HK1014881B - Epidural administration of therapeutic compounds with sustained rate of release - Google Patents

Description

Epidural administration of therapeutic compounds with a delayed release rate

Scope of the invention

The present invention relates to the controlled release of therapeutic compounds from drug delivery systems. More particularly, the invention relates to the epidural administration of therapeutic compounds from liposomal formulations at a delayed release rate. The invention also relates to a method of placing an epidural catheter in a living vertebrate.

Background

Post-operative pain control is a serious problem for patients and physicians, especially in recovery rooms, when the patient is recovering from anesthesia. Systemic administration of large doses of opioids to control pain may cause life-threatening respiratory depression. On the other hand, under-or late-dose post-operative analgesic drug therapy can lead to patient recovery during intolerable severe pain. Furthermore, poor control of post-operative Pain after abdominal or thoracic surgery inhibiting the ventilatory movements of the chest wall, abdomen and diaphragm has been shown to lead to atelectasis (P.R. Bromage, Textbook of Pain, P.D. wall, et al (Eds.): Churchill Livingstone, 1989, pp 744-753).

The presence of opiate receptors in the spinal cord was found in the seventies. After the initial clinical efficacy report in 1979 (M.Behar et al, Lancet 1: 527- & 529, 1979), epidural administration of opioids has become very popular for post-operative pain control (T.I.Ionescu et al, Act.Anaesth.Belg.40: 65-77, 1989; C.Jayr et al, Anesthesiology 78: 666- & 1993; S.Lurie, et al.European Journal of Obtristics and Gynecology and reproduction Biology 49: 147- & 153, 1993). Epidural administration of opioids has the advantage of achieving good local anesthesia at the spinal level without losing control of movement or vasomotor or reducing the degree of wakefulness.

Injectable opioids are widely used postoperatively and postnatally epidural. Post-operative and post-partum Pain usually persists for several days, however, injectable opioids have a short duration of action (W.G.Brose et al, Pain 45: 11-15, 1991; R.H.Drost et al, Arzneim-Forsch/Drug Res.38: 1632-. Thus, continuous or repeated infusion (J.W.KWan, am.J.Hosp.pharm.47(Suppl 1): S18-23, 1990; J.S.Anulty, International animal Medicine Clinics 28: 17-24, 1990; R.S.Sinatra, the Yale Journal of Biology and Medicine 64: 351-. In addition, repeated bolus injections (bolus injections) or continuous infusions can lead to respiratory depression.

Late phase onset of respiratory depression and asphyxia are the side effects of greatest concern in early studies (P.R. Bromage, Anesthesia and Analgesia 60: 461-. A recent prospective non-randomized study in 1085 patients undergoing thoracic, abdominal or orthopedic surgery estimated a "respiratory depression" rate of 0.9% following epidural morphine use (R. Stenseth et al, Acta Anaesthesio.Scand 29: 148-156, 1985). In contrast, the incidence of "life-threatening respiratory depression" was 0.9% in 860 patients given systemic morphine (PO, IV, IM, SC) (r.r. miller et al, drugs in hosptilized patents john Wiley & Sons, New York, 1976). Prospective, randomized studies comparing epidural to systemic administration of opioids (IM or IV) in high risk patients have shown that postoperative pain control with epidural opioids produces superior analgesic effects with reduced postoperative complications (N.Rawal et al, Anesth.Analg.63: 583. cake 592, 1984; MP.Yeager, et al.Anesth.60: 729. cake 736, 1987).

Intrathecal, subcutaneous and intraperitoneal administration in vitro and in animals and intrathecal administration in patients have been demonstrated to provide delayed release of various therapeutic agents after liposome (e.g., multivesicular liposomes) incorporation (S.Kim et al, J.Clin.Oncol.11: 2186. 2193, 1993; V.Ruspack et al, Ann neurol.34: 108. 112, 1993; and M.C.Chamberlain et al, Arch.neurol.50: 261. 264, 1993). However, delayed release of an epidural administered compound is heretofore unknown in the art.

Thus, there is a need for new and better methods of epidural administration of opioids and other therapeutic compounds in single doses in order to achieve effective therapeutic levels of delayed release rates. The present invention addresses the limitations of the prior art by providing an extended release formulation of a therapeutic agent, such as an opioid, which produces the greatest analgesic effect immediately after a single dose is given epidurally and gradually decreases in analgesic effect over the next few days.

Description of the drawings

Figure 1 is a set of four curves recording the analgesic effect in rats at a time after a dose of 10, 50, 175, or 250 μ g (from top to bottom panels) of disposable epidural morphine sulfate (DTC401) (open circles) or free morphine sulfate (filled circles). The analgesic intensity is expressed as the "percentage of maximum possible analgesic effect (% MPA)". Each data point represents the mean and Standard Error (SEM) of the mean of 5 or 6 animals.

Figure 2 is a graph showing the peak dose-response curve of analgesia measured in rats after a single epidural administration of DTC401 (open circles), free morphine sulfate (closed circles), or after a single subcutaneous administration of free morphine sulfate (closed squares). Mean peak% MPA ± SEM were obtained from 5 or 6 animals.

Figure 3 is a graph comparing the total analgesic effect [ as measured by area under the analgesic-time curve (AUC) ] of rats after topical administration of DTC401 (open circles) or free morphine sulfate (filled circles) to disposable hard films. Each data point represents the mean and Standard Error (SEM) of the mean of 5 or 6 animals.

FIG. 4 is a set of graphs comparing percent oxygen saturation of hemoglobin (SpO) in rats at times after administration of DTC401 (open circles) or free morphine sulfate (closed circles) in disposable hard films at doses of 10, 50, 175, 1000 or 2000 μ g (from upper to lower panels)₂) Five curves of (2). Each data point represents the mean of 5 animals and the Standard Error (SEM) of the mean, except for n-3 in the 50 μ g dose group.

Figure 5 is a graph showing the maximum respiratory depression dose-response curve in rats after administration of DTC401 (open circles) or free morphine sulfate (filled circles) outside of a disposable hard membrane. Will reach the lowest SpO₂The epidural morphine dose is plotted. Each data point represents the mean of 5 animals and the Standard Error (SEM) of the mean, except for n-3 in the 50 μ g dose group.

Figure 6 shows two curves comparing pharmacokinetics in cerebrospinal fluid (upper panel) and serum (lower panel) of rats after epidural administration of 250 μ g of DTC401 (open circles) or free morphine sulfate (filled circles). Each data point represents the mean and Standard Error (SEM) of the mean from 3 or 4 animals.

Summary of the invention

Epidural administration of a therapeutic compound in a drug delivery system produces a surprising, greater sustained release and sustained therapeutic effect than using the free therapeutic compound.

Thus, one aspect of the present invention provides a method for epidural administration to a vertebrate in need of such treatment using a drug delivery system, for delayed release of a therapeutic compound.

The vertebrate is preferably a mammal such as a human. In a different preferred embodiment, the drug delivery system is lipid based, in particular a multivesicular liposome (multivesicular liposome).

The invention features the ability to provide delayed release of various therapeutic compounds (which in preferred embodiments include opioid or opiate antagonists) in order to modulate the analgesic effect. Other embodiments may deliver therapeutic compounds such as neurotropic factors.

Furthermore, using the sustained release formulation according to the method of the invention simplifies and reduces the overall cost of epidural analgesia, and also reduces possible infection, by eliminating continuous infusion, multiple bolus injections, or placement of catheters. Even in the presence of an epidural catheter, it is very advantageous to reduce the number of injections.

Detailed description of the invention

The present invention proposes a lipid-based sustained release drug delivery system for the epidural administration of therapeutic compounds such as opioids with an epidural effect. By epidural administration, the compounds are released into the central nervous system and cerebrospinal fluid without puncturing the dura mater and at a delayed rate.

The term "sustained release" refers to the release of the therapeutic compound over a longer period of time when administered as a bolus encapsulated in a lipid-based formulation as compared to the same drug in free form administered as a bolus by epidural injection. It does not necessarily mean that the concentration of the therapeutic compound remains constant during the sustained release. Generally, patients experience a decrease in the level of pain over time after surgery or after delivery. Thus, after a certain time, the patient's need for analgesia is also decreasing. Using the method of epidural administration of the invention, a therapeutically effective concentration of the therapeutic compound in the cerebrospinal fluid and/or serum can be maintained over several days, preferably about 2 to about 7 days.

The term "therapeutic compound" as used herein refers to a compound that has the desired effect of modulating a biological process in order to achieve modulation or treatment of an undesirable condition in an organism. The term therapeutic compound includes chemical non-protein drugs such as antibiotics and analgesics as well as protein drugs such as cytokines, interferons, growth factors and the like.

Drug delivery systems are well known in the art. The present invention relates to any sustained release formulation such as synthetic or natural polymers and lipid matrices in the form of macromolecular complexes, nanocapsules, microspheres or globules, including water-including oil emulsions, micelles, mixed micelles, synthetic membrane vesicles (membrane vesicles) and re-encapsulated red blood cells. These systems are called dispersions. Dispersions are two-phase systems in which one phase is distributed in the form of particles or droplets in the second phase. Generally, the particles contained in the system have a diameter of about 20nm to 50 μm. The particle size range is such that it is dispersed in the drug solution and introduced into the epidural space using a needle or catheter and a syringe.

The materials used to prepare the dispersion are generally non-toxic and biodegradable. For example, collagen, albumin, ethylcellulose, casein, gelatin, lecithin, phospholipids and soybean oil may be used in the process. The polymer dispersion can be prepared by a process similar to coacervation or microencapsulation. The density of the dispersion can be adjusted by changing the specific gravity so that the dispersion is high or low, if necessary. For example, the density of the dispersion can be altered by adding iohexol, iodixanol, meglumine, sucrose, trehalose, glucose, or other molecules compatible with high specific gravity organisms.

One type of dispersion that may be used in accordance with the present invention includes a dispersion of the therapeutic agent in a polymer matrix. The therapeutic agent is released as a polymer matrix and is broken down or biodegraded into soluble products, excreted outside the body. Several types of synthetic polymers have been studied for this purpose, including polyesters (Pitt, et al in Controlled Release of Bioactive Materials, R.Baker, Ed., Academic Press, New York, 1980); polyamides (Sidman, et al, Journal of Membrane Science, 7: 227, 1979); polyurethane (Master, et al, Journal of Polymer Science, Polymer Symposium, 66: 259, 1979); polyorthoesters (Heller, et al, Polymer Engineering Science, 21: 727, 1981); and polyanhydrides (Leong, et al, Biomaterials, 7: 364, 1986). Much research has been done on PLA and PLA/PGA polyesters. This is of course for convenience and safety reasons. These polymers are readily available because they have been used as biodegradable sutures and they break down into non-toxic lactic and glycolic acids (see U.S. patent 4578384 and U.S. patent 4785973, incorporated herein by reference).

Solid polymer dispersions can be synthesized using Polymerization methods such as bulk Polymerization, interfacial Polymerization, solution Polymerization, and ring-opening Polymerization (Odian, G., Principles of Polymerization, 2 nd., John Wiley & Sons, New York, 1981). Using any of these methods, a variety of different synthetic polymers can be obtained with a wide range of mechanical, chemical and biodegradable properties; by changing the parameters: reaction temperature, reactant concentrations, solvent type, and reaction time to control differences in properties and characteristics. If desired, a large solid polymer dispersion can be initially prepared and then comminuted or otherwise processed into small particles sufficient to maintain a dispersion in an appropriate physiological buffer (see, e.g., U.S. patent 4452025, U.S. patent 4389330, and U.S. patent 4696258, incorporated herein by reference).

If desired, the therapeutic compound may be incorporated into a non-dispersed structure that is surgically or mechanically implanted epidurally. A non-dispersive structure is a structure having a certain shape, such as a plate, a cylinder or a sphere. Hopfenberg has described the mechanism of Release of therapeutic agents from biodegradable platelet, cylindrical and spherical structures (Controlled Release polymers, pp.26-32, Paul, D.R.and Harris, F.W., eds., American chemical Society, Washington, D.C., 1976). A simple expression describing the release of additives from these devices is (where release is controlled primarily by matrix degradation):

M_t/M_∞＝1-[1-k₀t/C₀α]ⁿwhere n is 3 for spheres, 2 for cylinders and 1 for plates. The symbol α represents the radius of a sphere or a cylinder or half the thickness of a plate. M_tAnd M_∞The mass of drug released at time t and infinity, respectively.

Any known lipid-based drug delivery system may be used in the practice of the present invention. For example, porous liposomes (MVLs), multilocular liposomes (also known as multilamellar vesicles or "MLV") unilocular liposomes, including small unilocular liposomes (also known as unilocules or "SUVs") and large unilocular liposomes (also known as large unilocules or "LUVs") may be used, provided that the rate of delayed release of the encapsulated therapeutic compound can be determined. However, in a preferred embodiment, the lipid-based drug delivery system is a porous liposome system. Methods for preparing controlled release porous liposomal drug delivery systems are described fully in the following references: U.S. patent application accession numbers 08/352342 (filed 12/7/1994) and 08/393724 (filed 2/23/1995) and PCT application accession numbers US94/12957 and US94/04490, all of which are incorporated herein by reference.

The composition of the synthetic membrane vesicles (membrane vesicles) is generally a composition of phospholipids, generally associated with steroids, in particular cholesterol. Other phospholipids or other lipids may also be used.

Examples of lipids used in the preparation of synthetic membrane vesicles include phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and dioleoylphosphatidylglycerol.

Variables such as the potency of the drug encapsulation, the instability of the drug, the homogeneity and particle size of the formed membrane vesicles, the drug to lipid ratio, the permeability, the instability of the formulation and the pharmaceutical acceptability of the formulation should be considered in the preparation of the therapeutic-containing membrane vesicles (Szoka, et al, Annual Reviews of Biophysics and Bioengineering, 9: 467, 1980; Deamer, et al, in Liposomes, Marcel Dekker, New York, 1983, 27; Hope, et al, Chem. Phys. lipids, 40: 89, 1986).

Others have conducted limited progress in the study of the use of lipid-based formulations of opioids and none have conducted studies of the epidural route. For example, studies on the preparation and in vitro activity of liposome-encapsulated opioids have been performed (F.Reig, et al., J.Microencapsis 6: 277-283, 1989), without any in vivo epidural studies. Furthermore, the anti-nociception (antinociception) and side effects of fentanyl tetrazolium encapsulated in liposomal formulations and administered by the spinal column in rats have been investigated (M.S. Wallace et al, Anesth.Analg.79: 778-535, 1994; C.M. Bernards et al, Anestthesiology 77: 529-535, 1992). However, the pharmacokinetics and pharmacodynamics of these compounds are not sufficiently different from those of standard opioids to demonstrate their effects in clinical practice. These studies have not explored sustained release formulations for administering opioids via the epidural route.

The lipid-based drug delivery system incorporating the therapeutic compound may be administered in a single dose, for example via an epidural catheter. However, in a preferred embodiment, the lipid-based drug delivery system is injected into the epidural space around the spinal cord in individual doses using a small gauge (gauge) needle, thereby avoiding the need for catheter placement. Preferably, an 18-25 gauge needle is used.

Representative compounds of therapeutic compounds for epidural administration include morphine opiates, hydromorphone, codeine, dihydrocodeinone, levorphanol, oxycodone, oxymorphone, diacetylmorphine, buprenorphine, naloxone, cyclobutyloxymorphone, tromethamine, methamphetamine, methadone, fentanyl, sufentanyl, and fentanyl tetrazolium (alfentanyl). In addition, opioid antagonists such as naloxone and naltrexone may be administered epidurally using the methods of the present invention to reverse or antagonize the effects of opioids.

Peptides and peptidomimetics (peptidomimetics) that bind to one or more neuroreceptors such as delta (delta) opioid receptors, mu (mu) opioid receptors, kappa (kappa) opioid receptors and epsilon (epsilon) opioid receptors are considered opioids and can be administered for treatment according to the methods of the present invention. Such compounds include enkephalins (enkephalins), endorphins (endorphins), casomorphins (casomorphins), kyotorphins (kyotorphins), and biologically active fragments thereof. The term "biologically active fragment" as used herein refers to any portion of a therapeutic compound that substantially retains the biological activity of the intact therapeutic molecule. One skilled in the art will know or can readily determine whether a fragment substantially retains the biological activity of the entire molecule.

In addition to opioids, a number of compounds having a therapeutic effect when administered epidurally at a delayed rate may also be used in the practice of the methods of the present invention. These compounds include neurotrophic factors such as insulin-like growth factor, ciliary neurotrophic factor and nerve growth factor; neurotransmitters and antagonists thereof such as dopamine, epinephrine, norepinephrine and gamma-aminobutyric acid; local anesthetics such as tetracaine, lidocaine, bupivacaine, and mepivacaine; substance P and related peptides; and alpha-2 receptor agonists such as clonidine and dexmedetomidine. In addition, supplemental local anesthetics such as lidocaine, bupivacaine, and tetracaine can increase the efficacy of epidural opioids.

In the present invention, lipid-based drug delivery systems incorporating opioids (e.g., morphine sulfate) were tested for maximum blood oxygen saturation or for maximum blood oxygen saturation compared to epidural administration of free drugBaseline values before administration of the drug were at hemoglobin oxygen saturation (SpO)₂) The percentage of the aspect reduction is shown to have the lowest possible respiratory depression. Those skilled in the art will be readily willing to accept the reality that blood oxygen levels can be readily measured using commercially available devices such as pulse oximeters.

It has also been shown that a single dose of a slow-release opioid formulated in a porous liposome composition and administered epidurally produces a long-lasting analgesic effect, and that the peak cerebellar medullary CSF concentration of the therapeutic drug occurs within 60 minutes after the single dose is administered epidurally and then gradually decreases over the next few days (e.g., up to 8 days). Although the peak CSF concentration decreased compared to that following epidural administration of free morphine sulfate, the overall analgesic effect produced (e.g., as shown by the area under the curve (AUC) in figures 1, 3 and table 1) increased many-fold compared to that following epidural administration of free morphine sulfate. For example, the peak blood concentration and CSF morphine concentration were reduced by 17-fold and 3.1-fold, respectively, in rats after epidural administration of 250 μ g of morphine sulfate encapsulated in porous liposomes (DTC401) compared to the same dose of unencapsulated morphine sulfate, whereas the CSF AUC increased by 2.8-fold.

Because of the reduction in peak blood and CSF concentrations of morphine with controlled-release epidural administration, there is no respiratory depression; while epidural administration of high doses of free morphine did cause respiratory depression.

The main advantages of the present invention are in three aspects. First, it is advantageous that the method of epidural administration of individual doses of the sustained release compound subjects the patient to a lesser risk of dose-related side effects (e.g., respiratory depression typically associated with large dose injections or infusions of therapeutic compounds). Second, by administering the therapeutic compound epidurally rather than directly into the cerebrospinal fluid, the therapeutic compound does not migrate throughout the brain and spinal cord and locally releases a therapeutically effective amount of the therapeutic compound into the epidural space over an extended period of time (e.g., up to 8 days). Finally, prolonged analgesia can be achieved without the need for multiple injections or continuous infusion.

Those skilled in the art will appreciate that the time period over which the release rate is maintained in the practice of the present invention will vary depending on the disease state being treated, the nature of the therapeutic compound and the slow release drug delivery system and the total amount of compound encapsulated and administered to the patient.

The term "therapeutically effective" as it relates to the compositions of the present invention, refers to the release of the therapeutic compound from the drug delivery system at a concentration sufficient to achieve the particular therapeutic effect to be achieved by the therapeutic agent. For example, if the therapeutic compound is an opioid, the desired therapeutic effect is analgesia without respiratory depression. The exact dosage will vary depending upon factors such as the particular therapeutic compound and the desired therapeutic effect, as well as patient factors such as age, sex, general condition, and the like. One skilled in the art can readily take these factors into account and use them to determine effective therapeutic concentrations without the need for further experimentation.

For example, a dosage range suitable for epidural administration of morphine sulfate to humans includes 1mg to 60 mg. More potent compounds may require doses as low as 0.01mg, while less potent compounds may require doses of 5000 mg. Although other dosages may be given in addition to the dosage ranges described above, the dosage ranges include all ranges of use for which epidural route administration is actually contemplated.

Previously reported methods for epidural procedures in rats involved drilling a hole through the lumbar bone and advancing the catheter 1cm into the epidural space. The present invention allows for placement of the catheter from above (i.e., from the neck) without injury from the surgical procedure. The catheter tip can also be placed anywhere along the spine rather than being limited to the waist as described in the prior art. The above method of placing a catheter can also be applied to animals other than rats, such as rabbits, dogs, and humans.

The following examples illustrate the practice of the process of the present invention. It is to be understood, however, that the examples are for the purpose of illustration and the invention is not to be construed as limited to the particular materials or conditions therein.

Example 1

A. Preparation of porous liposomes encapsulating morphine sulfate (DTC401) in the presence of hydrochloride

Step 1 to a clean 1 gram glass vial [1.3cm (inside diameter) × 4.5cm (high) ] was added a 1ml chloroform (Spectrum corp., Gardena, CA) solution containing 9.3 μ M dioleoyl lecithin (Avanti Polar Lipids, Alabaster, AL), 2.1 μ M dipalmitoyl phosphatidylglycerol (Avanti Polar Lipids), 15 μ M cholesterol (Avanti Polar Lipids) and 1.8 μ M triolein (Sigma). This solution is called a lipid fraction.

Step 2 to the above 1 g glass vial containing the lipid component was added 1ml of an aqueous solution containing 20mg/ml morphine sulfate (Sigma Chemical co., st. louis, MO) and 0.1N hydrochloric acid.

Step 3 to prepare a water-in-oil emulsion, the glass vial containing the mixture of "step 2" was sealed and held horizontally on the head of a vortex shaker (Catalogue # S8223-1, american scientific Products, McGaw Park, IL.) and shaken at maximum speed for 6 minutes.

Step 4 to suspend the chloroform spherules in water, the water-in-oil emulsion from "step 3" was divided into equal volumes and rapidly added with a small diameter sharp Pasteur pipette into two 1 gram glass vials [1.3cm (inner diameter). times.4.5 cm (height) ] each containing 2.5ml of water, glucose (32mg/ml) and free basic lysine (40mM) (Sigma). Then, each vial was sealed, mounted on the head of the same vortex shaker as used in "step 3" and shaken at maximum speed for 3 seconds to form chloroform globules.

Step 5 to obtain the porous liposomes, the chloroform pellet suspension prepared in the two vials of "step 4" was poured into the bottom of a 250ml Erlenmeyer flask containing 5ml water, glucose (32mg/ml) and free basic lysine (40 mM). The flask was incubated at 37 ℃ in a vibrating water bath and a nitrogen stream was passed into the flask at 7L/min for 10-15 minutes to slowly evaporate the chloroform. The liposomes were then separated by centrifugation at 600Xg for 5 minutes and washed three times with 0.9% sodium chloride solution.

B. Preparation of the formulation

The DTC401 formulation and control unencapsulated ("free") morphine sulfate were adjusted to 50 μ l containing 10, 50, 175, 250 or 1000 μ g doses prior to epidural injection. In addition, MVLs formulations containing morphine sulfate at a dose of 2000. mu.g for studying respiratory depression were formulated as 75. mu.l injections. The concentration of morphine in the different liposomal formulations was determined by dissolving 50 μ l of each formulation with 1ml of isopropanol and then diluting in water. Morphine concentration was determined by HPLC using a published method (S.P. Joel et al, Journal of Chromatography 430: 394-399, 1988). For the blank control, a blank porous liposome composition was prepared with glucose instead of morphine sulfate.

Example 2

A. Preparation of animals

Male Sprague-Dawley rats (Harlan Sprague-Dawley, San diego, Calif.) aged 6-8 weeks and weighing 205-. Animals were acclimated to the environment prior to each study. Each animal was studied only once. All Animals were housed according to the guidelines of the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council.

B. Epidural inserting catheter

Rat tail dura mater extrapolation catheter was performed as follows: halothane anaesthesia was induced and the animals were placed in a directed recumbent position at a height of 7 cm. The head was bent and care was taken to keep the animal breathing properly. A slightly beveled 19 gauge needleThe head is inserted into the spine at about 170 deg. to the centerline subtended by the curvature of the needle just caudal to the occipital ridge. The needle is advanced toward the C1 vertebra until the needle tip contacts the ratchet or C₁To the next layer. The needle tip is carefully moved to the ventral edge of the posterior layer. At this point, a little bit of flexibility is left and the needle is advanced a further 1-2 mm. Care was taken not to allow the needle to penetrate the dura. Accidental obstruction of the dura mater can be determined by extravasation of cerebrospinal fluid (CSF) through a needle sheath or a subcutaneously placed catheter. Subjecting a polyethylene pipe [ PE-10; the length is 12cm, and the inner diameter is 0.28 mm; volume 7.4. mu.l (Becton Dickinson, Sparks, MD)]Through the needle into the dorsal epidural space. The catheter was slowly advanced through the needle and stopped at about the L1 level from C18 cm. The exposed portion of the catheter was tunneled subcutaneously under the scalp and secured with a bag ligated 3-0 silk suture. Finally, the catheter was flushed with 10 μ l of physiological saline and stoppered with a stainless steel wire. The suturing process lasts about 10-15 minutes from the beginning of anesthesia. Animals were allowed to wake up and observed for 60 minutes. Only those animals that were fully recovered by the above procedure were used in the following study.

C. Anti-nociceptive effect

The baseline nociceptive values after placement of the epidural catheter were determined by placing the animals on a standard hot plate (52.5. + -. 0.5 ℃) according to the test method described by M.S.Wallace et al (Anesth.Analg.79: 778-786, 1994). Latency to nociceptive responses (expressed in seconds) was measured from when the animals were placed on the hot plate until they either licked their hind paw or jumped down. The baseline (pre-treatment) response latency value for each test animal was set to 0% of the Maximum Possible Analgesic (MPA) effect. Each animal was then epidurally injected with 50 μ l or DTC401 containing an epidural morphine dose ranging from 10-250 μ g, without encapsulation of morphine sulfate solution or control MVL white blood. The anti-nociceptive effect of morphine sulfate administered subcutaneously at a dose of 250 μ g to 1mg was also determined. After epidural administration of the test solution through the catheter embedded (emplant) as described above, the epidural catheter was flushed with 10 μ l of 0.9% sodium chloride.

The animals were then again subjected to a hot plate test to determine at a particular time point: antinociception was measured at 0.5, 1, 2, 3, 4, 6, 12 and 24 hours after administration of unencapsulated morphine sulfate and at 0.5, 1, 6 hours and 1, 2, 3, 4, 5, 6, 7 and 8 days after administration of DTC401 and MVL white blood. Antinociception was determined in 5 or 6 animals for each dose and each drug. To prevent tissue damage to the footpads, a 60 second cut-off was used. Thus, 100% MPA is stated as an antinociception lasting ≧ 60 seconds. Latency intervals of 10 + -2-60 seconds, respectively, corresponding to 0% -100% MPA were sensitive to showing dose-response within the studied dose range.

For each dose administered, a curve of efficacy and respiratory depression as a function of time was made. Hotplate response as percentage of maximal possible analgesic effect (% MPA) was calculated as described by Wallace et al (above):using the RSTRIP computer program [ Micromath, Salt Lake City, UT)]All areas under each curve are calculated by the trapezoid rule to the last data point.

One-way analysis of variance (ANOVA) was used in order to determine the dose dependence for different pharmaceutical formulations and routes of administration, respectively; and two-way ANOVA was used to compare different formulations at the same dose. Newman-Keuls test was performed on all ANOVA analyses to determine statistical significance; p < 0.05 was considered statistically significant for all experiments. All data are expressed as mean ± standard deviation of mean (SEM).

As shown by the data in fig. 1, epidural administration of DTC401 resulted in an analgesic effect of an allelic episode (equivalentonset), however, the duration of analgesia was significantly prolonged compared to epidural administration of free morphine sulfate. Epidural injection control MLV white blood showed no demonstrable antinociceptive effect (data not shown). As shown in figure 2, the peak analgesic effect of the epidural DTC401 and epidural and subcutaneous morphine sulfate was dose-dependent, and the peak analgesic latency of epidural free morphine sulfate was greater than that of the epidural DTC401, which was significantly greater than that of the subcutaneous free morphine sulfate (P < 0.05 for each comparison).

The significant prolongation of analgesic effect in animals given DTC401 epidural was readily seen in figure 1 and by the large area value (AUC) under the curve for DTC401 in figure 3. At a 250 μ g dose (which produces a peak effect close to 100% MPA for DTC401 and free morphine sulfate), the time to 50% MPA was 3.4 days for DTC401 compared to 0.17 days for morphine sulfate.

D. Respiratory depression

Respiratory depression was quantified using a pulse oximeter. Animals were transferred from the cages, placed in polystyrene rat confinement chambers (plates Labs, Lansing, MI) and allowed to acclimate for 5 minutes. At baseline and at specific time points after administration of a single dose of epidural bolus morphine sulfate or DTC401, a pulse oximeter probe was placed on the rat's right hind paw to measure oxygen saturation (Ohmeta Medical Systems, model 3740, Madison, WI). The dose range of DTC401 and free morphine sulfate is 10-2000 μ g. 5-6 animals were measured at each data point with a pulse oximeter, except that 3 animals were used at the 50 μ g dose. Continuous monitoring of hemoglobin oxygen saturation (SpO) over a practical period of time₂) Pulse oximeter value of percent. The maximum value obtained in the recording period of 3 minutes is specified as oxygen saturation.

FIG. 4 depicts oxygen saturation of hemoglobin (SpO) by pulse oximeter measurements with different doses of DTC401 and morphine sulfate₂) Time course of percent. As shown in figure 5, with increasing morphine sulfate dose, there was a dose-dependent increase in respiratory depression; with minimal respiratory depression produced by DTC401 at the same dose. On the other hand, within 1 hour after epidural administration of free morphine sulfate or DTC401, observed at SpO₂The greatest reduction in aspect and no delayed respiratory depression was observed for either formulation. The difference in peak respiratory depression between morphine sulfate and DTC401 was statistically significant (P)＜0.01)。

E. Pharmacokinetics

Pharmacokinetic studies were performed by measuring the concentration of morphine in the peripheral blood and in the CSF at appropriate time points after epidural administration of a 250 μ g dose of DTC401 or free morphine sulfate alone. Samples were taken 0.5, 1 hour and 1, 3, 5, 8, day after epidural administration of DTC401 described above and 0.5, 1, 3, 6, 12, 24 hours after epidural administration of free morphine sulfate. A group of 3-4 animals were anesthetized with halothane and CSF and blood samples were collected by cisternal puncture and cardiac puncture, respectively. The animals were then sacrificed with excess halothane. Serum was separated from blood by centrifugation and stored at-80 ℃ with CSF samples until further analysis by Radioimmunoassay (RIA).

Using a commercially available RIA apparatus [ Coat-A-Count ] highly specific to morphine as recommended by the manufacturer^TMSerum Morphine，Diagnostic Products Corp.，LosAngeles，CA]Morphine concentrations in serum and CSF were determined. All assays were performed in duplicate.

Figure 6 shows cerebellar medulla cisterna CSF and serum morphine concentrations in animals injected with 250 μ g free morphine sulfate or DTC 401. Table 1 summarizes the pharmacokinetic parameters. Peak CSF and serum morphine concentrations after epidural DTC401 administration were 32% and 5.9% after morphine sulfate administration, respectively. The end-point CSF half-life (β) of DTC401 was 82 hours, while morphine sulfate was 2.6 hours. For DTC401, the CSF Area (AUC) under the curve increased 2.7-fold compared to morphine sulfate, while the plasma AUC was very similar. The half-life is calculated for a bi-exponential function by fitting the pharmacokinetic curve. The rstip procedure was used in order to complete a curve fitted by repeated non-linear regression.

Example 3

larger-Scale preparation of DTC401

Step 1A 5ml chloroform solution containing 46.5. mu.M dioleoylphosphatidylcholine (Avanti Polar Lipids), 10.5. mu.M dipalmitoylphosphatidylglycerol (Avanti Polar Lipids), 75. mu.M cholesterol (Sigma Chemical Co.), and 9.0. mu.M triolein (Avanti Polar Lipids) was added to a 50ml clean stainless steel centrifuge tube. This solution is called a lipid component.

Step 25 ml of an aqueous solution containing 20mg/ml morphine sulfate pentahydrate (Mallinckrodt chemical inc.) and 0.1N hydrochloric acid was added to the above stainless steel centrifuge tube containing the lipid component.

Step 3 to prepare a water-in-oil emulsion, the mixture of "step 2" was stirred with a TK mixer (AutoHomoMixer, Model M, Tokushu Kika, Osaka, Japan) at 9000 revolutions per minute (rpm) for 9 minutes.

Step 4 to suspend the chloroform spherules in water, 25 ml of an aqueous solution containing 4% glucose and 40mM lysine was added to the water-in-oil emulsion of step 3, followed by mixing at 3500rpm for 120 seconds.

Step 5 to obtain the porous liposomes, the chloroform pellet suspension in the centrifuge tube was poured into the bottom of a 1000 ml Erlenmeyer flask containing 4% glucose and 40mM lysine in 25 ml aqueous solution. The flask was incubated at 37 ℃ in a vibrating water bath and a stream of nitrogen was passed into the flask at 7L/min for 20 minutes to slowly evaporate the chloroform. The liposomes were then isolated by diluting the suspension with 4-fold physiological saline and centrifuging the suspension at 600Xg for 5 minutes; the supernatant was decanted and the liposome particles were suspended in 50ml of physiological saline. The liposomes were again isolated by centrifugation at 600Xg for 5 minutes. The supernatant was decanted and the liposome particles were suspended in physiological saline.

The foregoing description of the invention is exemplary for the purposes of illustration and explanation. It will be understood that various modifications may be made without departing from the scope and spirit of the invention. It is, therefore, to be understood that the following claims are intended to cover all such modifications.

TABLE 1

Pharmacokinetic parameters after epidural injection of 250 μ g

	DTC401	MS
			Cmax(ng/ml)，CSF	1960±1280	6060±3590
Cmax(ng/ml)，Serum	86±20	1460±97
			t*α(hr)，CSF	5.0	0.85
t*β(hr)，CSF	82	2.6
			t*α(days)，Serum	0.48	0.68
t*β(hr)，Serum	49	5.0
			AUC(ng*days*ml-1)CSF	1170	432
AUC(ng*days*ml-1)Serum	53	58

MS, morphine sulfate; c_maxA maximum concentration; t is t_1/2α, onset half-life; t is t_1/2Beta, end point half

The stage of aging; AUC, area under the curve.

Claims (23)

1. Use of a porous liposome in the manufacture of a medicament for epidural administration for the treatment of a disease in a vertebrate, characterised in that a therapeutic compound is encapsulated in a porous liposome formulation containing said compound for a sustained release for 2-7 days, and said formulation is introduced into said vertebrate in a single epidural dose.

2. The use of claim 1, wherein the porous liposome comprises a synthetic membrane vesicle.

3. The use of claim 2, wherein the synthetic membrane vesicles are prepared from a material selected from the group consisting of: phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, gangliosides and suitable mixtures thereof.

4. The use of claim 2, wherein the synthetic membrane vesicles comprise at least one lipid associated with at least one steroid.

5. The use of claim 2, wherein the synthetic membrane vesicles comprise at least one phospholipid.

6. The use of claim 5, wherein the synthetic membrane vesicles are prepared from a material selected from the group consisting of: egg phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol, and suitable mixtures thereof.

7. The use of claim 1, wherein the vertebrate is a mammal.

8. The use of claim 7, wherein the mammal is a human.

9. The use of claim i wherein the therapeutic compound is an opioid.

10. The use of claim 9, wherein the opioid is morphine sulfate.

11. The use of claim 10, wherein the dose comprises 1-60 milligrams of morphine sulfate.

12. The use of claim 9 wherein the opioid is hydromorphone.

13. The use of claim 1 wherein the therapeutic compound is an opioid antagonist.

14. The use of claim 13 wherein the opioid antagonist is selected from the group consisting of naloxone and naltrexone.

15. The use of claim 1, wherein the therapeutic compound is a neurotrophic factor.

16. The use of claim 15, wherein the neurotrophic factor is selected from the group consisting of insulin-like growth factor, ciliary neurotrophic factor, nerve growth factor, dopamine, epinephrine, norepinephrine, gamma-aminobutyric acid, and neostigmine.

17. The use of claim 1, wherein the formulation is introduced into the epidural space via an epidural catheter.

18. The use of claim 17, wherein the epidural catheter is inserted from the neck down.

19. The use of claim 1, wherein the formulation is a dispersion.

20. The use of claim 1, wherein the therapeutic compound is a peptide or peptidomimetic.

21. The use of claim 1 wherein the therapeutic compound is selected from the group consisting of codeine, hydrocodone, levorphanol, oxycodone, oxymorphone, diacetylmorphine, buprenorphine, cyclobutyloxymorphone, methamphetamine, fentanyl, sufenadine, and fentanyl tetrazolium.

22. The use of claim 1, wherein the therapeutic compound is selected from the group consisting of enkephalins, endorphins, casomorphins, cotorubin, and biologically active fragments thereof.

23. The use of claim 1, wherein the pharmaceutical preparation is introduced through a hypodermic needle inserted into the epidural space.