ingredients of the present invention of the analgesic combination chemicals are ( percentage of weight ): the two ingredients ketanserin and propranolol should be evenly mixed according to the above proportions to get the analgesic combination . the ingredients ketanserin and propranolol in the combination arc available in the market . the combination is a drug that is made to treat pain . the powders in combination of the present invention are dissolved in injected vehicle to make injected drug . the components of this combination are dissolved in injected water to make injected drug which contains 1 - 50 mg of combination in 1 ml injected water . the use of the invention analgesia combination is to treat arthritic pain , muscleskeletal pain syndrome , general inflammatory pain and neuropathic pain . the ingredients prescribed in this combination are mixed according to the defined proportions and dissolved in the injected water ( 1 - 50 mg drugs , in 1 ml water ). then the mixed combination can be injected subcutaneously . the effective dose is 0 . 05 - 0 . 5 mg / kg , once or twice per day for 7 - 14 days depending on the severity of pain . however , period of treatment can be shorter or longer depending on the effect . rats received a subcutaneous injection of carrageenan ( 2 % in saline , 100 μl ) in the right hindpaw ( intraplantar injection ). drug or vehicle were administered intraplantarly ( i . pl ., 50 μl ) 1 hour after carrageenan injection and behavioral testing was performed at various time points . nociceptive threshold responding to heat was determined as described before ( williams et al ., 1990 ; wisden et al ., 1990 : jiang et al ., 2006 ). briefly , rat was placed in a device that held the body without restraining the head and legs . noxious heat stimulation was carried out by immersing the hindpaw up to the ankle joint into a slowly stirred water bath at 47 . 5 ° c . while holding the animal with the device . this temperature produced an average baseline pwl ( paw withdrawal latency ) of 7 - 10 s in naive paw . paw withdrawal latency for any test time point was measured twice at 3 - min intervals and the mean values were calculated . baseline latency was measured 10 min before drug or vehicle administration . result i - 1 effects of intraplantar injection of ketanserin on thermal hyperalgesia ( paw withdraw test ). effects of ketanserin on carrageenan - evoked hyperalgesia are illustrated in fig1 . paw withdrawal latencies in seconds were measured in plantar test at the various time points . data were normalized as percentage of pretreatment baseline ( 100 %) and expressed as the mean ± sem ( standard error of mean ). carrageenan ( 2 %, 100 μl was administered i . pl . in one hindpaw at 0 hour and 0 . 8 % dmso ( 50 μl , i . pl .) or ketanserin ( 2 , 6 and 20 μg in 50 μl , i . pl .) or prazosin ( 40 μg , i . pl .) at 1 hour . *** represents p & lt ; 0 . 001 compared with control ( carrageenan / 0 . 8 % dmso ). the means of pwls before injection of carrageenan were 6 . 6 - 7 . 9 sec . summary : local injection of the 5 - ht 2a antagonist ketanserin in the inflammed paw dose - dependently produces hypoalgesia in the inflamed area that displays lower nociceptive threshold than normal . the selective □ 1 - adrenergic receptor antagonist prazosin failed to alter the carrageenan - evoked response suggesting that ketanserin - induced hypoalgesia is specific . reversal of ketanserin - associated hypoalgesia in carrageenan - evoked inflammation by opioid receptor antagonist is shown in fig2 . paw withdrawal latencies in seconds were determined in plantar test at the various time points . data were normalized as percentage of pretreatment baseline ( 100 %) and expressed as the mean ± sem . carrageenan and ketanserin ( 20 μg ) were given i . pl . into unilateral hindpaw at 0 and 1 hour , respectively . then naloxone chloride ( 5 mg / kg ) or naloxone methiodide ( 10 mg / kg ) or saline ( 1 ml / kg ) was injected s . c . ( subcutaneous injection ) at 24 hours after carrageenan . * represents p & lt ; 0 . 05 and ** p & lt ; 0 . 01 compared with group of carrageenan / ketanserin / saline . conclusion : ketanserin - induced hypoalgesia in the inflammed paw is mediated by opioid analgesia mechanism . effect of way100635 on carrageenan - evoked response is demonstrated in fig3 . paw withdrawal latencies in seconds were examined at the various time points . data were normalized as percentage of pretreatment baseline ( 100 %) and expressed as the mean ± sem . ( a ) carrageenan ( 2 %, 100 μl ) was administered i . pl . at 0 hour and 0 . 8 % dmso ( 50 μl , i . pl .) or way100635 ( 3 , 10 and 30 μg , i . pl .) at 1 hour . * represents p & lt ; 0 . 05 , ** p & lt ; 0 . 01 and *** p & lt ; 0 . 001 compared with control ( carrageenan / dmso ). summary : local injection of the 5 - ht 1a antagonist way10063 in the inflammed paw dose - dependently produces hypoalgesia in the inflamed area that displays lower nociceptive threshold than normal . nociceptive response is higher but nociceptive threshold is lower . result i - 4 reversal of way1000635 - induced hyporalgesia by naloxone ( fig4 ) carrageenan and way100365 ( 30 μg ) were given i . pl . into unilateral hindpaw at 0 and 1 hour , respectively . then naloxone chloride ( 5 mg / ml / kg ) or naloxone methiodide ( 10 mg / ml / kg ) or saline ( 1 ml / kg ) was injected s . c . ( subcutaneous injection ) at 24hours after carrageenan . paw withdrawal latencies in seconds were measured at the various time points . data were normalized as percentage of pretreatment baseline ( 100 %) and expressed as the mean ± sem . *** represents p & lt ; 0 . 001 compared with group of carrageenan / way100365 / saline . conclusion : way100365 - induced hypoalgesia in the inflammed paw is mediated by opioid analgesia mechanism . result i - 5 enhancement effect of a combination of ketanserin + way1000635 ( fig5 ) carrageenan ( 2 %, 100 μl ) was administered i . pl . at 0 hour and saline ( 50 μl , i . pl .) or ketanserin ( 2 μg , i . pl . )+ way100635 ( 3 μg , i . pl .) at 1 hour . the results are illustrated in fig5 . paw withdrawal latencies in seconds were examined at the various time points . data were normalized as percentage of pretreatment baseline ( 100 %) and expressed as the mean ± sem . *** represents p & lt ; 0 . 001 compared with control ( carrageenan / saline ). summary : local injection of ketanserin plus way10063 with ineffective doses produces significant inhibition on hyperalgesia in the inflamed paw . arthritis model — arthritis was induced using a modification of the previously described method ( schaible & amp ; schmidt , 1985 ; pozo et al ., 1997 ; sluka , 1998 ; zhang et al ., 2002 ). briefly , rat was anesthetized with barbiturate ( nembutal ; 45 mg / kg ). a solution of kaolin ( 4 % in distilled water , 0 . 1 ml ) was injected into the synovial cavity of right knee joint . then , the knee joint was flexed and extended within the normal range of motion at regular intervals for 15 min . after that , 0 . 1 ml of a 2 % aqueous solution of carrageenan ( in saline ) was injected into the joint cavity . the knee joint was again manipulated by slightly rapid flexion and extension movements for 5 min . behavioral test was conducted before and after induction of arthritis by measuring paw withdrawal latency ( pwl ). a decrease in the pwl to noxious radiant heat in an animal with knee joint inflammation is indicative of secondary hyperalgesia and is used to assess arthritis ( coderre & amp ; wall , 1988 ; houghton et al ., 1998 ; sluka et al ., 1998 ; zhang et al ., 2002 ). thermal hyperalgesia was evaluated by using an analgesimeter ( plantar test , ugo basile , comerio - varese , italy ). on the testing day , each animal was placed in a plastic cage with a glass floor . after a 30 min habituation period , the plantar surface of the hind paw was exposed to a beam of radiant heat through the glass floor . the radiant heat source consisted of an infrared bulb . bulb intensity was adjusted so that the control latency was 8 - 10 s . a photoelectric cell detected light reflected from the paw and turned off the lamp when paw movement interrupted the reflected light . the pwl was automatically displayed to the nearest 0 . 1 s . the cut - off time was 20 s in order to avoid damaging dermal tissue . the average of the three trials with 3 - min interval was determined before and 4 hours after the induction of arthritis . thereafter , the pwl measurement was conducted daily for 2 weeks . the pwl before the injection in each rat served as its own control . to assess inflammation of the knee joint , the rat was anesthetized briefly with halothane and the circumference of the knee joint was measured using a flexible tape measure ( houghton et al ., 1998 ; yu et al ., 2002 ; zhang et al ., 2002 ; sluka et al ., 1998 ). musculoskeletal pain model — the model that is used in the previous study ( radhakrishnan et al ., 2003 ) has been modified in the present study . rats were given injection of lambda carrageenan ( type iv , 3 %, 100 μl , dissolved in sterile saline , sigma chemical company , st . louis , usa ) in the left gastrocnemius muscle belly . five days later , the same agent was re - injected in the gastrocnemius muscle using an identical injection protocol . pwl responding to heat stimulus ipsilaterally was measured . neuropathic pain model — the l5 ( l means lumbar ) spinal nerve of right side of the rat were tightly ligated ( kim & amp ; chung , 1992 ). thermal nociceptive threshold was evaluated by paw withdraw latency using an analgesimeter ( plantar test , ugo basile , comerio - varese , italy ). the detail has been described in the section of carrageenan model . mechanical threshold was measured in the hindpaw using an automated von frey type system ( dynamic plantar aesthesiometer 37400 , ugo basile , italy ). animals were acclimatized to the testing apparatus for 20 min for three days and briefly habituated to the test environment for 10 min on test day to minimize intra - and inter - individual variability of behavioral outcome measures . rats were placed on a metal mesh surface under a plastic enclosure . the stimulator unit was placed beneath the selected hindpaw with the filament below the plantar surface of the rat . a paw - flick response was elicited by application of an increasing force ( measure in grams ) using a stainless - steel filament ( 0 . 5 mm diameter ). when the unit was started , the electrodynamic actuator lifted the filament and exerted a force . the force was increased at a rate of 2 . 5 g / s until the rat moved its paw . a force of 50 g for 30 s was used as cut off to preclude possible damage to the paw . the force was defined as the mean of two measurements at 2 min - interval . the investigator was blind to the test drug conditions . result ii - 1 effects of subcutaneous injection of a combination ( ketanserin + way100635 ) on thermal hyperalgesia in a model of carrageenan - induced inflammation carrageenan ( 2 %, 100 μl ) was administered i . pl . in one hindpaw at 0 hour and saline or ketanserin plus way100635 was injected subcutaneously at 1 hour . the results are shown in fig6 . paw withdrawal latency was measured at the various time points . data were normalized as percentage of pretreatment baseline ( 100 %) and expressed as the mean ± sem . * represents p & lt ; 0 . 05 , ** p & lt ; 0 . 01 and *** p & lt ; 0 . 001 compared with control ( carrageenan / saline ). summary : systemic injection ( subcutaneous ) of ketanserin plus way10063 raises the nociceptive threshold to higher level than normal . result ii - 2 dose - dependent effects of subcutaneous injection of ketanserin or propranolol or combination on thermal hyperalgesia in the carrageenan model ( fig7 ). carrageenan ( 2 %, 100 μl ) was administered i . pl . in one hindpaw at 0 hour and saline or ketanserin ( a ) or propranolol ( b ) was injected subcutaneously at 1 hour in fig7 a or fig7 b . paw withdrawal latency was measured at the various time points . data were normalized as percentage of pretreatment baseline ( 100 %) and expressed as the mean ± sem . ** represents p & lt ; 0 . 01 and *** p & lt ; 0 . 001 compared with control ( carrageenan / saline ). carrageenan ( 2 %, 100 μl ) was administered i . pl . in one hindpaw at 0 hour and saline or combination ( ketanserin plus propranolol ) was injected subcutaneously at 1 hour in fig7 c or fig7 d . paw withdrawal latency was measured at the various time points . data were normalized as percentage of pretreatment baseline ( 100 %) and expressed as the mean ± sem . * represents p & lt ; 0 . 05 , ** p & lt ; 0 . 01 and *** p & lt ; 0 . 001 compared with control ( carrageenan / saline ). fig7 c illustrates that combination of ketanserin plus propranolol at low doses exerted synergistic effect which completely inhibited inflammatory hyperalgesia , fig7 d illustrates that ketanserin or propranolol alone at higher dose ( higher than those in fig7 c ) exerted stronger effect of on inflammatory hyperalgesia . however , it looks that the combination of these two drugs produced similar synergistic effect compared to fig7 c . summary : systemic ( subcutaneous ) injection of ketanserin or propranolol produces significant inhibition on the nociceptive threshold in the inflamed paw . injection of lower doses of ketanserin plus propranolol also produces the same effects . result ii - 3 effects of subcutaneous combination on pain and edema in arthritis model ( fig8 ) kaolin ( 4 %, 0 . 1 ml ) and carrageenan ( 2 %. 0 . 1 ml ) were injected into unilateral knee joint at 0 hour and the results are shown in fig8 a and fig8 b . the drugs were given subcutaneously daily starting on day 2 . paw withdrawal latency and knee circumference were measured at the various time points . data analyses were done between drug and saline groups . * represents p & lt ; 0 . 05 . ** p & lt ; 0 . 01 and *** p & lt ; 0 . 001 compared with control ( saline ) in fig8 a , and p & lt ; 0 . 05 - 0 . 001 after d5 compared to saline group in fig8 b . there are significant difference between combination treatment and saline groups after d3 in fig8 a . there are significant difference between combination treatment and saline groups after d5 in fig8 b . conclusion : systemic injection of ketanserin and propranolol produces significant inhibition on the nociceptive threshold in the arthritic paw and edema in the arthritic joint without tolerance . result ii - 4 effects of subcutaneous injection of combination in musculoskeletal pain model ( fig9 ). carrageenan ( 3 %, 0 . 1 ml ) was injected intramuscularly in the left gastrocnemius muscle belly at days 1 and 5 . then the drugs ( ketanserin plus propranolol ) or saline were given subcutaneously daily starting at day 6 . paw withdrawal latency ( plantar test ) was measured * represents p & lt ; 0 . 05 and ** p & lt ; 0 . 01 and compared with control ( saline ). conclusion : systemic injection of low doses of ketanserin + propranolol produces significant inhibition on the nociceptive threshold in the musculoskeletal pain model without tolerance . result ii - 5 effects of subcutaneous ketanserin on thermal threshold of the hindpaw in neuropathic pain ( snl stands for spinal nerve ligation ). spinal nerve ( l4 ) was ligated at day 0 and the drug or saline was injected subcutaneously starting at day 4 ( fig1 ). paw withdrawal latency was measured . conclusion : systemic injection of ketanserin dose - dependently inhibits thermal nociceptive threshold in the chung &# 39 ; s model of neuropathic pain without tolerance . result ii - 6 effects of subcutaneous combination on mechanical threshold of the hindpaw in neuropathic pain ( snl ) spinal nerve ( l4 ) was ligated at day 0 and the drug or saline was injected subcutaneously starting at day 4 ( fig1 ). then the drugs ( ketanserin plus propranolol ) or saline were given subcutaneously daily starting at day 6 . paw withdrawal latency responding to electronic von frey monofilaments ( dynamic plantar aesthesiometer 37400 , up basile , italy ) was measured . conclusion : systemic injection of low doses of ketanserin + propranolol produces significant inhibition on the mechanical nociceptive threshold in neuropathic pain ( chung &# 39 ; s model ) without tolerance . this invention proposes to locally or systemically ( subcutaneously ) inject a combination of ketanserin ( 5 - ht 2a receptor antagonist ) and propranolol ( 5 - ht 1a receptor antagonist ) to treat pain that is associated with inflammation and tissue injury . our study has showed that post - treatment with intraplantar ketanserin and propranolol in the inflamed site results in prolonging in paw withdrawal latency 24 hours after intraplantar injection of carrageenan . interestingly , pwl at this time was even significantly longer than the baseline level indicating that the blockade of peripheral 5 - ht 2a or 5 - ht 1a receptors produced hypoalgesia 24 hours after inflammation . this hypoalgesia was abolished by i . p . naloxone . this is a novel discovery showing that the blockade of 5 - ht 2a or 5 - ht 1a receptor in the inflamed site inhibits activation of nociceptor and enhances endogenous opioid analgesia . the advantage of this invention is to target peripheral receptors which play a pivotal role in mediating nociceptive responses . therefore , the side - effects can be avoided . the excellence and novelty of the present invention is that targeting 5 - ht 2a and 5 - ht 1a at the same time produces a synergistic effects for inhibition of nociception . the 5 - ht 2a and 5 - ht 1a receptors that the combination of ketanserin and propranolol acts on have not been taken as targets by the existing analgesics . importantly , the combination of these two drugs produces satisfied antinociception at low doses . therefore , the possible side - effects can be minimal , if not zero . in addition , the analgesia occurs rapidly and lacks tolerance . moreover , both local and systemic injections of the combination are effective . therefore , it is convenient to use . potential applications include arthritis , muscleskeletal pain syndrome and general inflammatory pain .
the present invention is a novel analgesic combination and its use . it is classified to he an invention in the field of medicine and technology . the purpose of this invention is to solve the problem that existing analgesics does not effectively attenuate pain or produces serious side - effects in clinic . the ingredients of the analgesic combination include ketanserin and propranolol . this combination is used to make a drug to treat pain . because the ingredients of the analgesic combination target peripheral receptors , the unwanted effects could be only minimal . the big advantage of this combination is that each of the ingredients acts on the different receptors producing the synergizing analgesic . therefore , the analgesia that the combination produces is remarkable and the effective doses are low .