MXPA98002560A - Transfer therapy of mioblastos to release pain and to treat perceptive and comparative abnormalities - Google Patents

Abstract

The present invention relates to a composition for delivering a peptide in vivo to a human such peptide is linked to an opioid receptor that interferes with the ligation of substance P to its receptor, comprising: (i) myogenic cells that have been obtained from a patient to be treated, a relative or another human, such cells contain heterologous DNA encoding said peptide, such that the myogenic cells express the peptide, and (ii) a pharmaceutically acceptable carrier such that the myogenic cells mature into muscle fibers or they are converted into fat cells and the peptide is produced in vivo, where the peptide is selected from the group consisting of enkephalins, beta-endorphins, alpha-endorphin, gamma-endorphin, delta-endorphin, Met sup 5, opioid peptides and analogues of substance

Description

TRANSFER THERAPY OF MIOBLASTOS TO RELEASE PAIN AND TO TREAT PERCEPTIVE ABNORMALITIES AND BEHAVIOR BACKGROUND OF THE INVENTION The present invention relates to a proposal to release pain and to treat perceptual and behavioral abnormalities by using myoblast transfer therapy to provide a continuous long-term supply of peptides in vivo that have analgesic activity. The modern theory of analgesia advanced significantly with a proposition by Pomeranz et al., Exp. Neurol. 54: 172 (1977), that a pituitary peptide similar to morphine mediates acupuncture analgesia. It was found that electroacupuncture reduces the responses in the spinal cord neurons to noxious stimuli in anesthetized cats and to increase the squeak threshold in awake mice. The prolonged time course, observed, involved a hormonal mechanism for the response. The spinal transaction, decerebration or hypophysectomy eliminated this effect of acupuncture, and the intravenous injections of naloxone, a morphine antagonist, also markedly reduced it. These results indicate that electroacupuncture stimulates sensory nerves, which activate the pituitary glands to release hormones (peptides) similar to morphine by effecting prolonged reduction in transmission along with nociceptive pathways. It is believed that this mechanism is a major mediator of generalized and localized analgesia. Morphine-like peptides have been identified, and receptors to morphine-like peptides and other opioid peptides have been found in the brain, intestines, pituitary gland, pancreas, and placenta. Hughes et al., Nature 258: 577 (1975); Pert et al., Science 179: 1011 (1979). These peptides are now known as β-endorphins and enkephalins. Cooper et al., THE BIOCHE ICAL BASIS OF PHAR ACOLOGY, 4th ed. (Oxford University Press, New York 1982). Additionally, stimulation of brain neurons with an opioid peptide, such as endorphin, produces analgesia. Fields ef al., Ann. Rev. Physiol. 40: 217 (1978). This effect can be reversed by naloxone. Opioid peptides, especially β-endorphins, are essentially neural transmitters or hormones, which reach all body tissue through diffusion. The presence of endorphin receptors in large numbers in different areas of the cerebral cortex and diencephalon suggests that conjugated opioid peptides play a role in analgesia, which goes beyond a simple modulator of pain perception. Covenas et al. Neuropeptides 30: 261 (1996); Bernstein et al., Neurosci. Lett. 215: 33 (1996); Bianchi et al., Brain Res. Bull. 40: 269 (1996). Actually, increases in plasma levels and cerobroespinal fluid of β-endorphins have been shown to modulate and optimize behavioral patterns exhibited in patients suffering from stress, psychiatric disorders, alcoholism, drug addiction, obesity and diabetes. Ryu er al., Am. J. Chin. Med. 24: 193 (1996); Odagiri et al., Int. J. Sports Med. 17: 325 (1996); Dalayeun ef al., Biomed. Pharmacother. 47: 311 (1993); Gianoulakis ef al., J. Psychiatry Neurosci. 18: 148 (1993). These increases also promote cytotoxicity mediated by natural killer cells. Jonsdottir ef al., Regul. Pept. 23: 113 (1996); Priest et al., Reg? L. Pept. 63: 79 (1996). Analgesia is also affected by the ligature of a pain mediator called "substance P" to its receptor. There are many similarities between the terminals of opioid neurons and the terminals of sensitive neurons of substance P. For example, both types of terminals mediate the sensation of pain in the spinal cord. Jessel et al., Nature 268: 549 (1977). As indicated, for example, in JP 3133998, the substance P receptors have been shown to act as analgesics by masking the activity of the substance P. According to the PCT application WO 92/16547, the NK-1 receptor preferentially binds substance P and can be used to treat pain, inflammatory disease, mental illness and tension. Patients afflicted with conditions such as tension, psychiatric disorders, alcoholism, drug addiction, obesity, and diabetes may obtain some measure of release of a higher than normal level of endogenous opioid peptides in their plasma. The clinical release of symptoms from these conditions have been associated with the ligation of opioid peptides with their receptors, which is directly correlated with the level of opioid peptides in the patient's plasma and cerebrospinal fluid. Patients may also benefit from increased levels of substance P receptors or P substance analogues. See WO 92/16547, supra, and PCT application WO 91/02745. To date, no adverse reaction has been associated with physiological increases in the levels of cerebrospinal fluid or plasma of ß-endorphins, enkephalins or P-substance receptors. The use of drugs to increase the production and / or secretion of opioid peptides can provide temporary release, but the uncontrollable metabolism of the drug and the strong dosage will eventually require too much of the "sick" neurons and their counterparts. Additionally, the side effects of medications are numerous and undesirable. Opioid peptides by themselves, and opioid peptide receptors have been administered as sedatives and analgesics, see U.S. Patent No. 4, 123,523, but the effects of such administrations are short lived. Xenogeneic tumor cells that secrete β-endorphin have been transplanted into the cerebrospinal fluid space of the spinal cord of rats, producing analgesic effects. Saitoh et al., Cell Trans. 4 (Supp.1): S13-7 (1995). The transplanted cells were reported to survive for a month, and in vitro studies indicated that the cells secrete β-endorphin for a month. AtT-20 cells and AtT-20 / hENK cells, which secrete β-endorphin and enkephalin, respectively, were implanted in the mouse spinal subarachnoid space to investigate their use as a therapy for pain. Wu et al., J. Neuroxci. 14 (8): 4806 (1994); J. Neural Transplant. Plast. 4 (1): 15 (1993). But these procedures are very aggressive and therefore very dangerous, since they involve the transplantation of cells directly into the spinal subarachnoid space or cerebrospinal fluid. Also, only a limited number of cells are transplanted, making the amount of opioid peptide provided by these methods, limited. Therefore, there is a need for a long period analgesic method by delivering a peptide that binds to opioid receptors or that interferes with the binding of substance P to its receptors in vivo over a long period of time. Such a method would be useful for treating chronic pain and psychiatric conditions involving abnormal perception, such as depression, chronic anxiety syndromes, paranoia, alcoholism, and drug addiction, and other diseases in which the terminals of substance P and opioid neurons play a paper.

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a method for treating psychiatric conditions involving abnormal perception, such as depression, chronic anxiety syndromes, paranoia, alcoholism, and drug addiction, chronic pain. , and other diseases in which opioid neurons and neurons sensitive to substance P play a role. It is also an object of the present invention to provide a composition for carrying out this method. In accordance with this and other objects of the invention, there is provided a method for continuously delivering in vivo a peptide that can bind to opioid receptors or that can interfere with the binding of substance P to its receptor comprising the steps of (a) transducing cells myogenic with DNA encoding the peptide, and (b) administering the myogenic cells transduced to the patient, so that the cells continuously produce the peptide. In one embodiment, the analgesic peptide is selected from the group consisting of an opioid peptide, a polypeptide that binds substance P and a substance analogue P. In one embodiment, the myogenic cells are selected from the group consisting of myoblasts, myotubes, and muscle cells. In another embodiment, the cells are transduced with DNA encoding multiple-copy sequences of the separated peptide by cutting sites. In another embodiment, the transduced cells are administered by intramuscular injection into a patient's paraspinal muscle. In yet another embodiment, long chondroitin-6-sulfate insulin or proteoglycan is administered with the transduced myogenic cells. The co-administration of an immunosuppressant is also preferred in some embodiments.

The invention also provides a method for continuously delivering in vivo an analgesic peptide that occurs naturally comprising the steps of (a) transducing myogenic cells with DNA containing a promoter for an endogenous structural gene encoding the peptide, and (b) administering the myogenic cells transduced to a patient, so that the cells continuously produce the peptide. The invention further provides a composition for continuously delivering in vivo a peptide that binds an opioid receptor or that interferes with the ligation of the substance P to its receptor, comprising the steps of (a) transducing myogenic cells containing heterologous DNA and expressing the peptide, and a pharmaceutically acceptable carrier. In one embodiment, the heterologous DNA comprises a gene encoding the peptide and a promoter. In another embodiment, the heterologous DNA comprises a promoter for an endogenous structural gene encoding the peptide. In another embodiment, the composition further comprises insulin or long chondroitin-6-sulfate proteoglycan. Further objects and advantages of the invention are set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practicing the invention.

DETAILED DESCRIPTION OF PREFERRED MODALITIES It has been found that genetically transduced myogenic cells can be employed to provide a continuous, long-term supply of a peptide having analgesic activity. This method is useful for treating chronic pain as well as psychiatric conditions that involve abnormal perception, such as depression, chronic anxiety syndromes, paranoia, alcoholism and drug addiction, and other diseases in which neurons that bind opioids and / or or neurons that bind substance P play a role. Such conditions have not been addressed so far by long-term administration of analgesic peptide in vivo. Analgesic peptides suitable for the invention are peptides that bind opioid receptors or that interfere with the binding of the substance P to its receptor. Among these peptides are the opioid peptides, the polypeptides that bind the substance P, and the peptides that are analogues of the substance P. In this context, the phrase "polypeptide that binds substance P" denotes a peptide or protein having affinity for the substance P such as, for example, example, the receptor protein of substance P or a peptide or peptide analogue derived from this receptor and retaining the ability to bind the substance P. Such peptides and proteins bind substance P and therefore interfere with the binding of the substance P with its receiver. The skilled artisan can test the ligation to substance P with an assay. Analogs of substance P act as analgesics by interfering with the ligation between the substance P and its receptor. For example, the PCT application WO 91/02745, supra, describes analogs that do not exhibit the natural activity of substance P but that act as competitive inhibitors of the substance P. According to the invention, myogenic cells are transduced ex vivo so that they express at least one of the peptides listed above, either while in the cell culture, or after differentiation in vivo. Cells that have been transduced with a gene encoding such a peptide are administered to the patient, for example, by injection into the patient's muscle or adipose tissue. The transduced cells can survive and grow in the recipient tissue. For example, cells injected into muscle tissue can form myotubes and mature in muscle fibers. The cells injected into adipose tissue can survive and be converted into fat cells. Transduced cells injected into both types of tissue can express the desired analgesic peptide continuously. The expressed peptide leaves the cell and travels through the blood to other areas of the body, including the spinal cord and the brain. Myoblast transfer therapy (MTT) has been used to treat muscle degeneration and weakness and is a useful technique for administering cells that express an analgesic peptide. See U.S. Patent No. 5, 130, 141, the contents of which are incorporated herein by reference. According to this method, the genetically normal myogenic cells are administered to a myopathic muscle of the patient, thereby increasing muscle function, locomotor patterns and respiratory function. It has been shown that normal myoblast transfer therapy produces missing protein dystrophin for up to six years in patients with Duchenne muscular dystrophy. Law et al., Cell Transplantation 6: 95-100 (1997). Although previous studies of myoblast transfer used muscle as the receptor tissue, other tissues can also be used. For example, myoblasts can grow after their injection or surgical implantation into adipose tissue, as described by Satoh et al., Transplantation Proceedings 24: 3017-19 (1992). The myoblasts have been transduced with genes for Factor IX, erythropoietin (EPO) and human growth hormone, and the Fas ligand to increase the circulation levels of these proteins. Thompson, Thromb and Haemost. 74 (1): 45 (1995); Hamamori ef al., J. Clin. Invest. 95: 1808 (1995), and Human Gene Therapy 5: 1349 (1994); Barr et al., Science 254: 1507 (1991); Dhawan et al., Science 254: 1509 (1991); Lau ef al., Science 273: 109 (1996). The success of these methods has varied. According to Thompson (1995), for example, preliminary data suggest that human myoblasts removed from the body survived less well in culture and progressively lost their ability to express factor IX. Lau et al. (1996), reports that Fas ligand expression was local and seemed to cease after 80 days. On the other hand, Hamamori et al. (1994) reports that in vivo implantation of a stable, high-level EPO-producing muscle cell clone resulted in high serum EPO levels sustained for three months, and Dhawan et al. (1991) states that transduced myoblasts continued to secrete hGH after they differentiated into myotubes, with no difference in secretion levels between myoblasts and myotubes. However, the transduced myoblasts have not previously been used to deliver an analgesic peptide continuously in vivo. In addition, although gene therapy has been studied as a means to deliver opioid peptides in vivo, the transduced cells were injected directly into the spinal subarachnoid space or cerebro-spinal fluid or spinal cord. Saitoh et al., Cell Trans.A (Supp.1): S13-7 (1995); Wu ef al., J. Neurosci., 14 (8): 4806 (1994); Wu ef al., J. Neural Transplant. Plast. 4 (1): 15 (1993). As discussed above, these methods are very aggressive, only a limited number of cells are transplanted, and the transduced cells expressed the opioid peptides for only one month. According to the present invention, in contrast, the transduced myoblasts are not injected into the central nervous system. Moreover, unlike the short-term expression of opioid peptides carried out in conventional gene therapies, the present invention provides a long-term, continuous supply of opioid peptides which lasts, for example, up to at least six years . These aspects of the present invention represent distinctive advantages that have not been appreciated. Myogenic cells that are suitable for the present invention include myoblasts, myotubes and muscle fiber cells. Myoblasts are particularly preferred, according to one embodiment of the invention. Myoblasts are mononuclear embryonic muscle cells that differentiate into multinucleated myotubes. Each nucleus of a myoblast contains over 100,000 genes, including genes for opioid peptides such as β-endorphins and enkephalins. Myoblasts are widely divided, migrate, naturally fuse to form syncytia, lose MHC-1 antigens rapidly after fusion and constitute approximately 50% of the dry body weight of humans. Myoblasts are unusual in that they are able to fuse natural cells between them and with mature muscle fibers. As a result of this fusion, a transduced myoblast transfers its nucleus and therefore all its genes to the cell with which it fuses, which can be a genetically normal or abnormal muscle cell. The myogenic cells can be obtained from a patient to be treated, from a relative or another human or another donor animal. In a typical procedure, 1 to 2 grams of skeletal muscle is harvested from a donor. The myogenic cells can also be cultured or produced by cloning methods known to those skilled in the art, as shown for example in U.S. Patent No. 5,130,141. According to one embodiment of the invention, the muscle cells of a human or animal donor are stimulated 0 to 3 days before being harvested to produce a pool of satellite cells that are reserves of myoblasts in mature muscles. The myogenic cells can be stimulated by, for example, injuring the cells with a number of needle probes or by sonication. According to one embodiment of the invention, the harvested cells are processed to obtain a pure culture of myoblasts. See Law et al. Cell Transplant. 1: 235 (1992); Cell Transplant. 2: 485 (1993); Muscle and Nerve 11: 525-33 (1988). For example, a muscle biopsy is dissociated with 0.1% collagenase and 0.2% crude trypsin in phosphate buffered saline at pH 7.3. The mixture is stirred for 45 minutes, with three changes of enzyme solution alternating with three changes of a neutralizing medium comprising 100 parts of Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 0.37% NaHCO3 and 4mM glutamine; 10 parts horse serum and 1% antibiotic-antifungal. According to one embodiment of the present invention, the harvested myogenic cells are transduced ex vivo with DNA encoding a peptide that either binds an endorphin receptor or inhibits the ligation of the substance P to its receptor. Peptides that are known to have adequate activity in this context are β-endorphin, a-endorphin, gamma-endorphin, delta-endorphin, Met sup 5 (a peptide of 5 amino acid residue with activity similar to endorphin), peptides of active endorphin comprising parts of the sequence of β-endorphin, enkephalin, an NK-1 receptor, a polypeptide that binds substance P or an analogue of substance P that competitively inhibits the binding of substance P to its receptor. The phrase "substance analog P" denotes a peptide comprising the sequence of five carboxy-terminal amino acids of the substance P (-Phe-Phe-Gly-Leu-Met) and which binds to the substance P receptor, inhibiting the activity of the substance P. See Payan, Ann. Rev. Med. 40: 341 (1989), and the PCT application WO 91/02745. Additional peptides can be found by kinetic experiments that reveal whether a given peptide either binds to an opioid receptor or competes to bind between substance P and its receptor. These experiments can be done routinely. See, for example, the PCT application WO 92/16547. The DNA sequences useful for the invention are known or can be designed by those skilled in the art from known amino acid sequences of the peptides. For example, Saitoh ef al., Cell Trans. 4 (Supp.1): S13-7 (1995), describes a DNA sequence encoding β-endorphin; Wu et al. (1993, 1994), supra, describes sequences for β-endorphin and enkephalin; U.S. Patent No. 4,123,523 discloses amino acid sequences of β-endorphin peptides; the PCT application WO 92/16547 describes a gene encoding the substance P NK-1 receptor; Japanese patent document JP 3133998 describes the amino acid sequence of a substance P receptor; and the PCT application WO 91/02745 describes the amino acid sequences of various analogs of substance P, such as addition and deletion mutants of P substance. According to one embodiment of the invention, the DNA encodes a plurality of copies of a peptide that produces analgesia. In a preferred embodiment, the peptide is an opioid peptide and DNA regions encoding multiple copies are separated by cut sites (see PCT application WO 96/17941). This embodiment can provide an amplified amount of a peptide that occurs naturally. Transduction of myogenic cells with a DNA sequence can be effected via known methods, such as those reported by Thompson (1995) and Hamamori et al. (1995), supra. Generally, a DNA construct is used which contains a promoter upstream of the structural gene encoding the desired peptide. Suitable promoters are described, for example, in U.S. Patent No. 5,618,698. According to another embodiment of the present invention, the harvested myogenic cells are transduced ex vivo with a DNA containing a promoter that can bind to and function (i.e., turn on or increase expression) with an endogenous gene within the nuclei of a myogenic cell. In this modality, the DNA comprising a regulatory sequence, an exon and a splice donor are introduced into a cell by homologous recombination in the cell genome at a preselected site. The introduction of this DNA results in the production of a new transcription unit in which the regulatory sequence, exon and splice donor site are operatively linked to the endogenous gene. The introduction of DNA is typically followed by the selection of cells that have received a promoter at a desired location, to ignite the desired gene. The applicable selection methodology is described, for example, in U.S. Patent Nos. 5,641, 670 and 5,272,071. Selection techniques are also described by Mansour et al., Nature 136: 348, 349 (1988). After selection, the cells, which express the desired gene are cultured and then introduced into a patient. The transduced myogenic cells are cultured to produce a sufficient amount of cells for administration to the patient by any of a variety of methods known in the art. For example, see Law ef al. (1988, 1992), supra. The amount of cultured cells will depend on the condition of the patient and the severity of the disease being treated. For example, from about 1 billion to about 100 billion myoblasts can be cultured for administration to a patient. According to one embodiment of the invention, the cells are cultured in the neutralizing medium described above, supplemented with two parts of chicken embryo extract. Cells are fed fresh growth medium every two days, and incubated in 7% CO2 at 37 ° C for 35-40 days. According to one embodiment of the invention, the transduced cells are administered to the patient by intramuscular injection. Law et al., Cell Transplant. 1: 235 (1992); loe cit. 2: 485 (1993); Law ef al. Exp. Neurol., Transplant Proc. 29: 2234 (1997). The amount of opioid peptides provided according to the present invention can be controlled by selecting the number of muscles injected and the number of cells injected. According to one embodiment of the invention, the direction of the injection is controlled to optimize the number of transduced cells delivered to the recipient muscle fibers. For example, it has been shown that injecting cells delivered diagonally through muscle fibers maximizes the resulting number of muscle fibers fused with the cells administered. In accordance with another embodiment of the invention, the transduced cells are administered to specific muscles which help to direct the cells to a location between laminae IV and V of the spinal cord. For example, transduced cells can be injected into the paraspinal muscles or neck muscles, such as the shoulder scapulae. Although transduced myogenic cells administered anywhere in the body will secrete peptides that will travel through the blood and reach the spinal receptors, directing the administration of the cells to the paraspinal muscles or neck muscles that are in proximity to the spinal cord will be it is expected to result in more peptides reaching receptors more rapidly, thereby increasing the effectiveness of the method. The transduced cells can also be administered by surgical implant in the patient. The cells can be implanted in, for example, adipose tissue. In a further embodiment, the patient is also provided with an effective amount of an immunosuppressant to minimize rejection of the transduced cells. See U.S. Patent No. 5,130,141 and Law et al. (1992, 1993), supra. For example, cyclosporin A, another immunosuppressant, or combinations of immunosuppressants, may be provided according to known procedures. Suitable dosage forms, dosage amounts and dosage schedules are known in the art. For example, cyclosporin A can be provided orally at a daily dose of about 7 mg / kg body weight. A typical dosage program comprises providing the daily dose in two divided doses, and the patient's whole blood can be monitored to maintain a channel level of approximately 250 mg / ml cyclosporin A. In accordance with one embodiment of the invention, the fusion of the transduced myoblasts is facilitated by administration of long chondroitin-6-sulfate proteoglycan (LCS6SP) as described in the US application cited above with serial No. 08 / 477,377. The trauma of injecting myoblasts into the extracellular matrix triggers the release of basic fibroblast growth factor and long chondroitin-6-sulfate proteoglycan. These released molecules stimulate the proliferation of myoblasts. Increasing the level of long chondroitin-6-sulfate proteoglycan at the injection site facilitates fusion of myoblasts and proliferation. According to this, in accordance with one embodiment of the invention, the long chondroitin-6-sulfate proteoglycan is preferably administered with the transduced myoblasts. According to one embodiment of the invention, the long chondroitin-6-sulfate proteoglycan is low-sulfated. See Hutchison et al., Devel. Biol. 1 15: 78-83 (1986). The long chondroitin-6-sulfate proteoglycan is believed to be synthesized in a low-sulfated pre-fusion, but becomes more highly sulfated after fusion. Id. As used herein, therefore, the phrase "low-sulfated chondroitin-6-sulfate long-term proteoglycan" denotes a degree of sulfation that is approximately the same as that observed in the long chondroitin-6-sulfate proteoglycan which occurs naturally from cells just before the fusion. In accordance with this aspect of the invention, the low-sulphated long chondroitin-6-sulfate proteoglycan is administered in a concentration between about 5 μM to about 5 mM. Chondroitin-6-sulfate can be administered together with the transduced cells, or it can be provided in a separate formulation as a separate injection. Insulin also facilitates the proliferation of myoblasts and promotes the development of myotubes. According to one aspect of the invention, therefore, insulin is administered with the transduced myocytes. For example, approximately 0.2mM of insulin may be provided, either as part of the same formulation as the cells, or as a separate formulation, given, for example, in a separate injection. According to one embodiment of the invention, undesirable effects of overproduction of the desired peptide are regulated by agonists such as naloxone or SP-40,40. Pomeranz ef al., Altern. Thor. Health Med. 2: 85 (1996); Choi-Miura et al., Biol. Pharm. Bull. 16: 228 (1993); Pomeranz et al., Exp Neurol. 54: 172 (1977). For example, if the endogenous level of the peptide becomes too high, naloxone or SP-40,40 can be administered to counteract the effects of the peptide. Typical symptoms of overproduction of an analgesic peptide include drowsiness, low respiratory velocity, cyanosis, low blood pressure, fixed, symmetric pupils, and reduced urine formation. A usual course of naloxone treatment involves providing small intramuscular or intravenous doses of naloxone (approximately 0.4 mg to approximately 0.8 mg). Symptoms often improve after the first dose, but may recur after 2-3 minutes, up to a total dose of approximately 10 mg.

As discussed before, the administration of transduced myogenic cells according to the present invention provides a long-term, continuous supply of an analgesic peptide in vivo. The peptide travels from the site of synthesis such as adipose tissue or muscle and reaches the sensory nerve endings, the spinal cord and the brain, where it combines with nerve cell receptors to produce analgesia. The analgesia produced by the peptide is useful for treating chronic pain and psychiatric conditions that involve abnormal perception, such as depression, chronic anxiety syndromes, paranoia, alcoholism and drug addiction, and other diseases in which opioid neurons and the terminals of substance P play a role. The continuous long-term supply of an analgesic peptide in vivo as a medical treatment offers a new methodology to treat these conditions. The invention also provides a composition that makes an analgesic peptide that binds opioid receptors or interferes with the ligation of substance P to its receptor in vivo. In one embodiment, the composition comprises myogenic cells containing heterologous DNA encoding an analgesic peptide together with one or more pharmaceutically acceptable carriers. Examples of acceptable pharmaceutical carriers include diluents, solvents, buffers and / or preservatives. An example of a pharmaceutically acceptable carrier is the phosphate buffer containing NaCl. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, salts, preservatives, buffers and the like, as described in REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed. Easton: Mack Publishing Co., pages 1405-1412 and pages 1461-1487 (1975) , and THE NATIONAL FORMULARY XIV., 14th ed. Washington: American Pharmaceutical Association (1975). Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include nutrient and fluid fillers. The preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components of the linker composition are adjusted according to routine skills in the art. See GOODMAN AND G ILMAN'S THE PHAR MACOLOGICAL BASIS FOR THERAPEUTICS (7th ed.). According to one embodiment, the composition comprises transduced myogenic cells, long chondroitin-6-sulfate proteoglycan, and a pharmaceutically acceptable carrier. According to another embodiment, the composition comprises transduced myogenic cells, insulin and a pharmaceutically acceptable carrier. The embodiments of the invention are further illustrated by the following examples, which show aspects of the invention in detail. These examples illustrate specific elements of the invention and should not be considered as limiting the scope thereof.

Example 1. Treatment of patients suffering from depression by injection into muscle tissue The skeletal muscles of a patient suffering from a psychiatric condition involving depression are stimulated by numerous needle probes to produce a pool of satellite myoblast cells. Three days later, the patient is placed under general anesthesia, and 2 g of skeletal muscle are harvested from the patient. The harvested muscle is processed to obtain a pure culture of myoblasts. The harvested muscle is dissected free of skin and other tissue, and the cells are dissociated with 0.1% collagenase and 0.2% crude trypsin in phosphate buffered saline at pH 7.3. The mixture is stirred for 45 minutes, with three changes of alternating enzyme solution with three changes of a neutralizing medium comprising 100 parts of Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 0.37% NaHCO3 and 4mM glutamine; 10 parts horse serum and 1% antibiotic-anti-fungal. These myoblasts are transduced with DNA containing a gene for enkephalin and a suitable promoter. The transduced myoblasts are then cultured in the medioneutralizer described above supplemented with 2 parts of chicken embryo extract. Cells are fed fresh growth medium every 2 days, and are incubated in 7% CO2 at 37 ° C for 40 days, when approximately 2 billion myoblast cells (progeny of transduced myogenic cells) are present. The patient is again placed under general anesthesia and the progeny of the transduced myogenic cells are injected intramuscularly into the patient's paraspinal muscles.

Within a subsequent week, the patient's symptoms should begin to improve.

Example 2. Treatment of a patient suffering from depression by injection into the adipose tissue Myoblasts are obtained and transduced as described in Example 1 to form approximately 10 trillion progeny myoblast cells. The patient's chest tissue is anesthetized and the cells are injected into the anesthetized tissue. Within a subsequent week, the patient's symptoms should begin to improve.

Example 3. Treatment of a patient suffering from alcoholism The skeletal muscles of a patient suffering from alcoholism are stimulated by sonication to produce a pool of satellite myoblast cells. Three days later, the patient is placed under general anesthesia, and 2 g of skeletal muscle is collected from the patient. The harvested muscle is processed to obtain a pure culture of myoblasts as described in Example 1 above, until 50 trillion cells are obtained.

The patient is placed again under general anesthesia and the progeny of the transduced myogenic cells are injected into the patient's paraspinal muscles. Within a week after the procedure, the patient's symptoms begin to be relieved.

Example 4. Composition to provide a continuous long-term supply of enkephalin in vivo The following composition is provided: 1 trillion myogenic cells transduced with enkephalin encoding DNA; and a phosphate buffer containing NaCl and human serum albumin as a pharmaceutically acceptable carrier.

Example 5. Composition to provide a long term continuous supply of β-endorphin in vivo The following composition is provided: 1 trillion myogenic cells transduced with DNA encoding a promoter of a human endorphin gene; and water as a pharmaceutically acceptable carrier. It will be apparent to those skilled in the art that various modifications and variations may be made to the processes and compositions of this invention. Thus, it is intended that the present invention cover the modifications and variations of this invention as long as they fall within the scope of the appended claims and their equivalents.

Claims (32)

1. A method for delivering in vivo a peptide that binds an opioid receptor or that interferes with the ligation of substance P to its receptor, comprising the steps of (a) transducing myogenic cells with DNA encoding the peptide, such that the myogenic cells they express the peptide, and then (b) administer the myogenic cells to a patient, so that the peptide is produced in vivo.

The method of claim 1, wherein the peptide is selected from the group consisting of an opioid peptide, a polypeptide that binds substance P, and an analogue of substance P.

3. The method of claim 1, wherein the myogenic cells are selected from the group consisting of myoblasts, myotubes, and muscle fiber cells.

4. The method of claim 1, wherein the myogenic cells are harvested from the skeletal muscle tissue of the patient.

The method of claim 1, wherein the myogenic cells are harvested from the skeletal muscle tissue of a normal donor.

6. The method of claim 4, wherein the skeletal tissue is stimulated prior to harvesting to produce a pool of satellite myoblast cells.

7. The method of claim 4, wherein the collected myogenic cells are processed to produce a purified sample of myoblast cells.

The method of claim 1, wherein the transduced myogenic cells are cultured to produce a progeny sample of transduced myogenic cells comprising at least one trillion cells.

9. The method of claim 1, wherein the peptide is an opioid peptide.

The method of claim 9, wherein the opioid peptide is selected from the group consisting of β-endorphin, α-endorphin, gamma-endorphin, delta-endorphin, Met sup 5 and enkephalin.

The method of claim 1, wherein the peptide is a polypeptide that binds the P substance.

The method of claim 1, wherein the peptide comprises the sequence Phe-Phe-Gly-Leu-Met.

The method of claim 1, wherein the DNA comprises two nucleotide sequences, each encoding the peptide, and a segment that separates the two nucleotide sequences, wherein the segment encodes a cleavage site.

The method of claim 1, wherein step (b) comprises administering the myogenic cells by intramuscular injection.

15. The method of claim 14, wherein the myogenic cells are injected into a paraspinal muscle of the patient.

16. The method of claim 14, wherein the myogenic cells are injected into a patient's levator scapula muscle.

The method of claim 14, wherein the myogenic cells are injected into a neck muscle of the patient.

18. The method of claim 1, wherein the long chondroitin-6-sulfate proteoglycan is administered with the myogenic cells.

19. The method of claim 1, wherein the insulin is administered with the myogenic cells.

The method of claim 1, further comprising the step of administering an immunosuppressant to the patient.

21. A method for continuously delivering a peptide that binds an opioid receptor in vivo, comprising the steps of (a) transducing a plurality of myogenic cells, at least some of which contain (i) a gene encoding the peptide and (ii) a flanking region associated with the gene under conditions favorable to homologous recombination, with DNA comprising a promoter and a segment that is homologous to the flanking region; (b) selecting from among the plurality of myogenic cells wherein the promoter and the gene are functionally linked; (c) multiplying the myogenic cells selected in step (b) to produce progeny cells; and (d) administering the progeny cells to a patient, such that the cells continuously produce the peptide.

22. A composition for delivering a peptide in vivo that binds to an opioid receptor or that interferes with the binding of substance P to its receptor, comprising (i) the myogenic cells that contain the heterologous DNA encoding the peptide, such that the cells myogenic cells express the peptide, and (ii) a pharmaceutically acceptable carrier.

23. The composition according to claim 22, wherein the heterologous DNA comprises a structural gene for the peptide and a promoter.

The composition of claim 23, wherein the heterologous DNA comprises multiple copies of a gene for the peptide.

25. The composition of claim 22, wherein the peptide is an opioid peptide.

The composition of claim 25, wherein the opioid peptide is selected from the group consisting of β-endorphin, α-endorphin, gamma-endorphin, delta-endorphin, and Met sup 5.

27. The composition of claim 25 , wherein the opioid peptide is selected from the group consisting of β-endorphins and enkephalins, and wherein the heterologous DNA comprises a promoter for an endogenous structural gene encoding the peptide.

28. The composition 22, wherein the peptide is a polypeptide that binds substance P.

29. The composition of claim 22, wherein the peptide is a substance P analogue comprising the sequence -Phe-Phe-Gly-Leu. -Met.

30. The composition of claim 22, further comprising long chondroitin-6-sulfate proteoglycan.

The composition of claim 30, wherein the long chondroitin-6-sulfate proteoglycan is low-sulfated.

32. The composition of claim 22, further comprising insulin.