analgesics. • Mice are placed into the cages leaving the tail exposed. A light beam is focused to the proximal third of the tail. Within a few seconds the animal flick the tail to escape. The time until this reaction occurred is measured.

19. Radiant Heat method • Procedure • Groups of 10 mice of both sexes (weight:18 to 22g) are used for each dose. Before administration of the test compounds or standard (SC/orally), the reaction time is determined. • The animal is put into a small cage with an opening for the tail at the rear wall. The tail is held gently by the investigator. • By the opening of the shutter, a light beam exerting radiant heat is directed to the proximal third of the tail. For about 6 s the reaction of the animal is observed. • The mouse try to pull the tail away and turns the head. With a switch the shutter is closed immediately upon reaction is observed.

20. Radiant Heat method The animal are submitted to the same testing procedure after 30, 60 and eventually 120 min. For each individual animal reaction time is noted • Evaluation: • The average value of reaction time after each intervals are calculated and compared with the pretest value by analysis of significance. • At each time interval only those animals which show a reaction time twice as high or heigher as the pretest value regarded as positive

21. 3. Hot Plate Method • Purpose and Rational • The paw of mice and rats are very sensitive to • heat at the temperature which do not damage • the skin. • The reaction to heat is characterized by jumping, withdrawal and licking of the paws. • The time until these response occur is prolonged after administration of centrally acting analgesic Procedure( by Woolfe and Mac Donald, 1944): The animal are placed in the hot plate consisting of electrically heat surface (Temp.: 55 to 56 oC) and time until licking or jumping occur recorded by stop watch. The latency is recorded before and after 20, 60 and 90 min following oral and s.c administration of standard or test compounds.

22. Cont- Hot plate Method Evaluation The prolongation of latency times comparing the values before and after administration of the test compounds or values of the control with experimental groups cab be used The hot plate tefor comparison using t-test. Critical Assessment of the test st has been used by many investigators and has been found to be suitable for evaluation of centrally acting analgesic. Limitation Sedative, muscle relaxant and psycho mimetic drugs cause false positive test.

23. 4. Tail Immersion Methods • Purpose and rationale • The method has been developed to be selective for morphine like compounds which are selectively capable to prolong the reaction time of the typical tail- withdrawal reflex in rats induced by immersing the end of the tail in the warm water of 55 oC. • Procedure • Young Female Wister rats (170-210 g) are placed into the individual cages leaving the tail hanging out freely. The animals are allowed to adopt to the cages for 30 min before testing. • The lower 5 cm of the tail marked. This part of the tail is immersed in a cup of freshly filled water of exactly 55oC. Within few second rats react by withdrawing the tail. The reaction time is recorded in 0.5 s units by stopwatch.

24. Cont- Tail Immersion method • The reaction time is determined before and periodically after either oral or subcutaneous administration of test substances. Eg. after 30, 60, 120, 180, 240 and 360 mins. • The cut-off time of immersion is 15 s. • The withdrawal time of untreated animal is • between 1 to 5.5 s. A withdrawal time more • than 6s is regarded as positive response. Evaluation: ED50 values can be calculated for each compounds and time response curves (onset, peak and duration of effect) be measured. All the morphine like analgesic have been shown to be active at dose which do not change the behavior. Critical Assessment of the test: The test is useful to differentiate central opioid like analgesic from peripheral analgesics.

25. 5. Formalin test in mice Purpose and Rational Formalin test consider as chronic pain model which is sensitive to centrally acting analgesic agents. Procedure: Male Wister rats (180-300 g) are administered 0.5ml 10% formalin solution into the dorsal portion of the front paw. The test drug simultaneously administered S.C or orally. Each individual rat placed into the clear plastic cage for observation. Pain response are observed, at 30 and 60 min, and indicated by elevation or favoring of paw or excessive licking or biting of the paw. Evaluation: Analgesic response or protection is indicated if both paws are resting on the floor with no obvious favoring of the injected paw.

26. The End