in one aspect , the invention relates to methods of preparing a long acting anesthetic . construction of the anesthetic will be described . experimentation to insure the anesthetic lasts over 30 hours will also be detailed . the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds , compositions , and biomaterials and / or methods claimed herein are made and evaluated , and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention . three compounds were used including bupivacaine hydrochloride : 1 - butyl - n -( 2 , 6 - dimethylphenyl )- 2 - piperidinecarboxamide and hyaluonan : ( also called hyaluronic acid or hyaluronate ) an anionic , non - sulfated glycosaminoglycan distributed widely throughout connective , epithelial , and neural tissues , and fibrinogenor coagulation factor i , which is a soluble 340 kd plasma glycoprotein required for normal platelet function and wound healing . the composite anesthetic composition was made with powdered bupivacaine ( 40 mg ) was mixed with fibrinogen ( 40 mg ) with a magnetic stir bar . hyaluronan ( 16 mg ) was then added to the bupivacaine / fibrinogen mixture and placed in an ultrasound sonicator for final mixing followed by a vacuum to remove excess air . special testing containers were constructed with a mid polyurethane shelf to centralize the compounds within a test tube , allowing them to bathe in two ml of release medium . simulated body fluid ( sbf ) was selected as the release medium , rather than serum , which contains potential binding proteins . each specimen was initially bathed with two ml of sbf . at the end of each time period , the specimen was withdrawn and replaced with fresh sbf . release fluid was replaced at every time point and saved for bupivacaine quantification analysis . release profile experiments were conducted on four bupivacaine composites . the composite combinations were selected based on their low energy bonding characteristics that would result in the best sustained - releasing profiles . the four groups were group 1 : bupivacaine + hyaluronan , group 2 : bupivacaine + hyaluronan + fibrinogen , group 3 : bupivacaine + heparin , and group 4 : bupivacaine + heparin + fibrinogen . in each of the four composites 40 mg of powdered bupivacaine was used . other combinations were added to the bupivacaine as follows : 16 mg of hyaluronan , 40 mg of heparin , and 40 mg of fibrinogen . initially , the powdered bupivacaine was mechanically mixed with one or two of the other compounds with a magnetic stir - bar . eight ml of normal saline was added to replicate a 0 . 5 % solution that is used in the clinical setting . each composite was then placed in an ultrasound sonicator , followed by a vacuum to remove excess air . the four composites were then ready for testing . all four composites were tested in an identical manner for three different trials ( n = 3 ). a control of 0 . 5 % bupivacaine without additives was also included in each trial . there were fifteen time periods for each of the 15 specimens , totaling 225 elute samples . after all samples were collected , an ultraviolet - visible spectrophotometer was used to determine the concentration of bupivacaine in each of the elute samples . specific wavelength for absorption of bupivacaine was found in the 280 nanometers range . serial bupivacaine dilutions were created in order to generate standard curves to assist in calculating accurate bupivacaine concentrations . this required a calculation of the linear regression analysis for best fit . once all concentrations were known , data interpretation was accomplished . statistical analysis was completed by anova f - test for all combinations . a student t - test was then run on those found to be significant after anova screening . statistical significance was determined by a p value & lt ; 0 . 05 . based on the releasing data , the best combination was the hyaluronan - fibrinogen - bupivacaine combination . statistical analysis comparing all combinations demonstrated a statistically significant difference between hyaluronan / fibrinogen / bupivacaine combination and the heparin / bupivacaine composite ( p & lt ; 0 . 0415 ). a separate method to analyze the releasing profile was completed . this data examined the total amount of bupivacaine released over the entire study period . a comparison between the control and the hyaluronan / fibrinogen / bupivacaine composite was made . the total concentrations released were nearly equal after comparing the areas under the curves . a similar amount of drug is released , maintaining the same toxicity potential ; however , bupivacaine is released at a slower rate in the hyaluronan / fibrinogen / bupivacaine composite .
the invention relates to structuring a combination of bioactive materials that are capable of controlled extended release of local anesthesia lasting over 30 hours .