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Development and characterization of a sustained release formulation of lidocaine using liposomes exhibiting… Mok, Miranda Jane 1997

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Development and Characterization of a Sustained Release Formulation of Lidocaine Using Liposomes Exhibiting a Transmembrane pH Gradient by Miranda Jane Mok B.Sc, McGill University, 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Pharmacology & Therapeutics We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January 1997 © Miranda T Mok, 1997 In presenting this thesis in partial fulfilment of. the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. -Department of ftlfl/rVTflCt>{QjLj f ~TheM2peUT]'CS The University of British Columbia Vancouver, Canada Date 07/02, /<? 7 DE-6 (2/88) Abstract Longer acting local anaesthetics which will provide prolonged nerve blockade are required for adequate management of both acute and chronic pain. It has been suggested that liposomes represent a potentially useful vehicle for sustained drug release following local administration. Thus, the aim of this thesis was to develop and characterize liposomal lidocaine by employing a transmembrane pH gradient to efficiently encapsulate lidocaine into large unilamellar vesicles (LUVs). The rate and extent of lidocaine uptake into LUVs exhibiting a pH gradient were determined and compared to drug association with control (no pH gradient) liposomes. While lidocaine was rapidly and efficiently accumulated into liposomes exhibiting a pH gradient, little uptake was seen for control vesicles. The in vitro release kinetics of lidocaine from liposomes were studied and the drug was shown to be slowly released from the carrier with drug efflux being controlled by the applied proton gradient. In addition, it was demonstrated that the rate of lidocaine release from pH gradient-loaded liposomes was only slightly increased in the presence of plasma. Using an in vivo model, the rates of clearance of lidocaine and the lipid carrier were monitored employing radiolabeled markers following intradermal administration. In the guinea pig cutaneous wheal model, the liposomal carrier was found to be cleared slowly from the site of administration, such that greater than 85% of the administered lipid dose remained at 48 hours. Similarly, clearance of liposomally encapsulated lidocaine from the site of administration was relatively slow compared to that of free drug. ii The guinea pig cutaneous wheal model was again used to investigate the efficacy of the liposomal local anaesthetic formulation. The sustained drug release afforded by liposomes exhibiting a pH gradient resulted in a two-fold increase in the duration of nerve blockade compared to free drug. A similar increase in duration of action was seen when these two formulations were compared in the presence of the vasoconstrictive agent, adrenaline (1:200,000). The results of the in vitro release kinetics showed that a significant amount of local anaesthetic remained within the liposomes after 24 hours, yet full recovery from nerve blockade was seen between 5-6 hours. As a result of this finding, the clearance and efficacy of smaller sized liposomes (200 nm vs. 600 nm) were determined using the guinea pig cutaneous wheal model. Again, only slow clearance of liposomes was observed, but encapsulated lidocaine was cleared much faster from the site of injection from 200 nm vesicles compared to the kinetics observed for 600 nm vesicles. However, the smaller sized vesicles did not provide longer nerve blockade compared to the 600 nm vesicles. The relative acute toxicities of free and liposomal lidocaine were also examined using intraperitoneal injections into mice to determine the CD 5 0 . Liposomally encapsulated lidocaine was far superior in providing a large margin of safety in contrast to free drug or no pH gradient liposomal lidocaine, since liposomally encapsulated lidocaine did not elicit any convulsions nor was it lethal. Clearly, the present results demonstrate that large unilamellar vesicles exhibiting a pH gradient can efficiently encapsulate lidocaine and provide a controlled drug release system. These systems reduce the rate of local anesthetic clearance following iii administration, resulting in a significant increase in duration of neural blockade and reduction of toxicity. iv Table of Contents Page Abstract ii Table of Contents v List of Tables vii List of Figures viii Abbreviations ix Acknowledgment x 1 Introduction 1.1 Historical Background 1 1.2 Rationale for Longer Acting Local Anaesthetics 2 1.3 Model Membrane Systems 3 1.3.1 Classification of Liposomes 4 1.4 Properties of Lidocaine 1.4.1 Structure 6 1.4.2 Mechanism of Action 7 1.4.3 Distribution and Metabolism 8 1.4.4 Toxicity 9 1.5 Previous Attempts to Prolong Local Anaesthesia 11 2 Uptake of Lidocaine into Large Unilamellar Vesicles in Response to a pH Gradient 2.1 Introduction 17 2.2 Materials and Methods 2.2.1 Materials 19 2.2.2 Preparation of Large Unilamellar Vesicles 19 2.2.3 Generation of the pH Gradient 20 2.2.4 Loading of Lidocaine into Liposomes 20 2.2.5 Determination of Trapped Volume 21 2.2.6 Efficiency of Lidocaine Uptake in Response to a pH Gradient 2.2.6.1 Gel Exclusion Chromatography 21 2.2.6.2 Centrifugal Filtration 22 2.2.7 Determination of Transmembrane pH Gradients 22 2.2.8 Analytical Procedures 23 2.3 Results 2.3.1 Kinetics of Lidocaine Uptake into LUVs 24 2.3.2 Centrifugal filtration 24 2.3.3 Transmembrane Lidocaine Distribution 25 2.4 Discussion 29 v 3 Release of Lidocaine from Large Unilamellar Vesicles 3.1 Introduction 32 3.2 Materials and Methods 33 3.3 Results 34 3.4 Discussion 39 4 Clearance Rate of LUVs and Lidocaine Following Intradermal administration 4.1 Introduction 41 4.2 Materials and Methods 41 4.3 Results 43 4.4 Discussion 45 5. Duration of Nerve Blockade In Vivo 5.1 Introduction 46 5.2 Materials and Methods 47 5.3 Results 49 5.4 Discussion 55 6 Influence of Liposome Size 6.1 Introduction 57 6.2 Materials and Methods 57 6.3 Results 58 6.4 Discussion 63 7 Toxicity 7.1 Introduction 64 7.2 Materials and Methods 66 7.3 Results 67 7.4 Discussion 74 8 Summary 76 9 Bibliography 80 vi List of Tables Page 1. Influence of lidocaine uptake on residual pH gradient 26 2wazzu . Statistical comparison of neural blockade duration as a function of lidocaine formulation 52 3. Statistical comparison of neural blockade duration as a function of lidocaine formulation 54 4. Statistical comparison of neural blockade duration as a function of lidocaine formulation 62 5. Observed convulsions and death due to different formulations of lidocaine (2%) 73 vii List of Figures Page 1. Model membrane systems 5 2. Structure of lidocaine and amide local anaesthetics 6 3. Lidocaine distribution into liposomes with and without a pH gradient 26 4. Lidocaine uptake into liposomes exhibiting a pH gradient 27 5. Lidocaine distribution into 600 nm vesicles in response to a pH gradient 28 6. Release kinetics of lidocaine (2%) in vitro from DOPC:Chol vesicles 36 7. Release kinetics of lidocaine (2%) in vitro from POPC:Chol vesicles 37 8. Influence of serum (25% total volume) on lidocaine release from liposomes exhibiting a pH gradient 38 9. Clearance of lidocaine and lipid carrier after intradermal administrations into Dunkin Hartley guinea pigs 44 10. Duration of nerve blockade produced by free and liposomal lidocaine 51 11. Duration of nerve blockade produced by co-administration of lidocaine with adrenaline (2%) 53 12. Clearance of liposomal lidocaine (2%) from 200 nm vesicles 60 13. Comparison of nerve blockade using 200 nm and 600 nm vesicles 61 14. Comparison between free drug, no pH gradient liposomal lidocaine and liposomal lidocaine exhibiting a pH gradient versus percent convulsions 69 15. Probit of convulsion activity versus log of 2% lidocaine and no pH gradient liposomal lidocaine 70 16. Comparison between free drug, no pH gradient liposomal lidocaine and liposomal lidocaine exhibiting a pH gradient versus percent death 71 17. Probit of death versus log dose of 2% lidocaine 72 viii Abbreviations CHE Cholesteryl hexadecyl wazzu ether Choi Cholesterol CNS Central nervous system DOPC Dioleoly phosphatidylcholine DPPC Dipalmitoyl phosphatidylcholine DSPC Distearoyl phosphatidylcholine HBS Hepes Buffered Saline HEPES [4-(2-Hydroxyethyl)]-piperazine ethansulfonic acid LUV Large Unilamellar Vesicle mg Milligram ul Microlitre ml Millilitre MLV Multilamellar Vesicle mM Millimolar mmole Millimole M.W.C.O. Molecular weight cut off nm Nanometre nmole Nanomole pHj pH inside the liposome PH 0 pH outside the liposome PL Phospholipid POPC Palmitoyloleoyl phosphatidylcholine rpm Revolutions per minute SUV Small Unilamellar Vesicle ix Acknowledgments I have come across many people who have been of great assistance through my years as a graduate student. First and foremost, I must thank my supervisor Dr. Thomas Madden. His encouragement, support, and constant attention has been greatly appreciated. In addition to promoting our knowledge in the field of liposome research and being so patient with us, he has taught us how to scale walls and stuffed us at his annual dinners! Other individuals whom deserve recognition include: my colleagues, Mac, Cliff, Edward, and Gitanjali for making the lab an interesting and pleasant environment in which to work; Jeffrey Mowat who was especially helpful with my animal experiments; and Dr. Bernard MacLeod for his many suggestions and advice as to different models of nerve blockade and for the use of his laboratory space. Individuals aside, I would also like to extend my gratitude to the Medical Research Council of Canada. Our project and my studentship could not have been realized without their financial support. Last but not least, my family and my husband, to whom I am wholly indebted. Their support and encouragement have helped me through all my endeavours. Al l of you have been instrumental in making this thesis possible. Thank you. 1 Introduction 1.1 Historical Background It has long been known that chewing on coca leaves will produce a numbing effect on the tongue and mouth, and for centuries South American Indians have been consuming coca leaves in spiritual rituals and in every day life to reduce fatigue.1 However, the importance of this observation was not duly noted until 1884 when an ophthalmologist named Carl Koller discovered the local anaesthetic action of cocaine. He demonstrated that reversible cornea anaesthesia could be produced by dropping cocaine directly into the eye. Since Koller's discovery, great efforts have been made to improve local anaesthetics' characteristics but with only limited success. Lidocaine is a widely used short-acting local anaesthetic with a plasma half-life of approximately two hours.1 It was synthesized by Lofgren in and introduced as a replacement for procaine in 1948. Lidocaine possesses faster onset, increased duration of action and longer local anaesthesia than an equivalent concentration of procaine. 1 1.2 Rationale for Longer Lasting Local Anaesthetics Local anaesthetics are used to produce a reversible loss of sensation when applied locally to nerve tissue in appropriate concentrations. By blocking impulse conduction along a nerve fibre, local anaesthetics provide temporary and complete suppression of pain. A large proportion (40%) of post-operative patients experience severe pain, as do many cancer and burn victims. Excellent pain control can be achieved by using local anaesthetics, but unfortunately, they are limited by a relatively short duration of action and can manifest deleterious side effects. Repeated administrations has been known to result in tachyphylaxis4, and placement of indwelling catheters is not without potential complications, including blockage or breakage of the catheter, systemic toxicity resulting from elevated plasma drug levels, migration of the catheter subdurally (in the case of epidural catheterization) and infection.5'6 Clearly the development of a local anaesthetic with properties, such as fast onset of action, prolonged analgesic duration after a single administration, selective blockade of nerve fibres and reduced toxicity, would be advantageous in the clinical setting. This need has prompted the investigation of slow-release formulations based upon microdroplets7, microcrystals8, lipospheres9, microspheres10, and liposomes.11"14 Systems such as these can be utilized to improve upon currently used local anaesthetics to allow for slow release of the drug. Some systems have been demonstrated to prolong the duration of action and reduce peak plasma concentrations of local anaesthetics, resulting in reduced toxicities.15 2 1.3 Model Membrane Systems The pioneering work on liposomes began with Bangham and his co-workers in the mid-1960s.16 Liposomes are microscopic spheres composed of a phospholipid bilayer encapsulating an aqueous core. The phospholipid itself consists of a hydrophilic head group and a hydrophobic tail containing two fatty acyl chains. It was demonstrated that liposomes can be used as models for biological membranes and upon hydration of dry phospholipids, liposomes spontaneously formed.16 Research on biological membranes has made the use of model membrane systems which are composed of naturally occurring or synthetic lipids. In order to generate a membrane system, the lipid of choice must first be isolated or synthesized, then an appropriate model constructed using that lipid. The final step would be to incorporate a non-lipid component, such as protein or a drug of choice, to determine its influence on the system. Liposomes have been used as delivery systems for water soluble agents entrapped in their aqueous compartments and lipid soluble agents in their bilayers. For example, liposomes have been demonstrated to enhance the therapeutic properties of a number of 17 antineoplastics, antifungal agents, antibiotics and local anaesthetics. Depending on the type of phospholipid used, vesicles possessing a variety of properties can be produced. 3 1.3.1 Classification of Liposomes Liposomes may be classified into three main types depending on their size and structure. Hydration of a dry phospholipid film results in the spontaneous formation of multilamellar vesicles (MLVs). MLVs are large, ranging from 1 to 20 urn in diameter18, and are composed of a series of concentric bilayers with aqueous channels between the different lamellae. Ions and other solutes are not distributed uniformly in between these aqueous channels but by employing a freeze-thaw technique, solute distributions can be enhanced. Large unilamellar vesicles (LUVs) are vesicles composed of only one bilayer with 18 diameters ranging from 0.1 to 1 um. They can be formed by forcing MLVs, under high pressure, through filters of a defined pore size. LUVs are superior as model membrane systems when compared to MLVs because LUVs, like most biological membranes, consist of a single bilayer separating two aqueous spaces. Small unilamellar vesicles (SUVs) are formed by mechanical disruption of MLVs. 18 They range in size from 20 to 30 nm in diameter. As a consequence of their size, the curvature of the bilayer formed is highly strained and thus their lipid packing instability renders SUVs more leaky than LUVs and not suitable for use in permeability studies. The trapped volume is the amount of solute encapsulated within the aqueous core of liposomes and it is usually expressed as ul solute per umole phospholipid. This trapped volume will be influenced by the type of lipid used but more importantly, by the size of vesicle produced. Since this research project deals with the utilization of liposomes as drug delivery systems, it would be beneficial to maximize the volume of drug encapsulated within 4 the aqueous core. LUVs possess trapped volumes ranging from 1 to 20 pi per pmole phosphoslipid19, in general, the largest of all three types of liposomes. Multilamellar Vesicles (MLVs) (1-20 u.m diameter) Large Unilamellar Vesicles (LUVs) (0.1 - 1 um diameter) Small Unilamellar Vesicles (SUVs) (20 - 30 nm diamter) Figure 1. Model membrane systems 5 1.4 Properties of Lidocaine 1.4.1 Structure Local anaesthetics are composed of an amine portion and an aromatic region separated by an intermediate alkyl chain. The linkage to the hydrophobic region is either an ester or an amide and is an important determinant of their pharmacological properties. Amide local anaesthetics, such as lidocaine and bupivacaine, are much longer lasting than ester-linked local anaesthetics because the ester link is readily hydrolyzed by plasma esterases. In addition, hydrophobicity of the aromatic region will dictate potency and the duration of action of the local anaesthetic. It enhances partitioning of the drug into its site of action and decreases the rate of hydrolysis by plasma esterases, but hydrophobicity 1943 also increases potential toxicity. Lidocaine Bupivacaine O C H 3 N H C Prilocaine Figure 2. Structure of lidocaine and other amide local anaesthetics 6 1.4.2 Mechanism of Action Local anaesthetics prevent the generation and conduction of the nerve impulse. They bind to the cytoplasmic side of sodium channels and block sodium conductance across the axon thus preventing its depolarization. The blockade of sodium channels by most local anaesthetics is both voltage- and time-dependent. Channels in the resting state, which predominate at more negative membrane potentials, have a much lower affinity for local anaesthetics than activated (open state) and inactivated channels, which predominate at more positive membrane potentials. Therefore, local anaesthetics are more effective in blocking rapidly firing axons than resting axons. Modification of the calcium and potassium concentrations can alter the membrane potential. Calcium increases the surface potential of the membrane and this favours the low-affinity resting state, while the elevation of extracellular potassium depolarizes the membrane and this favours the higher affinity inactivated state. Thus, high levels of extracellular potassium enhance the effects of local anaesthetics. Although local anaesthetics have been shown to block a variety of other channels, including chemically gated synaptic channels, there is no convincing evidence that such actions play an important role in the clinical effects of these drugs. Since local anaesthetics exert their action on the cytoplasmic side of the sodium channel, the degree of protonation of the anaesthetic is very important. The pK a 's of most amide local anaesthetics fall in the range of 7.8-8.2. The local anaesthetics must be able to cross the neuronal bilayer and this predominantly occurs as the uncharged species. This means that if the extracellular pH is low, as occurs with local infections, there will be a 7 greater proportion of drug in the protonated state. Under these conditions, the local anaesthetic will be unable to cross the bilayer and will be less effective. It should be noted that the pH inside of the axon is also very important in determining drug action because local anaesthetics bind to the sodium channel in the charged form. Therefore, the local anaesthetic must cross the neuronal bilayer as the uncharged species and, once inside, it must become protonated in order to bind to the inactivated sodium channel to block propagation of the neuronal action potential. 1.4.3 Distribution and Metabolism The amide local anaesthetics are distributed widely after bolus intravenous administration. The initial rapid distribution phase probably indicates uptake into highly perfused organs such as the brain, liver, kidney and heart. Uptake into the muscles and gut follow in the slower distribution phase. Metabolism does not play an important role in determining the duration of action of local anaesthetics. The metabolism of the amide local anaesthetics involves enzymes of the hepatic endoplasmic reticulum. Lidocaine is dealkylated in the liver followed by hydrolysis. 8 1.4.4 Toxicity Local anaesthetics are relatively safe and are widely used to provide pain relief. However, accidental intravenous injections of these agents can cause severe CNS toxicity, 20 cardiotoxicity and even death due to cardiac failure. Local anaesthetic-induced cardiovascular depression occurs less frequently but tends to be more serious and more difficult to manage. The most common toxicity due to overdosage of local anaesthetics is related to central nervous system effects. Local anaesthetics cross the blood brain barrier and may cause stimulation of the CNS, manifested as restlessness, tremor, and can be as severe as clonic convulsions, followed by central nervous depression. Local anaesthetics act upon cortical inhibitory pathways to exert their depressant effects, so that excitatory pathways are unopposed. This state is followed by a generalized depression of central nervous system function and without medical intervention, death may occur. Signs of CNS toxicity due to local anaesthetics include minor symptoms such as lightheadedness and can be as severe as grand mal convulsions, coma and apnea. Local anaesthetics block sodium channels in the myocardium where they act to decrease electrical excitability, conduction rate and force of contraction; cardiovascular collapse and death may occur as a result of either ventricular fibrillation or a change in the action of the pacemaker. With increasing plasma concentrations of lidocaine, hypotension, bradycardia and respiratory depression will occur. The depressant effects exerted by local anaesthetics are directly related to their potency. Comparison of the cardiovascular effects of bupivacaine and lidocaine demonstrates that at high concentrations, bupivacaine blocks 9 sodium channels in a fast on - slow off manner, whereas; lidocaine at high concentrations acts in a fast on - fast off manner. Thus, bupivacaine is more potent at producing block than lidocaine at physiological heart rates. This is one reason why bupivacaine is the most toxic local anaesthetic in clinical use. Local anaesthetics usually cause a reversible loss of sensation; however, the return of function may be slow or incomplete as a result of trauma, ischaemia, or the persistent effects of the local anaesthetics. In addition, allergic reactions to the ester linked local anaesthetics have been reported. Hypersensitivity occurs to p-aminobenzoic acid derivatives and may manifest as contact dermatitis or a typical asthma attack. 10 1.5 Previous Attempts to Prolong Anaesthesia Various attempts have been made to prolong the duration of action of local anaesthetics,7"14 but unfortunately these have met with only limited success. Manipulation of molecular structure is effective, but beyond a certain point the compounds produced are tissue irritants or neurotoxic.22'23 The addition of vasoconstrictors such as adrenaline to local anaesthetics delays clearance, but these agents are not without adverse effects and they do little to prolong the duration of the longer acting agents. Dextran and low molecular weight (LMW) dextran have been added to local 24 28 anaesthetics in an attempt to prolong nerve block.z""zo The increase in duration of action is thought to be a result of the increased viscosity of the test solutions or a result of complexation of the additive with local anaesthetic.24 When the mechanism by which dextran and LMW dextran prolonged nerve blockade was investigated, no evidence of 25 binding by LMW dextran was found using filtration or dialysis techniques. In addition, conflicting results have emerged from those studies. Some studies demonstrated a 26 27 prolongation of analgesia ' although others found no significant effect upon addition of 24 28 dextran to local anaesthetic solutions. ' The duration of block using 1% lidocaine was 25 unaffected by LMW dextran and no significant effect was shown for dextran on bupivacaine24; however, it appears that dextran may extend the duration of action of anaesthetics such as 2-chloroprocaine and prilocaine. Furthermore, dextran was found to be immunogenic and it therefore should not be used as an exipient.24 Microspheres are synthetic, biodegradable polymers capable of sustained release over an extended period of time. Although ultra-long duration of blockade was 11 demonstrated for dibucaine microspheres in vivo , the use of microspheres has its drawbacks. Preparation of microspheres containing local anaesthetic is labourious since both the local anaesthetic and microspheres must first be dissolved in organic solvent and then added dropwise to 1% gelatin or 1% sodium alginate, all the while stirring at a constant 10 29-31 rate. ' The solvent must then be evaporated and microspheres collected by filtration or centrifugation. This technique of preparing microspheres with local anaesthetic does not allow for a large quantity of drug to be incorporated; the drug content of microspheres is low, no more than 50%.10'29"31 In addition, aggregation of the microspheres causes a decrease in drug diffusion because of a reduction in surface area. Controlled release of drug is not possible with this technique because diffusion and disintegration of microspheres govern the kinetics of drug efflux. Volatile anaesthetics encapsulated in microdroplets were examined to determine whether or not they could be used for local anaesthesia. Although all anaesthetics examined produced local anaesthesia, ultra-long duration of local anaesthesia was demonstrated with lecithin-coated methoxyflurane microdroplets.7 Using a rat tail block model, the researchers were able to produce local anaesthesia lasting 23 hours after only one injection7; however, the disadvantage of these systems was that the onset of anaesthesia was slow. Lecithin-coated tetracaine microcrystals have also been demonstrated to produce prolonged analgesia lasting for 43 hours in a rat tail block model. Microcrystals are able to provide a large dose of tetracaine and no evidence of systemic toxicity was observed for 10% microencapsulated tetracaine in comparison to free drug. To produce the microcrystals, local anaesthetic and egg lecithin was mixed and sonicated. This method produces a non-uniform mixture of particles, 100 nm to 500 nm in diameter, which might make studying the 12 kinetics of drug release difficult. Particles of different sizes will invariably release drug at different rates; therefore, a non-uniform release of local anaesthetic will result. This could mean either too little or too much drug efflux at any one time, causing discomfort or too much anaesthesia. Iodophendylate is an iodinated fatty acid used for radiography of the spinal cord. Langerman et al have used this lipid carrier with procaine, lidocaine, and tetracaine to 11 32 prolong epidural and spinal anaesthesia. ' Tetracaine showed the longest duration of analgesia and this phenomenon was believed to be due to its hydrophobicity. The more hydrophobic the anaesthetic, the longer it will remain associated with the lipid carrier and the longer its duration of action. The use of iophendylate as a lipid carrier is somewhat controversial as aseptic arachnoiditis was reported to occur after intrathecal injection.11 Sustained release of drugs from biodegradable polymer matrixes has become a useful method of prolonged delivery of certain drugs such as the Norplant birth control system, which releases progestin over five years. In vivo trials using implants consisting of a biocompatible polymer local anaesthetic matrix (PLAM) that biodegrades at the site of implantation has been used by Masters et al to demonstrate prolonged analgesia lasting 2 to 33 6 days in a rat model. The cleavage of polyanhydride bonds allows the release of local anaesthetics in this system. Histological examination of sections of sciatic nerve adjacent and proximal to the implants showed no nerve damage. However, one drawback is the formation of fibrous capsules around the polymer pellets. This may slow release of drug to below the anaesthetic blocking dose and impede drug from reaching the nerve. In addition, the biodegradable implants must be surgically inserted. 13 Efforts have been made to prolong the action of local anaesthetics by using liposomes and their performance has been studied by several groups over the past decade. Lidocaine and bupivacaine have been studied in various ways, but each group has had to produce its own preparation which makes comparisons of drug loading and efflux difficult. Previously, Djordjevich et al prepared MLVs associated with bupivacaine.34 They demonstrated that MLVs with bupivacaine extended the duration of drug action compared to free drug when injected into the rat tail. The liposomal formulation used in this study did not allow for drug encapsulation; the local anaesthetic was hydrophobically associated with 35 13 the MLVs. Similarly, in studies done by Legros et al , Boogaerts et al and Mashimo et al14 local anaesthetic association with the liposomal carrier relied on hydrophobic interactions. In those previous studies, liposomal local anaesthetics were formulated such that the drug was added at the time of liposome formation and was either partially encapsulated in the aqueous core or associated hydrophobically, as the free base, with the liposomal bilayer. Liposomes, composed of phosphatidylcholine with or without cholesterol, and the local anaesthetic would then be dissolved in an organic solvent such as chloroform and the solvent subsequently removed under high vacuum.1 3'1 4'3 4'3 5 The dry lipid/local anaesthetic film 13 35 would then be rehydrated with a buffer to form MLVs ' which in some cases were then sonicated to form SUVs. 1 4 ' 3 4 Drug efflux rates from MLVs containing local anaesthetic might be slower than from LUVs because the drug must traverse through the "onion-like" layers of the vesicles to reach the outside. SUVs are small in size compared to MLVs and do not provide much trapped volume with which the local anaesthetic can be hydrophobically associated.14'34 Although these MLVs and SUVs containing local 14 anaesthetics do prolonge the analgesic effect compared to free drug, the liposomal systems employed were relatively unsophisticated, drug encapsulation was relatively insufficient and no mechanism was provided to control local anaesthetic release rates.13'35 Along with the promise of longer duration of action, liposomes may also provide an extended margin of safety.14'34'35 Liposomes, when injected epidurally into rats, remained at the site of injection for over 24 hours.36 Thus, slow release of drug from the liposome depot15 altered the pharmacokinetics of these drugs and significantly prolonged the duration of nerve block and reduced the toxicity.1 4'3 4'3 5 Each system has its benefits and drawbacks; thus, it is difficult to say which of the systems used to prolong the action of nerve blockade is best. Comparisons are not feasible since a variety of local anaesthetics at different concentrations were used, animal models used were not consistent, analgesia was assessed on different scales, and nerve block was performed on different locations. However, taking into account the current technology available, liposomes show the potential to be considered for further study, due to the flexibility of their composition, size, and ability to alter pharmacokinetics. In this thesis, liposomes of a defined size prepared by the extrusion method37 were studied. These large unilamellar vesicles allow for greater quantities of local anaesthetic to be trapped within the aqueous core.19 In previous methodologies, drugs were either hydrophobically associated with the bilayer or passively entrapped in the intravesicular medium. In this research project, a transmembrane pH gradient was employed to promote the uptake of lidocaine, the local anaesthetic under investigation, into the liposomes. Using this technique, drugs which are weak bases or weak acids redistribute in accordance to the 38 Henderson-Hasselbalch equation. This "remote loading" procedure allows for very 15 efficient drug accumulation and represents a mechanism whereby lidocaine release from the carrier can be controlled. 16 2 Uptake of Lidocaine into Large Unilamellar Vesicles in Response to a pH Gradient 2.1 Introduction Previous studies have shown that lidocaine, and many other pharmaceutical agents, can be accumulated into large unilamellar vesicles in response to a transmembrane pH gradient.38'44"47 This technique has been termed "remote loading" because drug uptake occurs into pre-formed liposomes and can be achieved immediately prior to clinical administration. The remote loading method of drug encapsulation has advantages over rehydration of lipid/drug film employed by other research groups. Commonly, the rehydration method yields low drug recoveries, especially since drug is either partially encapsulated in the aqueous core or associated hydrophobically, as the free base, with the liposomal bilayer. Further, drug loss is apparent when the rehydrated lipid/drug mixture is centrifuged and the filtrate is removed during washing; any drug not associated or encapsulated is lost. The remote loading technique of drug encapsulation uses a pH gradient to drive the drug into the aqueous core. Lidocaine (pKa of 7.9) is present in both the protonated and deprotonated forms outside the liposome with the neutral species in equilibrium on both sides of the bilayer. When the interior core is acidic and the exterior is at physiological pH, a pH gradient exists. As a result, deprotonated lidocaine crosses into the aqueous core in its neutral form and once inside becomes protonated because of the acidic interior. The protonated species is impermeable to the bilayer and is concentrated within the liposome (Figure 3). Lidocaine will not be released from the aqueous core until the concentration of lidocaine outside of the liposome is decreased. In addition, the ratio of the protonated and 17 deprotonated species is dictated by the pK a and the pH. This technique effectively sequesters a large proportion of drug into the interior of the liposomes where it is retained until the drug concentration outside the liposome decreases. Lidocaine uptake into large unilamellar vesicles will be examined in this section. In order to provide prolonged analgesia, efficient and stable uptake of lidocaine must be achieved. Techniques to characterize uptake of lidocaine into LUVs include: determination of the trapped volume, to ensure an adequate volume is available within the interior; gel exclusion chromatography and filtration, to separate entrapped lidocaine from free drug so that the percentage of lidocaine entrapped within liposomes may be determined. The transmembrane pH gradient before and after lidocaine uptake was also quantitated to determine the residual gradient. This residual gradient will determine the initial rate of lidocaine efflux from the vesicles following administration. 18 2.2 Materials and Methods 2.2.1 Materials l,2-Dioleoyl-s«-glycero-3-phosphocholine (DOPC) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Cholesterol (standard for chromatography) and lidocaine (>99% pure) were obtained from Sigma Chemical Company, St. Louis, MO. Radiolabeled [carboxy-l4C]-lidocaine hydrochloride (51.7 mCi/mmol), [14C]-methylamine (48 mCi/mmol), [3H]-dipalmitoyl phosphatidylcholine (115 mCi/mmol), and [3H]-cholesteryl hexadecyl ether (51.5 mCi/mmol) were supplied by New England Nuclear (Mississauga, Ontario, Canada). Al l salts and reagents used were of analytical grade. 2.2.2 Preparation of Large Unilamellar Vesicles Mixtures of DOPC and cholesterol (55:45 molar ratio) (including [ H]-cholesteryl hexadecyl ether or [ H]-dipalmitoyl phosphatidylcholine where appropriate) were co-solubilized in benzene:methanol (95:5 v/v), frozen in liquid nitrogen and then lyophilized under vacuum (<100 mtorr) for a minimum of 6 hours, protected from light. The lyophilized lipid mixture was then hydrated with 300 mM citrate buffer, pH 3.0 and dispersed by vortexing. The resulting MLVs were subjected to five freeze-thaw cycles employing liquid nitrogen and subsequent thawing in a water bath at 37°C. This procedure was employed to maximize liposome trapped volume.39 Large unilamellar vesicles (LUVs) were then prepared by extrusion of the frozen and thawed MLVs ten times through two 19 stacked 600 nm pore size polycarbonate filters (Nuclepore Inc.) employing an Extruder (Lipex Biomembranes, Vancouver, B.C.) as described previously.37'40 2.2.3 Generation of the pH Gradient A transmembrane pH gradient was established by dialyzing the LUVs suspension (Spectra Por 2 tubing, moi. wt. cut off 12-14,00) against 500 volumes of 150 mM NaCl, 25 mM HEPES, pH 7.4 (HBS) at 4°C for 48 hours with one change of external solution after 24 hours. Large unilamellar vesicles without a pH gradient were prepared as described above except the lyophilized lipid mixture was hydrated in 150 mM NaCl, 25 mM HEPES, pH 7.4. 2.2.4 Loading of Lidocaine into Liposomes A 20% stock solution of lidocaine was prepared by dissolving lidocaine into distilled water and titrating the solution to pH 7.4. Local anaesthetic was added to LUVs, prepared as described in sections 2.2.2 and 2.2.3, then the samples were incubated at 37°C for 30 minutes. The final drug and lipid concentrations were 85.4 mM (2%) and 100 mM, respectively. 20 2.2.5 Determination of Trapped Volume Large unilamellar vesicles were prepared as described in section 2.2.2, except that the lyophilized lipid mixture was hydrated in 300 mM citrate, 1 mM sucrose pH 3.0 containing [14C]-sucrose (0.3 uCi/ml). Following dialysis against HBS, as described in 2.2.3, the trapped volume was determined and expressed as ul/umole DOPC. 2.2.6 Efficiency of Lidocaine Uptake in Response to a pH Gradient 2.2.6.1 Gel Exclusion Chromatography The kinetics of drug uptake by LUVs exhibiting a pH gradient (pH 3.0 inside/pH 7.4 outside) were determined using LUVs (120 nm av. diameter) prepared by extrusion as described above but using two stacked 100 nm pore size polycarbonate filters. Liposomes of 600 nm will not elute efficiently down a Sephadex G-50 gel exclusion column; therefore, liposomes were reduced to 100 nm and the lipid concentration was lowered from 78.6 mg/ml to 50 mg/ml. The pH gradient was established by passage of the LUVs, prepared in 300 mM citrate buffer, pH 3.0, (50 mg phospholipid/ml) down a Sephadex G-50 (medium) column (1.5 x 10 cm) equilibrated in 150 mM NaCl, 25 mM HEPES pH 7.4. This technique differs from the method described in section 2.2.3. The LUVs suspension (5 mM DOPC) was then incubated at 25 °C with 1 mM or 2 mM lidocaine and at various times aliquots (100 ul) taken. Drug uptake was determined by passage of the aliquot down 1 ml Sephadex G-50 "minicolumns" centrifuged at 2,500 rpm for 3 minutes.41 Phospholipid and drug concentration were determined by liquid 21 scintillation counting for [3H]-DPPC and [14C]-lidocaine employing a Beckman LS 3801 instrument. Uptake kinetics were also determined using LUVs without a pH gradient (pH 7.4 inside/pH 7.4 outside) as a control. 2.2.6.2 Centrifugal Filtration Microcentrifuge filtration tubes, moi. wt. cut off 30, 000 (Costar Scientific Corp., Cambridge, MA), were used to determine lidocaine association with LUVs at high drug and phospholipid concentrations. This procedure allows for undiluted vesicles 600 nm in size to be used, this being an equilibrium method for determining loading efficiency, compared to the non-equilibrium method using minicolumns. Aliquots (lOOpl) were placed in the filtration tubes and centrifuged for 1 hour at 15,000 rpm in a Sorval MC 12V microcentrifuge (DuPont). Lidocaine concentrations in the filtrate were then determined by liquid scintillation counting, as described above, and compared to the initial sample. Control experiments confirmed that no liposomal lipid was present in the filtrate. 2.2.7 Determination of Transmembrane p H Gradients The weak base [14C]-methylamine was used to quantitate transmembrane pH gradients in the presence and the absence of lidocaine using an equilibrium dialysis procedure. Large unilamellar vesicles (100 mM DOPC) exhibiting a pH gradient (pH 3.0 inside/pH 7.4 outside) were incubated at 37°C for 60 minutes in the absence of lidocaine or with 10 mg/ml (1%) or with 20 mg/ml (2%) lidocaine. The weak base [14C]-methylamine was then added to each sample (2 ml) and placed in a dialysis bag (Spectra Por 2, moi. wt. 22 cut off 12-14,000) and dialyzed against 7 ml HBS at 37°C in a shaking water bath. At various times, aliquots of both the liposome suspension and external solution were taken and [14C]-methylamine determined by liquid scintillation counting. 2.2.8 Analytical Procedures Phospholipid concentrations were determined by phosphate assay following perchloric acid digestion.42 Vesicle size distributions were determined by quasi-elastic light scattering using a Nicomp 370 submicron particle sizer as described previously.43 The raw data were fitted to a Gaussian distribution and the analyses weighted on the basis of vesicle volume. 23 2.3 Results 2.3.1 Kinetics of Lidocaine Uptake into LUVs Large unilamellar vesicles (5 mM phospholipid) exhibiting a pH gradient (pH 3.0 inside/pH 7.4 outside) rapidly accumulated lidocaine at concentrations of 1 mM or 2 mM following incubation at 25°C. As illustrated in Figure 4, equilibrium drug uptake levels were seen at the earliest time-point, 5 minutes, and lidocaine uptake was stable over the 90 minute time-course. Liposomal accumulation of the local anaesthetic was very efficient with essentially 100% uptake observed at 1 mM lidocaine (drug to lipid ratio 0.2) and approximately 80%) uptake at 2 mM lidocaine (drug to lipid ratio 0.4). Control experiments were also conducted in which liposomes without a pH gradient (pH 7.4 inside/pH 7.4 outside) were incubated with 1 mM or 2 mM lidocaine. As shown schematically in Figure 3, under these conditions only low levels of the drug are associated with the vesicles. 2.3.2 Centrifugal Filtration An ultracentrifugation method was employed to confirm that similar drug encapsulation efficiencies could be obtained at lidocaine concentrations used clinically. Free and liposomally encapsulated local anaesthetic at 2% lidocaine was analyzed for uptake into 100 mM DOPC:Chol LUVs. Figure 5 illustrates diagramatically the distribution of lidocaine into 600 nm sized liposomes in response to a pH gradient. Liposomes exhibiting a pH gradient encapsulated 76% of 85.4 mM lidocaine for a molar ratio of 0.85. These 24 equilibrium distribution data are in good agreement with the results shown in Figure 4 for 120 nm LUVs incubated with lidocaine at a drugdipid ratio of 0.4. 2.3.3 Transmembrane Lidocaine Distribution Accumulation of lidocaine in response to an imposed pH gradient will result in partial dissipation of the gradient due to protonation of the local anaesthetic with the hydrogen ions in the internal aqueous compartment of the liposome.38 The extent to which the proton gradient is dissipated will be determined by the buffering capacity of the intravesicular medium and the level of drug accumulated. It is this residual gradient that will determine the initial rate of lidocaine efflux from the vesicles following administration and therefore, the transmembrane pH gradient before and after lidocaine uptake was quantitated using [14C]-methylamine as a pH probe.44 As shown in Table 1, [1 4C]-methylamine redistribution indicates a pH gradient of 3.7 units prior to lidocaine uptake (in reasonable agreement with the imposed gradient of 4.5 units) and this decreases to 2.5 pH units and 2.1 pH units following incubation with 1% and 2% lidocaine, respectively. 25 Figure 3. Lidocaine distribution into liposomes with and without a pH gradient. Lidocaine crosses the bilayer in the deprotonated form and is accumulated within the aqueous core in response to a pH gradient. When there is no pH gradient, lidocaine is in equilibrium on both sides of the bilayer. HB + pH7.4 IB4S HB + pH Gradient Liposomes [HB ]taside » [HB ]ontside Non pH Gradient Liposomes [HB ]inside = [HB ]outside Table 1. Influence of lidocaine uptake on residual pH gradient. Lidocaine Concentration Initial pH Gradient pH Gradient after Uptake 1% 3.7 units 2.5 units 2% 3.7 units 2.1 units 26 Figure 4. Lidocaine uptake into liposomes exhibiting a pH gradient. Uptake of local anaesthetic into DOPC:Chol LUVs (5 mM phospholipid) was determined at two lidocaine concentrations, 1 mM (•) and 2 mM (•). In addition, association of lidocaine with control liposomes (no pH gradient) was determined; 1 mM (O), 2 mM (•). O Q_ O Q o E c 0 o c o O <D c 'ro o o -o 400 350 300 U 250 o 200 150 100 50 0 0 10 20 30 40 50 60 Time (minutes) 70 80 90 27 Figure 5. Lidocaine distribution into 600 nm vesicles in response to a pH gradient. The liposome represents the entrapped lidocaine in a 1.0 ml sample of liposomal lidocaine. The interior aqueous core represents 0.3 ml while the exterior volume represents 0.7 ml. 28 2.4 Discussion The uptake studies using gel exclusion chromatography and ultracentrifugation demonstrated that lidocaine is rapidly and efficiently loaded within DOPC:Choi LUVs employing a pH gradient; even at the highest drug concentrations used clinically. When both the external and internal media were of equal pH, very little encapsulation of local anesthetic was seen. However, when the internal environment was more acidic, pH 3.0, and the external environment was pH 7.4, the local anesthetic was efficiently encapsulated into the aqueous core. The local anaesthetic is driven into the liposomes in its deprotonated form with the aid of the proton gradient. Once inside the liposome, the local anaesthetic becomes protonated due to the acidic interior and does not permeate across the lipid bilayer. Drug redistribution in response to a pH gradient appears to be consistent with the concentration differentials predicted by the Henderson-Hasselbalch equation where the ratio of the concentration of drug inside/outside the liposome is equal to the ratio of the proton 38 concentration inside/outside the liposome. In the present work, encapsulation was determined to be efficient; values for percent encapsulation of 2.0% lidocaine into liposomes include 76% for 85.36 mM lidocaine into 100 mM vesicles (649 nmole umole"1 DOPC), 80% for 2 mM lidocaine (320 nmole umole"1 DOPC) and approximately 100% for 1 mM lidocaine into 5 mM vesicles (200 nmole umole" 1 DOPC). Higher drug to lipid ratios exhibited lower efficiencies of drug uptake. When lidocaine enters the liposome it becomes protonated, which raises the internal pH of the vesicle. The rise in pH as lidocaine is accumulated reduces the pH gradient. As a consequence, lower mole ratios of lidocaine are more efficiently encapsulated than larger 29 mole ratios for the same concentration of pH gradient liposomes. Further, a comparison of vesicles exhibiting a pH gradient and those without demonstrates the incredible ability of the pH gradient to drive encapsulation of lidocaine. Control (no pH gradient) liposomes show only very low levels of drug association with vesicles, whereas pH gradient liposomes rapidly and efficiently accumulate lidocaine. There are limitations to using the gel exclusion chromatography method for determining drug uptake. Due to the nature of the column, lower concentrations of lipid and smaller diameter liposomes must be used to study uptake since larger, more concentrated liposomal formulations will be retarded and will not elute efficiently from the column. The data generated from the 100 nm sized vesicles however, may not reflect the actual uptake in 600 nm systems. The trapped volume of 600 nm vesicles was determined to be 3 pi per pmol of phospholipid. This is approximately three fold greater than the trapped volume for 100 nm vesicles. In vesicles with larger trapped volumes, there is a greater aqueous compartment per mole of lipid for the drug to be entrapped. Therefore, at equal molar concentrations, there is a greater sink available for lidocaine storage. In addition, larger trapped volume liposomes will have greater buffering capacities per mole lipid since the aqueous core is larger. The gel exclusion method of drug uptake demonstrated approximately the same efficiency of uptake, even though the drug to lipid ratio was lower than that found by the ultracentrifugation method. The difference can be explained by the fact that when lidocaine-filled LUVs are passed down a gel exclusion column, lidocaine is likely to be released. The design of the liposomes used in these experiments will release lidocaine as more lidocaine is lost outside of the vesicles. As the liposomes pass down the column, lidocaine will be 30 released and lost as free drug is removed as a result of equilibration across the bilayer. This will result in an underestimation of the true efficiency of encapsulation using the gel exclusion method. Regardless, the gel exclusion chromatography method demonstrates the efficient and rapid uptake of lidocaine into pH gradient liposomes. The ability to load large quantities of local anaesthetic into liposomes is one of the key requirements for prolonged analgesia. The ease of preparation is another attribute of the liposomal local anaesthetic system used in these studies. Incubation of pH gradient liposomes, prepared outside the hospital setting, with free local anaesthetic is all that is needed to formulate liposomal lidocaine. [14C]-Methylamine redistribution, as shown in Table 1, can be correlated to the H+/lidocaine stoichiometry. Thus, before uptake of lidocaine, the concentration of hydrogen ions inside the liposomes was approximately 5000 more than that on the outside (take the inverse log of 3.5 pH units). After uptake of 1% lidocaine, the concentration of H + ions was approximately 300 fold greater on the inside and approximately 125 fold greater after 2% lidocaine uptake. This can be correlated to the amount of lidocaine inside the liposomes using the Henderson-Hasselbalch equation: [ H B J i n s i d e = [HJinside [HB ] o u t side [H ]outside where the ratio of the concentration of lidocaine inside/outside the liposome is equal to the proton concentration inside/outside the liposome. Thus, lidocaine concentration inside the liposomes is 300 fold greater for 1% lidocaine and 125 fold greater for 2% lidocaine. 31 3 Release of Lidocaine from Large Unilamellar Vesicles 3.1 Introduction Sustained local anaesthetic delivery is required if prolonged analgesia is desired. To accomplish this, release kinetics of the drug from lipid carrier must be such that therapeutic levels are maintained for the duration of nerve blockade. Such a goal may be attained by utilizing a transmembrane pH gradient. The pH gradient provides a' mechanism whereby drug release rates, and hence duration of action, can be controlled. Using an in vitro model of release, it can be demonstrated that lidocaine release kinetics are determined by the transmembrane pH gradient. In vitro models are used so that interpretation of the in vivo models can be understood without confounding variables such as serum proteins, macrophages, blood components, etc. Lidocaine-loaded vesicles were placed in a dialysis bag and drug efflux was induced by dialyzing the sample against a large excess of HEPES buffered saline solution. Samples were taken of both the liposomal solution and dialysis solution over time to determine the rate of lidocaine release. 32 3.2 Materials and Methods Materials used are as stated in section 2.2.1. In vitro release rates of lidocaine from liposomal systems, subsequent to the procedures described in sections 2.2.2, 2.2.3, and 2.2.4, were determined by placing the drug-loaded vesicles within Spectra Por 2 dialysis tubing (molecular cut off weight =12-14,000) and dialyzing drug-loaded liposomes against 500 volumes of HBS at 37°C. The external solution was exchanged after 4 and 8 hours and at various time-points lidocaine concentrations both within the dialysis bag and in the external solution were determined based on liquid scintillation counting of [14C]-lidocaine. Control experiments also examined the rate of efflux of free lidocaine and lidocaine in the presence of liposomes without a pH gradient. In some experiments nigericin (10 uM) and valinomycin (lOpM) were added to the vesicles to collapse the pH gradient. The influence of plasma proteins on lidocaine release kinetics from pH gradient-loaded liposomes was also determined. DOPC([3H]-DPPC):Chol liposomes were prepared as described in sections 2.2.2, 2.2.3, and 2.2.4. Lidocaine (2%) -loaded pH gradient liposomes were dialyzed in the presence or absence of mouse serum (25% total volume) against 500 ml of HBS, pH 7.4 at 37°C. 33 3.3 Results The kinetics of drug release were examined for LUVs exhibiting a pH gradient. Lidocaine-loaded vesicles were placed in a dialysis bag and drug efflux was induced by dialyzing the sample against a large excess of HBS. Figure 6 shows the rates of lidocaine efflux from control and liposomal formulations, specifically drug-loaded liposomes (pH gradient) and a control whereby lidocaine was incubated with non-pH gradient liposomes (pH 7.4 inside/pH 7.4 outside). As expected, fairly rapid lidocaine efflux was seen from control (no pH gradient) liposomes with less than 10% of the initial drug concentration remaining within the dialysis bag by 8 hours. In contrast, liposomal systems exhibiting a pH gradient show an initial rapid efflux phase during which approximately 50% of the local anaesthetic is released, followed by a much slower phase of lidocaine release. The lidocaine remaining within the dialysis bag at 24 hours represents drug retained as a result of the transmembrane gradient. This can be shown by lidocaine collapsing the gradient with the ionophores nigericin and valinomycin. As shown in Figure 6 (indicated by the arrow), rapid drug efflux occurs once the proton gradient is dissipated. With regard to the free drug, it was rapidly released from the dialyses bag and 100% of the drug was released by 6 hours. Lidocaine release rates from POPC lipid mixtures exhibiting a pH gradient were also examined. As shown in Figure 7, lidocaine release from POPC:Chol LUVs is slightly different to that of DOPC:Chol (cf. Figure 6). The difference in release rate is likely due to differences in bilayer fluidity. Vesicles composed of POPC possess one 16 carbon chain and one 18 carbon chain with an unsaturated bond while DOPC systems are composed of two unsaturated bonds and two 18 carbon acyl chains. The double bonds in DOPC produce a kink in the hydrophobic chains which force neighbouring phospholipids to be further apart. 34 This increases the permeability of the DOPC bilayer; thus, the release rate of DOPC systems are slightly faster that POPC systems. Plasma proteins can interact with liposomes and under certain circumstances can cause leakage of aqueous contents.49'50 Therefore, the influence of serum on lidocaine release from pH gradient-loaded liposomes was examined. As can be seen in Figure 8, the rate of lidocaine release is only slightly increased in the presence of serum. 35 Figure 6. Release kinetics of lidocaine (2%) from DOPC:Chol vesicles in vitro. Lidocaine efflux was followed for samples consisting of liposomes exhibiting a pH gradient (•), control liposomes (pH 7.4 in/pH 7.4 out) ( O ) , and free lidocaine (•). For the pH gradient liposomal sample, valinomycin (10 uM) and nigericin (10 pM) were added at 24 hours (arrow) to collapse the proton gradient. Time (hours) 36 Figure 7. Release kinetics of lidocaine (2%) from POPC:Chol vesicles in vitro. Lidocaine efflux was followed for samples consisting of liposomes exhibiting a pH gradient (•), control liposomes (pH 7.4 in/pH 7.4 out) (•). Figure 8. Influence of serum (25% total volume) on lidocaine release from liposomes exhibiting a pH gradient. Samples consisted of LUVs exhibiting a pH gradient (pH 3.0 in/pH 7.4 out) loaded with lidocaine (2%) and incubated at 37°C in the presence (•), or absence (O) of serum. (0 CD CO CO CD CD rr CD c CO o o "D 10 15 Time (Hours) 38 3.4 Discussion The rate of lidocaine efflux from liposomes was shown to be dependent on the pH gradient. Both the free drug and control (no pH gradient) liposomes released lidocaine much more rapidly than pH gradient liposomal lidocaine. Without a pH gradient, cannot be loaded; therefore, it diffuses freely and quickly from the dialysis bag. However, drug from liposomes exhibiting a pH gradient definitely showed different release rates. The release of lidocaine from pH gradient liposomes consist of two phases: a rapid initial release phase followed by a much slower sustained phase. The initial rapid release phase is due to approximately 30% of the drug which is not loaded, thereby diffusing easily out of the dialysis bag. In addition, when the drug is loaded to the maximum extent , the internal pH is 3.7 pH units; therefore, a greater proportion of the encapsulated lidocaine will be in the unprotonated (membrane permeant) form. This initial rapid release can be very useful as an initial loading dose of local anaesthetic to provide rapid onset of analgesia. The second phase is characterized by a slow sustained release. As lidocaine diffuses away from the external solution surrounding the liposomes, more drug from within the aqueous core crosses the bilayer to replace that which is removed. Drug redistribution in response to a pH gradient appears to be consistent with the concentration differentials predicted by the Hederson-Hasselbalch equation: [HB^] i n s i d e = [H ]inside [HB ] o u t s jde [H Lutside where the ratio of the concentration of lidocaine inside/outside the liposome is equal to the proton concentration inside/outside the liposome. As deprotonated lidocaine leaves the vesicle, a proton is released inside the liposome causing the internal pH to become more 39 acidic. As this process advances, the rate of release slows because the ratio of unprotonated lidocaine over protonated lidocaine decreases and hence the concentration of the membrane permeant species decreases. Thus, lidocaine efflux slows as a result of decreased internal pH leading to reduction of the permeable species. The magnitude of the pH gradient will affect several components of the liposomal system. Altering the internal pH will influence drug uptake and release rates. If the magnitude of the pH gradient is reduced, uptake will be less efficient, and release will occur more readily. As the pH gradient is increased, slower release kinetics will be seen. In addition, highly acidic buffers inside the liposome will catalyse the hydrolysis of ester bonds linking the phospholipid glycerol backbone with the hydrophobic acyl chain. Free fatty acids and lyso-lipids are known to destabilize the bilayer, increase membrane permeability, and decrease formulation shelf life. Therefore, it is advantageous to choose a pH gradient that allows both efficient uptake and reasonable release kinetics to provide a slow, sustained release of lidocaine over an extended period of time. 40 4 Clearance of Lidocaine and Large Unilamellar Vesicles In Vivo 4.1 'Introduction Two critical requirements for providing prolonged analgesia using liposomal local anaesthetic are that the liposomal carrier should be cleared only slowly from the site of administration and that the lidocaine release from the carrier should maintain therapeutic levels of free local anaesthetic over an extended period of time. The clearance rates of liposomes and lidocaine following intradermal administration to guinea pigs were therefore examined. The non-exchangeable, non-metabolizable lipid marker, [ H]-cholesteryl hexadecyl ether, was used to monitor liposome distribution48 while [14C]-lidocaine hydrochloride was used to quantitate the local anaesthetic. 4.2 Materials and Methods Dunkin Hartley guinea pigs were ordered from the Animal Care Facility, University of British Columbia. All other materials as per section 2.2.1. Male Dunkin Hartley guinea pigs (450-500 gm) were housed in pairs and provided with food and water ad libitium. One day prior to the clearance studies, dorsal areas were shaved. The area of the guinea pig back can accommodate five injections to be made per animal. Different formulations of free drug and liposomal lidocaine were administered intradermally using a 27Vi gauge needle. At set time-points, injections of 100 ul DOPC:Chol (100 mM phospholipid) liposomes without lidocaine, lidocaine in liposomes exhibiting a pH gradient, or lidocaine with control (no pH gradient) liposomes were made. 41 The margins of the wheals were marked and the final injections of each sample were made immediately prior to sacrificing the animal in order to obtain a zero time-point. Clearance of the liposomes was followed using [3H]-cholestryl hexadecyl ether, a non-exchangeable, non-metabolizable lipid marker and lidocaine was followed using [14C]-lidocaine hydrochloride. Injection sites were excised, weighed and digested with Solvable (4 ml) at 65°C for 12 hours. Muscle tissue below the injection sites and the dermal layers surrounding the wheal were also dissected in order to confirm that injections were made intradermally and that drug or lipid did not migrate beyond wheal margins. Digested samples were cooled to 4°C and decolourized with 500 pi of hydrogen peroxide (30%), given in two 250 pi aliquots. Samples were then divided into 1 ml aliquots and 15 ml of scintillation fluid was added to the samples. After overnight storage in the dark, the samples were counted using a Beckman LS 3801 scintillation counter. 42 4.3 Results Clearance rates of after liposomes and lidocaine after intradermal administration were examined to determine whether the lipid carrier remains at the site of injection and how rapidly lidocaine diffuses from this site. As shown in Figure 9, greater than 90% of the [3H]-CHE activity remained at the injection site, even after 48 hours. This indicates that the liposomes are cleared only slowly from the wheal sites. Any loss of the lipid carrier occurred within the first eight hours. Lidocaine clearance rates in vivo following administration of either the free drug, local anaesthetic in pH gradient liposomes, or with control liposomes (no pH gradient) are also presented in Figure 9. As would be anticipated based on the in vitro drug release kinetics, administration of liposomally encapsulated lidocaine results in much slower drug clearance rates compared to either free drug or lidocaine co-administered with control liposomes (no pH gradient). Free drug and lidocaine co-administered with control liposomes (no pH gradient) behaved identically with very little local anaesthetic remaining at the injection site after 2 hours. The clearance of liposomally encapsulated lidocaine is similar to the in vitro kinetics of release in that it is comprised of two phases. Again the initial rapid release of drug is seen up to 4 hours at which time over 40% of the administered drug remains at the wheal site. A slower, more sustained release follows and almost all of the drug is released by 48 hours. Based on this model of drug clearance, it might be anticipated that nerve blockade could last up to two days. 43 Figure 9. Clearance of lidocaine and liposomal carrier following intradermal administration to Dunkin Hartley guinea pigs. Clearance rates are shown for the liposomal carrier (•), lidocaine administered in pH gradient liposomes (•), with control (no pH gradient) liposomes (•), and as free drug (O). 100 CO o Q "D CD CO 'E T J < CD O CD t_ CO <D O CD c c o o o "D 80 60 40 20 \ 9 IK - o — a - j _ ii 10 20 30 Time (hours) 40 50 44 4.4 Discussion 51 52 In agreement with reports by Turner et al and Allen et al , very little clearance of liposomes following subcutaneous administration was observed. Liposomes were shown largely to remain at the site of injection with over 90% present 48 hours. Within the first 8 hours, some clearance of liposomes was observed. This is probably due to clearance of liposomes which were much smaller than 600 nm in size. Smaller diameter liposomes have been previously shown to be cleared much more readily than larger systems. There appears to be a cut-off of approximately 120 nm in diameter above which the lipoosmes fail to appear in blood to any significant extent.52 As would be anticipated based on the in vitro and in vivo drug efflux studies, administration of liposomally encapsulated lidocaine exhibiting a pH gradient results in slower drug clearance rates compared to either free drug or liposomal lidocaine without a pH gradient. Interestingly, following intradermal injection, control liposomes do slightly delay lidocaine clearance relative to administration of free drug alone. This effect may reflect lidocaine interaction with the liposomal carrier or decreased diffusion or lymph flow due to the presence of liposomes in the interstitial matrix. 45 5. Duration of Nerve Blockade In Vivo 5.1 Introduction The duration of nerve blockade produced by free and liposomal lidocaine was compared using the cutaneous wheal model in the guinea pig back. This model reduces inter-animal variation since the guinea pig back is large enough to accommodate up to five injections, including a control. Intradermal injections were made and the duration of analgesia was measured. Susceptibility to nerve blockade by local anaesthetics is related to the size and myelination of the nerve fibres. Typically, unmyelinated, small diameter nerve fibres such as type C fibres, which are responsible for pain and temperature, are blocked more readily than lightly myelinated type B fibres and long before myelinated large diameter type A (alpha) nerve fibres, which are responsible for motor function. Fortunately, the sensation of pain is usually the first modality to disappear, then followed by the sensations of cold, warmth, touch and deep pressure, although individual variation is considerable. Anatomical position plays a role in the early stages of anaesthetic action. The fibres in the mantle region are the first to be exposed to the anaesthetic as it diffuses inward, while those in the core are exposed last. Hence, blockade starts with the fibres of the mantle region and then moves inward towards the core. The sensitivity to local anaesthetics is also determined by the distance between nodes. These distances are critical lengths of axons which must be exposed to local anaesthetic before blockade can occur. Narrow fibres, with their shorter internodal distances and thus shorter critical lengths, are blocked before larger fibres. In addition, the use-46 dependent mechanism of local anaesthetics, which block more efficiently at higher frequencies, will dictate which types of fibres are blocked first. Sensory fibres, especially pain fibres, fire at higher frequencies and have longer action potential durations than motor fibres so that they are blocked before motor fibres. In present clinical usage lidocaine is often co-administered with adrenaline to prolong the duration of analgesic action; adrenaline acts as a vasoconstrictor reducing the clearance rate of drug from the injection site. The addition of adrenaline to free drug and liposomal lidocaine samples was examined to determine whether a further prolongation of nerve blockade is possible. 5.2 Materials and Methods Male Dunkin Hartley guinea pigs were acquired from Animal Care Facility, University of British Columbia. Al l other materials as per section 2.2.1. Dunkin Hartley guinea pigs were used to compare the duration of nerve blockade produced by free and liposomal lidocaine. To reduce stress, the animals' backs were shaved the day before the experiment was conducted. Wheals were formed by injecting 100 ul of the sample, using a 27/4 gauge needle, into the intradermal layer of the guinea pigs' back. As a control, a saline or "empty" liposomes sample was also injected into each animal. Wheals were marked and pricked five times using a 18/4 gauge needle attached to a 5 ml syringe. The syringe was placed inside a plastic sheath so that the needle was exposed at one end but could move freely up and down within the sheath. This system ensured that 47 when the needle was touched to the guinea back a constant pressure was applied. Upon application of the needle to the wheal site, a positive response was scored if the back muscle twitched. Inter-animal variation was reduced by testing all four samples on the same animal in each experiment. Each animal was used three times with a minimum 48 hour interval between experiments and wheals were never generated in the same location twice. A random number generator was used to assign the formulations to various regions on the back and to determine the order of testing each wheal. In the first study, intradermal injections consisted of free lidocaine (2%), lidocaine in pH gradient liposomes (100 mM DOPC, 2% lidocaine), and lidocaine with control (no pH gradient) liposomes. The above experiment was repeated using intradermal injections of free lidocaine (2%), free lidocaine with adrenaline (1:200,000), pH gradient liposomal lidocaine (100 mM, 2% lidocaine), and pH gradient liposomal lidocaine with adrenaline (100 mM DOPC, 2% lidocaine, 1:200,000 adrenaline). The person testing the response to probe stimuli was blind to the contents of the sample injected at each site. 48 5.3 Result The guinea pig wheal model was used to compare the duration of nerve blockade resulting from intradermal administration of free lidocaine, lidocaine encapsulated in liposomes exhibiting a pH gradient, lidocaine co-administered with control liposomes (no pH gradient), and free or liposomal lidocaine co-administered with adrenaline. It must be emphasized that in these experiments the same total amount of local anaesthetic was administered for each formulation. While administration of free lidocaine (2%) produces a rapid local anaesthetic effect, this is of relatively short duration with half maximal response being recovered by about 90 minutes (Figure 10). Full recovery was seen by 3.5 hours. Co-administration of lidocaine with control liposomes (no pH gradient) also resulted in rapid onset of nerve block but in this case the duration of drug action was significantly prolonged relative to free drug alone. Recovery of half maximal response was not seen until about 2.5 to 3 hours following injection with full recovery at about 4.5 hours. A further prolongation of local anaesthetic action was seen for lidocaine administered in liposomes exhibiting a pH gradient. Again, this formulation produced a rapid onset of nerve block and half maximal response did not occur until about 4 hours after administration with full recovery seen by 5.5 to 6 hours. When the durations of nerve blockade produced by these three formulations were compared using an ANOVA model, the differences between all three were found to be statistically significant (Table 2). In order to decrease lidocaine clearance rates, this local anaesthetic is often administered with adrenaline. The effects of this vasoconstrictive agent on nerve blockade 49 in the guinea pig wheal model were examined for both free and liposomal drug formulations. As shown in Figure 11, co-administration of adrenaline with lidocaine produced a significantly prolonged effect in comparison to free drug alone. Recovery of half maximal responses was not seen until approximately 3 hours following injection, with full recovery after 5-6 hours. This increase in duration of local anaesthetic action was also seen when adrenaline was co-administered with lidocaine encapsulated in pH gradient liposomes. Recovery of half maximal response was now shifted from 4 hours to about 8 hours (Figure 11). Again as shown in Table 3, the difference is statistically significant, as are the respective comparisons of free and liposomal lidocaine in the presence and absence of adrenaline. The squared multiple r is a number which represents how well the model has been designed to test for differences. This value is indicated for Tables 2, 3 and 4. 50 Figure 10. Duration of nerve blockade produced by free and liposomal lidocaine (2%). Recovery of tactile response was determined following administration of free lidocaine (•), lidocaine in control (no pH gradient) liposomes (•), or lidocaine in pH gradient liposomes (•). Error bars indicate standard error and n = 12. 0 1 2 3 4 5 6 7 8 Time (hours) 51 Table 2. Statistical comparison of neural blockade as a function of lidocaine formulation. Statistical significance was determined for different formulations of lidocaine; free drug, liposomal lidocaine without a pH gradient (pH; & pH 0 = 7.4), and liposomal lidocaine exhibiting a pH gradient (pHj = 3.0, pH 0 = 7.4). 2.0% Lidocaine P Squared Multiple R Free Drug vs. No pH Gradient <0.0005 0.877 Free Drug vs. pH Gradient <0.0005 0.854 No pH Gradient vs. pH Gradient <0.008 0.611 52 Figure 11. Duration of nerve blockade produced by co-administration of adrenaline with lidocaine (2%). Recovery of tactile response was determined following intradermal injection of free lidocaine (•), free lidocaine and adrenaline (•), lidocaine in pH gradient liposomes (•), and lidocaine in pH gradient liposomes with adrenaline (O). Error bars indicate standard error and n = 6. Time (hours) 53 Table 3. Statistical comparison of neural blockade duration as a function of lidocaine formulation. Statistical significance was determined for different formulations of lidocaine; free drug, free drug with adrenaline, liposomal lidocaine exhibiting a pH gradient (pHj = 3.0, pH 0 = 7.4), and liposomal lidocaine exhibiting a pH gradient (pHj = 3.0, pH 0 = 7.4) with adrenaline. 2.0% Lidocaine P Squared Multiple R Free Drug vs. Free Drug with Adrenaline <0.014 0.846 Free Drug vs. Liposomal Lidocaine <0.001 0.932 Free Drug vs. Liposomal Lidocaine with Adrenaline O.0005 0.933 Free Drug with Adrenaline vs. Liposomal Lidocaine <0.185 0.719 Free Drug with Adrenaline vs. Liposomal Lidocaine with Adrenaline O.0005 0.749 Liposomal Lidocaine vs. Liposomal Lidocaine with Adrenaline <0.003 0.69 54 5.4 Discussion When the duration of lidocaine-induced neural blockade is examined in the same model used to compare liposome and drug clearance rates, a correlation is seen between tissue clearance kinetics and duration of analgesia. While half maximal recovery from nerve blockade is seen by about 90 minutes following injection of free lidocaine, this recovery takes 2.5 hours for drug administered with control liposomes (no pH gradient) and about 4 hours for liposomally encapsulated lidocaine. The significant prolongation of neural blockade seen for local anaesthetic with control (no pH gradient) liposomes compared to free drug is, however, correlated with a reduction in clearance rate (Figure 9). This discrepancy likely reflects the different time-frames over which the clearance and neural blockade studies were conducted. At the earliest time-point used for the clearance study (2 hours), recovery from nerve block is largely complete for the free drug. A reliable comparison between free lidocaine and lidocaine in control liposomes would therefore require examination of drug clearance over a much shorter time-period. Further, while the prolongation of local anaesthetic action by liposomes exhibiting a pH gradient is impressive, inspection of the drug clearance data for this formulation reveals that a significant fraction (40%) of the administered lidocaine is still present at the injection site at 8 hours. This suggests that drug release from the liposomal carrier may not be sufficiently rapid to maintain therapeutic levels of free drug and hence neural blockade. By increasing the rate of lidocaine release at these latter time-points; therefore, it should be possible to further prolong the duration of analgesia. Such an increased rate of drug efflux could be achieved, 55 for example, by reducing the buffering capacity of the intravesicular medium and hence the magnitude of the residual pH gradient following lidocaine uptake. Clinically, local anaesthetics are often co-administered with adrenaline to increase their duration of action. This vasoconstrictive agent reduces blood flow at the injection site and thereby decreases the rate of local anaesthetic clearance. As shown in Figure 11, this effect results in an approximately two-fold increase in the duration of action of free lidocaine. A similar increase in duration of neural blockade is seen when adrenaline is co-administered with liposomal lidocaine. Interestingly, it has been shown previously that adrenaline can also be accumulated by liposomes in response to a pH gradient.45 It appears likely, therefore, that a proportion of the added adrenaline will be taken up by the lidocaine-loaded vesicles on initial mixing. In view of the relativly low molar concentration of this vasoconstrictive agent, however, it would not be expected to significantly displace lidocaine from the vesicles. Further, as the concentration of adrenaline in the interstitial medium was reduced, efflux from the vesicles would occur in the same manner as for lidocaine. This system, therefore, provides sustained release of both the local anaesthetic and vasoconstrictor. In the guinea pig back, it is uncertain which fibres responded first to the applied stimulus when the anaesthetic wore off. Touch and pressure will elicit a muscle twitch response, indicating stimulation of type A fibres. However, these fibres are the slowest to be blocked and the first to recover when anaesthetic wears off; therefore, pain fibres, which are blocked at much lower concentrations and are slower to recover, might still be blocked even though the guinea pig can feel touch and pressure. This leads to an underestimation of "pain" block. 56 6 Influence of Size on Liposome Clearance and Efficacy 6.1 Introduction From the results of the in vitro release kinetics, a significant amount of local anaesthetic remained within the liposomes after 24 hours. Further, as much as 40% of the administered lidocaine was still present at the injection site after 8 hours in the drug clearance studies, yet full response from the nerve blockade was recovered between 5-6 hours. As a result of this finding, a formulation that would allow for faster delivery of the local anaesthetic between 2 and 10 hours was examined. The influence of size on liposome clearance was determined using 200 nm vesicles and comparing it to the clearance of 600 nm vesicles. The duration of nerve blockade produced by lidocaine encapsulated into 200 nm vesicles ) was examined using the guinea pig wheal model and compared to the efficacy of 600 nm vesicles. It was hoped that faster drug delivery between 2 and 10 hours would result in increased duration of nerve blockade compared to the 600 nm vesicles. 6.2 Materials and Methods Liposomes were prepared as per sections 2.2.2, 2.2.3, and 2.2.4, except two stacked 200 nm pore size polycarbonate filters were used to extrude the 200 nm vesicle sample. Clearance studies were conducted as described in section 4.2 and comparison of nerve blockade elicited by 200 nm and 600 nm vesicles were conducted as described section 5.2. 57 6.3 Results The clearance of 200 nm vesicles was monitored using the guinea pig cutaneous wheal model. Very little clearance of "empty" liposomes was observed, even after 48 hours greater than 85% of the [3H]-CHE activity remained at the site of administration, which is comparable to the results in section 4.3. As shown in Figure 12, the clearance of lidocaine (encapsulated in 200 nm liposomes) from the injection site is much faster than that seen for drug administered in 600 nm vesicles (Figure 9). At 8 hours approximately 20% remains at the wheal site compared to about 40% for the larger liposomal formulation. More drug is cleared as a result of the decrease in size of the liposome which decreases the trapped volume available to encapsulate the local anaesthetic. While co-administration of lidocaine with 200 nm control liposomes (no pH gradient) produces a rapid local anaesthetic effect, this is of relatively short duration with half maximal response being recovered by about 90 minutes (Figure 13). Full recovery was seen by 3.5 hours. These results are in very good agreement with the results from administration and efficacy of free lidocaine (2%) previously demonstrated in section 5.3. Control liposomes (no pH gradient) of 600 nm size co-administered with lidocaine slightly prolonged the duration of nerve blockade compared to the 200 nm control sample. This effect may reflect lidocaine interaction with the larger liposomal carrier. Surprisingly, a further prolongation of local anaesthetic action was not seen for lidocaine administered in 200 nm liposomes exhibiting a pH gradient. Again, this formulation produced a rapid onset of analgesia but half maximal response recurred at 90 minutes. Full recovery, however, was considerably prolonged and did not occur until 6 hours. Again, an impressive prolongation 58 of analgesic action was seen for lidocaine administered in 600 nm liposomes exhibiting a pH gradient. This formulation produced rapid onset of nerve block with recovery of half maximal response seen at 4.5-5 hours. Full recovery was seen between 7 and 8 hours. Using an ANOVA model, the differences between pH gradient 200 nm liposomes and pH gradient 600 nm liposomes were found to be statistically different (Table 4). 59 Figure 12. Clearance of liposomal lidocaine (2%) from 200 nm vesicles. Clearance rates are shown for the 200 nm liposomal carrier (•) and for lidocaine administered in pH gradient liposomes of 200 nm size (O). Error bars indicate standard error and n = 6. Figure 13. Comparison of nerve blockade using 200 nm and 600 nm sized vesicles. Recovery of tactile response was determined following administration of free lidocaine (•), lidocaine in control (no pH gradient, 200 nm sizedliposomes (O), lidocaine in control (no pH gradient, 600 nm sized) liposomes (•), lidocaine in pH gradient 200 nm liposomes (•), and lidocaine in pH gradient 600 nm liposomes (A). Error bars indicate standard error and n = 9. 0 1 2 3 4 5 6 7 8 Time (hours) 61 Table 4. Statistical comparison of neural blockade duration as a function of lidocaine formulation. Statistical significance was determined for different formulations of lidocaine; free drug, liposomal lidocaine exhibiting a pH gradient (200 nm vesicles), liposomal lidocaine without a pH gradient (200 nm vesicles), liposomal lidocaine exhibiting a pH gradient (600 nm vesicles), and liposomal lidocaine without a pH gradient (600 nm vesicles). 2.0% Lidocaine P Squared Multiple R Free Drug vs. 200 nm pH Gradient Liposomal Lidocaine <0.977 0.352 Free Drug vs. 600 nm pH Gradient Liposomal Lidocaine <0.028 Free Drug vs. 200 nm No pH Gradient Liposomal Lidocaine <0.437 Free Drug vs. 600 nm No pH Gradient Liposomal Lidocaine <0.977 200 nm pH Gradient Liposomal Lidocaine vs. 600 nm pH Gradient Liposomal Lidocaine <0.03 200 nm pH Gradient Liposomal Lidocaine vs. 200 nm No pH Gradient Liposomal Lidocaine <0.42 200 nm pH Gradient Liposomal Lidocaine vs. 600 nm No pH Gradient Liposomal Lidocaine <0.953 600 nm pH Gradient Liposomal Lidocaine vs. 200 nm No pH Gradient Liposomal Lidocaine O.004 600 nm pH Gradient Liposomal Lidocaine vs. 600 nm No pH Gradient Liposomal Lidocaine <0.026 200 nm No pH Gradient Liposomal Lidocaine vs. 600 nm No pH Gradient Liposomal Lidocaine <0.455 62 6.4 Discussion Liposomes of 200 nm size were shown to remain at the site of administration, as was seen previously with 600 nm vesicles. Any loss of liposomes was probably due to clearance of smaller vesicles present within the Gaussian population. The clearance of liposomally encapsulated lidocaine is similar to the in vitro kinetics of release in that it is comprised of two phases. Again the initial rapid release of drug is seen up to 4 hours with less than 20% of the drug remaining at 8 hours. A slower more sustained release follows and almost all of the drug is released by 48 hours. With both studies on drug clearance and the study on nerve blockade, it would be anticipated that nerve blockade using lidocaine administered in 200 nm liposomes exhibiting a pH gradient would surpass the prolongation produced by the 600 nm system. Interestingly, 200 nm vesicles containing lidocaine did not result in a prolongation of analgesia compared to 600 nm vesicles. Although recovery of half maximal response occurred very early for lidocaine administered in 200 nm liposomes exhibiting a pH gradient, full recovery was seen only after a time interval similar to that of the 600 nm liposome sample (6 hours). This modest prolongation of nerve blockade cannot be a consequence of liposome clearance from the site of administration since greater than 85% of the 200 nm sized vesicles remained at the injection site. It is probable that less anaesthetic is taken into the pH gradient liposomes because of the smaller trapped volume of the 200 nm vesicles. Control liposomes (no pH gradient) co-administered with lidocaine (2%) for 600 nm sized vesicles did prolong nerve blockade compared to free drug and 200 nm vesicles co-administered with 2% lidocaine. 63 7 Toxicity 7.1 Introduction Local anaesthetics are relatively safe and are widely used to provide pain relief. However, accidental intravenous injections of these agents can cause severe CNS toxicity (the most common side effect), cardiotoxicity or even death due to cardiac failure. Local anaesthetic-induced cardiovascular depression occurs less frequently but tends to be more serious and more difficult to manage. In general, local anaesthetic potency is directly related to its lipid solubility, which dictates toxic effects associated with the local anaesthetic. Bupivacaine, for example, exhibits greater cardiotoxicity compared to lidocaine as a result of its high lipid solubility and sodium channel blockade in the heart. Lidocaine, which is less lipid soluble than bupivacaine, dissociates rapidly from sodium channels while bupivacaine dissociates much more slowly or incompletely from resting channels. The y-aminobutyric acid (GABA) receptor complex controls chloride channels at the synaptic cleft and GABA opens channels to hyperpolarise membranes and cause an inhibition of transmission. Lidocaine, however, closes channels and potentiates transmission, leading to convulsions. Signs of CNS toxicity due to local anaesthetics include minor symptoms such as numbness of the tongue and mouth and lightheadedness, and can be as severe as grand mal convulsions, coma, and apnoea. In the previous sections, it has been demonstrated that liposomal systems which allow slow release of local anaesthetic provide a prolonged duration of neural blockade relative to either free drug or liposomal systems without a proton gradient. Along with the 64 advantage of longer duration of action, liposomal local anaesthetics may also provide an extended margin of safety. The design of the liposome delivery system is intended to alter the normal biodistribution of the free drug by slowly releasing the local anaesthetic over a prolonged period of time. Thus, the drug would be expected to be cleared or metabolized before the parent compound can accumulate to toxic levels when released at a sufficiently slow rate. Acute toxicity produced by three different formulations of lidocaine was evaluated after administration by the intraperitoneal route. Injections made by the intraperitoneal route result in the anaesthetic entering the portal system and passing through the liver, before reaching the systemic circulation. The intraperitoneal route of administration was used to determine toxicity instead of the intravenous route because we can determine the onset of symptoms using this model. The onset of convulsions and deaths are too rapid to record using the i.v. route of administration. Both the convulsive dose (CD 5 0) and the lethal dose (LD 5 0) were determined by regression analysis. 65 7.2 Materials and Methods See sections 2.2.1, 2.2.2, 2.2.3, and 2.2.4. Liposomally encapsulated lidocaine (pH gradient) was compared to control (no pH gradient) vesicles, control "empty" liposomes and free drug. Lidocaine dose ranges used included 75 mg/kg to 155 mg/kg for free drug, 100 mg/kg to 200 mg/kg for control non-pH gradient liposomal lidocaine, and 150 mg/kg to 250 mg/kg for pH gradient liposomal lidocaine. Female CD-I mice from Charles River, Quebec were given intraperitoneal injections to determine the dose at which 50% of the sampled mice convulsed (CD 5 0). Subsequent to convulsions, the mice frequently died because the convulsive dose is close to the lethal dose; therefore, the lethal dose (LD 5 0) was also determined if it could be obtained from the data. Injections were completed between the hours of 10 am to 1 pm to avoid • • 53 circadian rhythm effects. The mice were observed for a minimum of 30 minutes post-injection to score any adverse effects. If at this time the mice were not fully recovered, they remained under observation until they appeared normal. Al l survivors of the experiment were observed for at least 24 hours to determine whether or not any delayed toxicities occurred. Convulsions and righting motions were distinguished according to the criteria stated by deJong and Bonin.metabolites 5 4 The onset of ataxia, aimless running, respiratory distress and time of recovery or death were recorded. In addition, convulsions were graded according to their severity. The experiment was conducted using a randomized, double blind protocol to minimize experimental bias. Each mouse was given a number on its tail and randomly selected for injection using a random number generator. Both the order of injection and the 66 type of sample given were randomized. In addition, the person scoring the mice for convulsions was blind to the formulation administered. Statistical analysis of the different formulations of lidocaine was performed using the Spearman-Karber method. This method uses the dose-response curve generated by the data to calculate the effective dose at which 50% of the sample subjects respond. From this method, the CD 5 0 , L D 5 0 and the 95% confidence intervals were determined. The 95% confidence intervals are not presented in log units; therefore, they are not equal on both sides of either the C D 5 0 or L D 5 0 values. Further, regression analysis of the probit plots was undertaken and these values are presented in the Results section. 7.3 Results As shown in Figures 14 and 15, no toxicity was observed for liposomal lidocaine even at doses of 250 mg/kg. In comparison, the C D 5 0 for the free drug and no pH gradient liposomal lidocaine was calculated as 84.4 mg/kg and 149.6 mg/kg, respectively, from Figure 15 by regression analysis. Thus, liposomally encapsulated lidocaine provides a much larger margin of safety compared to free drug or lidocaine with control (no pH gradient) liposomes. Control liposomes did not elicit any toxicities and mice were observed to be cleaning and grooming themselves normally. The onset of toxicity was approximately 3 minutes for higher doses of free drug and close to 9 minutes for the lowest dose (75 mg/kg). The types of responses observed for free drug progressed from dragging of front and hind 67 paws to loss of balance, ataxia, aimless running, and finally convulsions. Table 4 summarizes the observed results of the mouse toxicity study. Similarily, where it is possible to determine, from the available data, the drug doses at which lethality is observed, lidocaine encapsulated in pH gradient liposomes was much less toxic than either free drug or lidocaine with control (no pH gradient) liposomes. The L D 5 0 for free drug, as calculated from Figure 16, was 125.1 mg/kg. This is in good agreement with the value of 133.1 mg/kg reported by deJong and Bonin.5 4 Although convulsions were elicited at 149.6 mg/kg, samples of no pH gradient liposomal lidocaine did not result in death for 5 out of 8 mice until the highest dose at 200 mg/kg. Thus, the L D 5 0 for this formulation of lidocaine is calculated as 196.1 mg/kg, using the Spearman-Karber method for determining L D 5 0 . As might be anticipated from the convulsion data, pH gradient liposomal lidocaine did not cause death even at doses of 250 mg/kg. This remarkable decrease in toxicity as a result of liposomal encapsulation of local anaesthetic would clearly be of considerable benefit in the clinical setting for pain management. Surviving mice kept for observation post-injection were all healthy after one week. Toxicity due to the local anaesthetic lidocaine was dramatically reduced as a result of liposomal encapsulation. The range of doses for lidocaine was chosen to encompass reported doses which cause convulsions and death. The mice behavednormally, cleaning and grooming themselves. Table 4 summarizes the results of different formulations of 2.0% lidocaine. 68 Figure 14. Comparison between free drug, no pH gradient liposomal lidocaine and liposomal lidocaine exhibiting a pH gradient versus percent convulsions. Convulsions were scored after intraperitoneal administration of free lidocaine (•), no pH gradient liposomal lidocaine (•), and liposomal lidocaine exhibiting a pH gradient (A). 75 100 125 150 175 200 225 250 Dose (mg/kg) 69 Figure 15. Probit of convulsion activity versus log dose of 2% lidocaine. Regression analysis was used to determine the C D 5 0 for free lidocaine (•) and no pH gradient liposomal lidocaine (•). The C D 5 0 for liposomal lidocaine exhibiting a pH gradient was not determined because mice given this formulation did not convulse. 70 Figure 16. Comparison between free drug, no pH gradient liposomal lidocaine and liposomal lidocaine exhibiting a pH gradient versus percent death. Percentage death was determined after administration of free lidocaine (•), no pH gradient liposomal lidocaine (•), and liposomal lidocaine exhibiting a pH gradient (A). 71 Figure 17. Probit of death versus log dose of lidocaine. The L D 5 0 was determined using regression analysis for free lidocaine (•). The L D 5 0 for no pH gradient liposomal lidocaine was not determined because death was not observed until the last dose (200 mg/kg) in 5 out of 8 mice. The L D 5 0 for liposomal lidocaine exhibiting a pH gradient was not determined since none of the mice died even at the highest dose of 250 mg/kg. 72 e '5 u o e "5 E im a 3 to °E > O • M <D 3 •o S3 CU TS "O s es tn S O 3 >• C o w "O in £i O irl <u 2 es H c c IO CD cn IO c , c IO I * co o c o IO 00 CN cn cn oo CN oo CN O m o m CN cn CN m 00 in m CN CN 00 m r -0fj| CD co i n o oo o II cu c 100 C i 6 00 cn wo cn cn cn m vo vo CN m OO 00 c o o 00 CD CU u. 00 00 00 ON I I in cn m m m vb ^ —( vo VO 00 00 o o m CN O m in o o cu g '3 o o B o co O ex c 1— O ex o 00 00 00 o m in 0 m O r- 0 CN m CN CN CN g "3 O o -0 s o cn O ex c a t-. O ex 73 7.4 Discussion Liposomal lidocaine exhibiting a pH gradient has been shown to be superior in terms of reduced toxicity compared to free drug and no pH gradient liposomal lidocaine. The reason for the diminished lethality is twofold: first, a large portion of the local anaesthetic is encapsulated within liposomes; second, the local anaesthetic is released at a slow rate. These conclusions are consistent with previous sections on liposomal lidocaine which demonstrated slow release of local anaesthetic for in vitro and in vivo studies. It can be speculated that the reduced toxicity would be a result of the slow rate of release such that free drug is cleared or transformed into safer before the parent compound can accumulate to toxic levels. The C D 5 0 value for free lidocaine, 84.4 mg/kg, falls between literature values ranging from 64 mg/kg to 100 mg/kg. Differences in C D 5 0 values might be a result of age, sex, strain and the time of day at which injections were made.54 In accordance with deJong and Bonin5 4, convulsions were assessed as fast movements of the limbs where the mouse is stiff compared to the righting movement where the mouse looks as if it is running but its body is limp. Again, one would expect the free drug and no pH gradient samples to behave in the same manner. However, there are differences in the C D 5 0 and the L D 5 0 values for these formulations. It is believed that the differences are due to non-specific binding of the drug to the liposomes, thereby slightly reducing the toxicity for the no pH gradient liposomal lidocaine formulation. The L D 5 0 in mice given intraperitoneal injections of free lidocaine was determined to be 125.1 mg/kg, which is similar to deJong and Bonin's value of 133.1 mg/kg and falls 74 within the range of literature values from 120 to 180 mg/kg. The maximum dose of pH gradient liposomal lidocaine (250 mg/kg) did not induce convulsions nor was it lethal, since no animal receiving liposomal lidocaine convulsed or died; thus, both the C D 5 0 and the L D 5 0 could not be determined. If the C D 5 0 for free lidocaine was hypothesized to be greater than 250 mg/kg, comparing the C D 5 0 values for free lidocaine to liposomal lidocaine (pH gradient), it is clear there is a greater that threefold increase in the amount of liposomal lidocaine which can be given safely in contrast to free drug. This noteworthy increase in safety for administration of local anaesthetic to control pain is a result of sequestration of the drug into liposomes. Presumably, only a small portion of the local anaesthetic enters the systemic circulation as a result of the slow, constant release of local anaesthetic from liposomes coupled with metabolism by hepatic enzymes after intraperitoneal injection. The amount of local anaesthetic which is released can be cleared or metabolized without any adverse effects. As was seen with the pH gradient liposomal formulation used in this study, there was a significant reduction in acute toxicity compared to free drug. Prolonged analgesia with reduced toxicity is of considerable importance in the clinical management of pain. This study has demonstrated a significant reduction of toxicity in a murine model using pH gradient liposomal local anaesthetic. Previous sections have shown that slow liposomal release of lidocaine produced a sustained analgesic effect after administration of a single dose, and I have now demonstrated that systemic local anaesthetic toxicity is greatly reduced. These findings might be of help in the armament to control pain, such that, long-lasting post-operative analgesia could be achieved without sophisticated infusion pumps or labour intensive efforts by hospital staff. 75 8 Summary The studies presented in this thesis illustrate the use of liposomes as carriers for the local anaesthetic, lidocaine. Liposomes exhibiting a transmembrane pH gradient provide a convenient technique for drug encapsulation and a mechanism whereby lidocaine release from the carrier can be controlled. This results in a system which may have a significant therapeutic advantage for long term neural blockade and reduced toxicity as compared to free drug or liposomes without a pH gradient. Liposomes exhibiting a pH gradient have been demonstrated to rapidly and efficiently encapsulate HdocaineEncapsulation is achieved simply by mixing and incubating the drug and liposomes together. This technique is termed "remote loading" because drug uptake occurs into pre-formed liposomes and can be achieved immediately prior to clinical administration. Uptake of lidocaine at clinical concentrations (2%) was rapid and stable for pH gradient liposomes with 76% of the local anaesthetic encapsulated. In contrast, there were only very low levels of drug association in control experiments where no pH gradient liposomes were used. The remote loading procedure represents a means whereby lidocaine release from the carrier can be controlled. The concentration of free lidocaine outside the liposomes will be dictated by the Henderson-Hasselbalch equation allowing maintenance of a fairly constant free drug concentration at the nerve site. As local anaesthetic diffuses away from the injection site or is carried away via the local vasculature, more drug will be released from the liposomal carrier to maintain the equilibrium transmembrane drug gradient. As long as the pH gradient is stable, this constant drug release should continue until the intravesicular 76 local anaesthetic pool is depleted. Liposomal systems exhibiting a pH gradient demonstrated a rapid efflux phase during which approximately 50% of the local anaesthetic was released, followed by a much slower phase of release. Drug remaining within liposomes was shown to be a result of the transmembrane gradient; thus, transmembrane pH gradients can be adjusted to control drug release rates. The main objective of this research project was to develop a liposomal local anaesthetic preparation to provide prolonged analgesia in the management of pain. Using the guinea pig wheal model, it was demonstrated that pH gradient liposomal lidocaine produced the longest blockade. Recovery of half maximal responses was not seen until 4 hours, whereas half maximal response for free drug was seen by 90 minutes. This impressive prolongation of analgesia was also seen for liposomal lidocaine with co-administration of adrenaline. Clinically, adrenaline is used as a vasoconstrictive agent to decrease the loss of lidocaine from the injection site. The design of the liposomal system allows for the encapsulation of both lidocaine and adrenaline; therefore, sustained slow release of adrenaline and the local anaesthetic can be achieved. This effect results in an approximately twofold increase in duration of analgesic action when adrenaline is co-administered with liposomal lidocaine. Alhough the prolongation of analgesia is convincing from the in vivo animal studies, results of the in vivo release kinetics illustrate a significant amount of local anaesthetic remaining after 24 hours. A formulation that would allow for faster delivery of the local anaesthetic was examined by investigating the influence of size on liposome clearance. It was observed that both 200 nm and 600 nm vesicles remained at the site of injection, while lidocaine was released more rapidly from the 200 nm vesicles. In addition to releasing 77 lidocaine more quickly, smaller diameter LUVs are less osmotically sensitive than larger vesicles and would therefore be more durable. It was hoped that the smaller vesicles with their faster release of local anaesthetic would result in a prolongation of nerve blockade. However, it was demonstrated using the guinea pig wheal model that pH gradient liposomal lidocaine encapsulated within 200 nm vesicles is no different than free drug (p < 0.977) according to Fisher's statistical test, with respect to analgesic duration. Not only do pH gradient liposomes increase the duration of anaesthetic action, they also provide an extended margin of safety. The maximum dose of pH gradient liposomal lidocaine tested did not induce convulsions nor was it lethal. This is a considerable improvement in terms of acute toxicity compared to free drug which had a C D 5 0 of 84.4 mg/kg and a L D 5 0 of 125.1 mg/kg. The decrease in lethality comes as a result . of a large proportion of the drug being encapsulated and the drug being released at a slow rate. This presumably allows for clearance or transformation of the free drug into safer metabolites at a rate which the hepatic system can handle. The objectives set forth for development of a liposomal local anaesthetic with prolonged duration of action and decreased toxicity have been accomplished in this thesis. However, several areas of this research project warrant further investigation. Firstly, studies using different lipid compositions should be investigated to determine if the duration to action can be extended still further. It was demonstrated that a large portion of local anaesthetic remained within the liposomes, yet full twitch response was recovered. A bilayer with more permeable characteristics and a larger trapped volume might provide 78 additional benefits, given that smaller vesicles did not seem to prolong the duration of nerve blockade as desired. The guinea pig wheal model is a good model to use since it allows for several formulations to be tested in a single animal, thus reducing inter-animal variation. However, one major disadvantage is the uncertainty as to which class of nerve fibres are being blocked. Studies investigating blockade of the sciatic nerve would resolve such a problem. The sciatic nerve model is more widely accepted as a standard for nerve blockade. The relative contributions of sensory and motor blockade can be determined by using the paw pinch to distinguish sensory blockade. Motor nerve blockade can be determined by the ability of the experimental animal to use the anaesthetized leg to grab and climb upon a metal mesh. It would be interesting to determine whether the present liposomal system can selectively prolong sensory nerve blockade as compared with motor nerve blockade. The work presented in this thesis demonstrates a significant prolongation of analgesia with an impressive reduction of toxicity using pH gradient liposomal lidocaine. These factors are of utmost importance in the treatment of pain in the clinical setting and might be of help in the armament to control pain. 79 9 Bibliography 1. Ritchie JM, Greene NM: General pharmacology of local anesthetics. In: The Pharmacological Basis of Therapeutics (8th edition). Edited by Goodman Gilman A, Rail TW, Nies AS, Taylor P. New York, Pergamon Press, 1990, pp 311-331. 2. Koller C: On the use of cocaine for producing anaesthesia on the eye. Lancet 1884; 2:990. 3. Khun S, Cooke K, Collins M , Jones JM, Mucklow JC: Perceptions of pain after surgery. Br Med J 1990; 300:1687-1690. 4. Simcock MJ: Bupivacaine for regional analgesia in labour. 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Moore PA: Circadian variations in the convulsant response and pharmacokinetics of lidocaine. Thesis, University of Pittsburgh, 1977 Taken from deJong and Bonin, Anesth Analg 1980; 59(6):401-405. 54. deJong RH, Bonin JD: Deaths from local anesthetic induced convulsions in mice. Anesth Analg 1980; 59(6):401-405. 55. Wieldling S: Xylocaine: the pharmacological basis of its clinical use. (2nd edition) Stockholm. Almqvist & Wiksell, 1964 Taken from deJong and Bonin, Anesth Analg 1980; 59(6):401-405. 84 

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