Liposomal mixture of local anesthetics and manufacturing method thereof Technical Field Transdermal delivery of local anesthetics which became effective in thirty minutes and prolong the duration upto four hours Formulation and evaluation of liposomal mixture of local anesthetics for topical anesthesia Background Art Patients undergoing venipuncture or intraveneous characterization often experience pain which makes minor procedures difficult for both the patient and the health care personnel. For patients compliance it is necessary to have effective topical anesthesia. Currently the most widely used topical anaesthesia is EMLA. It is a eutectic mixture of lidocaine and prilocaine and has been introduced clinically for surface anesthesia of intact skin. Although it is considered to be effective, the onset of anesthesia is slow. For administration EMLA should be occluded at least for an hour to be effective. The depth of penetration and degree of topical anesthesia attained following EMLA application depends on the duration of the application.

Liposomes are known to deliver more drugs into the dermis by increasing the rate of dermal penetration of drug as described by Adrienn et al., Anesthesia and Analgesia 67: 1079-81 (1988). Tetracaine was considered the most suitable drug candidate for topical anesthetic for its high permeability coefficient in vitro. While lidocaine is the most widely used local anesthetic due to its safe property.

Accordingly it is the object of the present invention to provide a method for manufacturing effective topical anesthesia by formulating liposomes containing mixture of local anesthetics such as tetracaine and lidocaine.

Disclosure of Invention Tetracaine base (Sigma Chemical Co.) 1 wt%, lidocaine base (Sigma Chemical Co.) 4wt% were encapsulated into multilamellar phospholipid vesicles. The lipid phase, consisting of 2: 5: 3 egg yolk phosphatidylcholine (Sigma Chemical Co.), polyoxyethylene (Sigma Chemical Co.) and cholesterol (Sigma Chemical Co.) was dissolved in 2ml chloroform (Sigma Chemical Co.) in a pear-shape flask. The solvent was evaporated to dryness in a rotary evaporator under reduced pressure at 10-20 torr for 2 hours until a smooth, thin lipid film was obtained on the surface of the flask.

The dried thin film lipid was hydrated in lml (O. lwt%) Tris-EDTA (pH 7.4 Sigma Chemical Co.) buffer on a vortex mixer and sonicated for 30 minutes in ultra-sonicator at 25 °C. Then it was followed by freezing and thawing three times. All chemicals purchased from Sigma Chemical Co., were used as received.

Brief Description of Drawings Figure 1: topical anesthetic effect of lipo-MLA (Mixture of Local Anesthetics) and EMLA (Eutectic Mixture of Local Anesthetics) as control from trial 1.

Figure 2: topical anesthetic effect of lipo-MLA (Mixture of Local Anesthetics) and EMLA (Eutectic Mixture of Local Anesthetics) as control from trial 2.

Figure 3: topical anesthetic effect of lipo-MLA (Mixture of Local Anesthetics) and EMLA (Eutectic Mixture of Local Anesthetics) as control from trial 3.

Figure 4: topical anesthetic effect of lipo-MLA (Mixture of Local Anesthetics) and EMLA (Eutectic Mixture of Local Anesthetics) as control from trial 4.

Figure 5: topical anesthetic effect of lipo-MLA (Mixture of Local Anesthetics) and EMLA (Eutectic Mixture of Local Anesthetics) as control from trial 5.

Best Mode for Carrvins out the Invention Preparation of Liposomes Tetracaine base (Sigma Chemical Co.) 0.5%-1%, lidocaine base (Sigma Chemical Co.) 2.5%-4% were encapsulated into multilamellar phospholipid vesicles. The lipid phase, consisting of different types of phospholipids and cholesterol was dissolved in chloroform in a pear-shape flask. The solvent was evaporated to dryness in a rotary evaporator under reduced pressure for 2 hours until a smooth, thin lipid film was obtained on the surface of the flask.

The dried thin film lipid was hydrated in Tris-EDTA (pH 7.4) buffer on a vortex mixer and sonicated for 30 minutes in ultra-sonicator at 25 °C. Then it was followed by freezing and thawing three times.

In vivo study This study was carried out in accordance with human research standards in Kyungpook National University Hospital ethics committee.

Thirty healthy adult volunteers (20 male, 10 female) participated in the trial after giving written informed consent. Liposomal preparation was compared to a commercial preparation, EMLA (Astra Pharma Inc, Mississauga, Ontario). Following the obtainment of informed consent, volunteers'forearms were cleaned with alcohol swab and allowed to dry. A template having two open area of lcm x lcm square was placed on the volar forearms. 50mg of liposomal preparation and EMLA respectively were spread over the skin surface within the open area using a clean spatula. The test preparations were left on the forearms for specific period of 30 and 60 minutes.

Following the application period, the test preparations were wiped from the skin with clean dry gauze.

Pinprick testing (as described by Chan AW et al, J Neurol Neurosurg Psychiat 1992; 55; 56-594) was used to assess the topical anesthesia. The needle weighed 5g was used to produce pinprick stimulus. The test was performed at 30,60,90,120,180, and 240 minutees following application of test preparations. The 10-scaled pain score (as described by Jong Young et al, The Kor. J. of Occup. Med. 1994 ; 6 ; 2 ; 342-347) was applied to quantitatively evaluate the topical anesthetic effect indicating complete topical anesthesia as 0, intact sensory 10. During the test procedure, the pinprick apparatus was applied three times to each test area on the volar forearms.

Data analysis All data were expressed as meanSEM. Data were analyzed using the repeated measured ANOVA and multiple comparison t-test. Values of *p<0.05 versus control (EMLA) was considered statistically significant.

Results Liposomal mixture of local anestheics was found to produce long lasting anesthesia and of the intact skin and the depth of anesthesia of lipo-MLA was stronger than EMLA. Anesthesia was present after application of liposomal mixture of local anesthetics for shorter than one hour. Pain score analysis indicated that onset time of reliably deep anesthesia was between thirty and one hour. By mild heating the applied site for a few minutes, the onset time can be reduced to less than thirty minutes. The difference between the anesthetic effects of lipo-MLA and EMLA were statistically significant at every time point. The anesthetic effect lasted upto 4 hours. In fact volunteers reported that the numbness at the test sites persisted a few hours after the testing had been completed.

In the case of high concentration, the depth and duration of analgesia were found to be deeper and longer than lower concentration. EMLA cream was less effective. The whole volunteers were entirely recovered with no side effect.

Example 1 After forming a lipid film with 0.5% of tetracaine, 2.5% of lidocaine, 5 : 3: 2 egg yolk phosphatidylcholine (lecithin), cholesterol and phosphatidylcholine dioleoyl, the lipid film was hydrated with the Tris-EDTA buffer. Subsequently, liposome was obtained by repeatedly freezing and thawing the hydrated lipid film. A comparison was made after applying 502mg of liposome and EMLA, respectively, to an square test site of lcm-on the flexural part of a forearm. After a specific time elapsed, the applied agent was wiped off from the test site. Subsequently the pain was measured by carrying out the weighted needle pinprick test (5g) at an interval of 30 minutes for 3 hours. The 10-scaled pain score was adopted where the state of pain on a normal site to which no agent was applied is 10 and the state of anesthesia is 0 (zero). The results were illustrated in Fig. 1.

In Fig. 1, the pain was measured by carrying out the needle pinprick test (5g weighted needle) at an interval of 30 minutes for 3 hours after applying the agent for 60 minutes and wiping off it. The left-hand bar indicates the pain score of liposomal local anesthetic, and the right-hand bar does that of EMLA.

Example 2 The liposome was prepared in a manner similar to that of Example 1 with 0.5% of tetracaine. 2.5% of lidocaine, 5: 3: 2 egg yolk phosphatidylcholine (lecithin), cholesterol and phosphatidylcholine dioleoyl. 502mg of liposome and EMLA, respectively, were applied to the square test sites of 1cm2 on the flexural part of a forearm. The pain was measured in a manner similar to that of Example 1 except that occlusion was performed. The results were illustrated in Fig. 2.

In Fig. 2, the pain was measured by carrying out the needle pinprick test (5g weighted needle) at an interval of 30 minutes for 3 hours after applying the agent for 60 minutes and wiping off it. The left-hand bar indicates the pain score of liposomal local anesthetic, and the right-hand bar does that of EMLA.

Example 3 The liposome was prepared in a manner similar to that of Example 1 except that 1% of tetracaine and 4% of lidocaine were used. 502mg of liposome and EMLA, respectively, were applied to the square test sites of 1 cm on the flexural part of a forearm. The pain was measured in a manner similar to that of Example 1. The results were illustrated in Fig. 3.

In Fig. 3, the pain was measured by carrying out the needle pinprick test (5g weighted needle) at an interval of 30 minutes for 3 hours after applying the agent for 60 minutes and wiping off it. The left-hand bar indicates the pain score of liposomal local anesthetic, and the right-hand bar does that of EMLA.

Example 4 The liposome was prepared in a manner similar to that of Example 1 with one group containing 0.5% of tetracaine, 2.5% of lidocaine, 5: 3: 2 egg yolk phosphatidylcholine (lecithin), cholesterol and phosphatidylcholine dioleoyl, and the other group containing 1% of tetracaine, 4% of lidocaine, 5: 3: 2 egg yolk phosphatidylcholine (lecithin), cholesterol and phosphatidylcholine dioleoyl. 502mg of liposome and EMLA, respectively, were applied to the square test sites of lcm'on the flexural part of a forearm. The pain was measured in a manner similar to that of Example 3.

The results were illustrated in Fig. 4.

In Fig. 4, the pain was measured by carrying out the needle pinprick test (5g weighted needle) at an interval of 30 minutes for 3 hours. The left-hand bar indicates the pain score of liposomal local anesthetic, and the right-hand bar does that of EMLA.

Example 5 The liposome was prepared in a manner similar to that of Example 1, using 0.5% of tetracaine and 2.5% of lidocaine. The pain was measured in a manner similar to that of Example 1 except that the test site was heated for 5 to 10 minutes. The results were illustrated in Fig. 5.

In Fig. 5, the test site was preheated for 0.5 to 2 minutes before application of the agent, and subsequently heated for 2 to 10 minutes after application of the agent. The pain was measured by carrying out the needle pinprick test (5g weighted needle) at an interval of 30 minutes for 3 hours.

The left-hand bar indicates the pain score of liposomal local anesthetic, and the right-hand bar does that of EMLA.