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Title:  Pharmacotherapeutic process and composition for central nervous system disorders

United States Patent:  6,491,939

Issued:  December 10, 2002

Inventors:  Kubek; Michael J. (Indianapolis, IN)

Assignee:  Advanced Research and Technology Institute, Inc. (Indianapolis, IN)

Appl. No.:  897179

Filed:  July 2, 2001

Abstract

Methods and compositions are disclosed for providing prolonged-release of therapeutic agents by way of in situ stereotaxic implantation in specific loci, including pathways, to treat known disorders. One or more microstructures comprising therapeutic agents and pharmaceutically acceptable carriers are implanted, for example, through a cannula. The microstructures are of a sufficient size and shape to prevent dispersion from the implant site.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following portion of the specification sets forth the preferred embodiments of the present invention. The embodiments of the invention disclosed herein include the best mode contemplated by the inventor for carrying out the invention in a commercial environment although it should be understood that various modifications can be accomplished within the parameters of the present invention.

In accordance with the present invention, microstructures containing a therapeutic agent, such as TRH and/or TRH analogs, and a nontoxic carrier that is biodegradable at body temperature can be used singly or in concert with other microstructures containing the same or other components at specific central nervous system loci. The microstructures of the present invention have significant utility, for example, in the treatment of many neurodegenerative disorders caused by excessive glutamate or aspartate release, such as stroke, epilepsy, ischemia, trauma, sclerosis, Alzheimer's disease and others.

Critically, the microstructures form a size and shape that is sufficiently large to prevent dispersion of the microstructure from one or more selected implant loci while also providing the necessary surface geometry to provide a relatively constant rate of release of the drug by surface erosion to the desired in situ site. The microstructures of the present invention include any shape in which, during erosion, the surface area of the microstructure decreases at a rate less than that of a microsphere, as described in more detail below. In a most preferred embodiment, such non-spherical microstructures may be in the form of microdisks.

While the thickness and diameter of the microdisks and other non-spherical microstructures can vary, the microdisks are preferably compatible with commercially available needles having relatively small diameters, for example, 24 to 15 gauge needles. The most ideal size of the microdisks and other non-spherical microstructures will differ depending upon the application but is selected in order to prevent dispersion, as noted above, and must also not be so large as to damage cells during implantation. By way of example, microdisks can have a diameter ranging from approximately 0.3 millimeters to approximately 1.5 millimeters and larger diameters can be inserted through other known stereotaxic methods particularly up to 5 millimeters, and can have an exemplary thickness ranging from about 0.1 millimeters to about 5.0 millimeters, most preferably about 0.2 millimeters. Microstructures having diameters significantly larger than the thickness or having a thickness significantly larger than the diameter are most preferred. Accordingly, the microdisks and other non-spherical microstructures of the present invention can be made larger than the microspheres of the prior art and can therefore avoid the possibility of dispersion in extracellular spinal fluid and they are therefore less susceptible to nonspecific uptake and delivery to more distant sites in the brain by CSF, glia and retrogradely by neurons. In addition, the microdisks and other non-spherical microstructures of the present invention can optimize the rate of drug delivery to the in situ sites. Advantageously, the TRH microdisks of the present invention, for example, provide sustained release as demonstrated by in vitro tests which show that the sustained release can exceed 70 hours, as seen in FIG. 1. This sustained release is important in view of the mechanism for inhibiting neurotransmitter release, as described in more detail herein below.

The TRH, TRH analogs, and/or other active therapeutic agents can comprise from about 1 percent to about 90 percent by weight of the polyanhydride microstructure such as a microdisk. Preferably, the microstructure comprises from about 1 to about 60% of the therapeutic agent in order to optimally control delivery of the drug through the biodegradable matrix, and more preferably, the therapeutic agent comprises from about 1% to about 10% of the microstructure. Also, dose effects can vary depending upon the desired applications, as well. For example, lower doses of TRH and/or TRH analogs can be sufficient to inhibit glutamate release, but in higher doses, the microstructures containing TRH and/or TRH analogs can more effectively eliminate glutamate and aspartate.

It is to be noted that non-spherical microstructures such as microdisks are also advantageously desired over other methods for providing sustained release such as minipumps. For example, drug delivery by microstructures is not susceptible to the increased risk of infection found in the use of minipumps. In addition, minipumps are also relegated to one site, whereas microstructures of the present invention can be advantageously placed in several sites. Further, the microstructures of the present invention have the advantageous capability of sinusoidal delivery. In this regard, the microstructures can be formed with a porous structure, as desired, which can be designed to degrade at differing rates in order to control the release of drug, for example, by selecting differing high and/or low concentration release cycles.

As noted above, the non-spherical microstructures such as microdisks can be multiply implanted but single microstructures can also be implanted if desired. It is most preferred to perform a single implantation at a specific locus in which one or more microstructures are implanted at one time by way of a cannula, and the cannula is then removed with the microstructures then left to biodegrade, without any toxic products, at body temperature of about 37oC C. Alternatively, repeated application is also contemplated under the present invention in which the microstructures are applied over time. In this regard, the delivering cannula remains available for microstructures to be implanted at various times and the cannula can be stereotaxically adjusted as desired.

The present invention is not limited by the specific locus selected for drug delivery. For example, TRH can have efficacy in any part of the central nervous system but is more applicably efficacious in regions where the density of TRH receptors is high, particularly, in the amygdala, the hippocampus and other limbic and neocortices, as well as in the spinal cord and the optic retina in the eye.

The carrier facilitates sustained release and eliminates the possibility of burst release in which there is a large loading dose in which for example, 90 percent of the drug is released quickly. In contradistinction, the carriers of the present invention are selected to release a relatively constant amount of active therapeutic agent by erosion from the surface over time. More specifically, over a preselected period of time for sustained release, the rate of change of the surface area of non-spherical microstructures such as microdisks can be designed to change relatively slowly, as opposed to the microspheres of the prior art, which will erode so that the surface area decreases quickly, and are therefore subject to a burst release. This problem of burst release is compounded when the microspheres increase in size. Whereas in an idealized model, the surface area of a sphere will erode at a rate of 8 .pi.r (dr/dt), where r is the radius and (dr/dt) is the time rate of change of r, the surface area of the microstructures of the present invention will decrease with erosion at a rate less than 8 .pi.r (dr/dt), preferably at a rate less than about 3.5 .pi.l (dl/dt), where l is a characteristic size of the microstructure and (dl/dt) is the time rate of change of l. In this regard, the term "characteristic size" refers to a size representative or typical of the microstructure and, in the case of a microsphere, refers to the diameter of the microsphere, while in the case of a microdisk having thickness much less than radius, refers to the diameter of the microdisk.

Referring now to the mechanism of action, the present invention has particular utility in providing an agonist that can modulate release of endogenous compounds, such as neurotransmitters, neuropeptides or hormones, by way of a novel mechanism of desensitizing a heterologous receptor by downregulating G-proteins common to both an agonist, or homologous, receptor and the heterologous receptor that is selected for desensitization. A number of conditions are important in this mechanism for achieving prolonged heterologous receptor desensitization. For example, homologous and heterologous receptors must be highly expressed in the same cell such as a neuron. In addition, the homologous and heterologous receptors must utilize the same G-protein signaling system, for example, Gi or Gq, The homologous receptor must be downregulated, that is, effectively reduced, by its transmitter/modulator and agonists. Also, the downregulation of the homologous receptor must be associated with downregulation of its specific G-protein. Critically, sustained receptor exposure with agonist is required for prolonged desensitization to occur.

As an example, the following discussion refers to modulation in the form of inhibition of glutamate release, but it will be appreciated that this discussion is merely exemplary and is not limiting to the present invention. It will be appreciated that the mechanism of the present invention will also function to modulate second messenger systems, including increase in release.

In accordance with one aspect of the present invention, metabotropic glutamate receptors (mGluRs) make up a small portion of the much larger superfamily of G-protein coupled receptors consisting of seven transmembrane spanning regions coupled to second messenger systems, such as adenylyl cyclase/cAMP, phospholipase-C (PLC)/DAG, IP3, by a class of GTPases termed G-proteins. One of ordinary skill in the art will appreciate that G-proteins are heterotrimeric and composed of .alpha., .beta., and .gamma. subunits encoded by a distinct gene. In particular, G-protein .alpha. subunits are subdivided into four main classes termed Gs, Gi, Gq, and G12. In addition to diversity among .alpha.-chains, there are also multiple genes encoding at least 4 .beta.- and at least 6 .gamma.-subunits. The .alpha. subunits appear to be the most important in regulating the signal cascade wherein both fast transmission (ionic) and long-term (Ca++ -dependent immediate early gene activation) events can be modulated. The .alpha. subunits of the Gq -like G proteins (Gq/11) have been observed to play a key role in the regulation of intracellular Ca++ levels and in the generation of second messenger systems. Therefore, this effector system is found among the metabotropic glutamate receptors as opposed to the ionotropic receptors (iGluRs) which are the second major category of glutamate receptors. It is to be noted that the iGluRs have been pharmacologically characterized by selective agonists and antagonists into three major classes, NMDA, AMPA, and Kainate. Activation of these receptors results in gating of cations (Na+, Ca++) from the extracellular fluid, through a specific ion channel. This ligand-dependent ion gating renders the interior of the target cell less negative, and thus resultant depolarization enhances cell excitability. Several genetic variants of each class of ionotropic receptor have been cloned but none of the ionotropic glutamate receptors are coupled to the G-protein effector pathways.

It is now recognized that a large proportion of the neurotransmitters (glutamate, GABAB, acetylcholine, dopamine, etc.), neuropeptides (TRH, neuropeptide-Y (NPY), cholecystokinin (CCK), neurotensin (NT), etc.), and hormones (glucagon, melatonin, etc.), act through G-protein linked receptors. Presently, eight different mGluR subtypes (mGluR1-8) have been cloned and subsequently expressed in various cell lines. The mGluRs have been classified into three groups based on amino acid sequence similarity, agonist/antagonist pharmacology and signal transduction pathways to which they couple. More specifically, group I mGluRs (mGlu1 and mGlu5) stimulate phospholipase-C/DAG, IP3 through G.alpha.q/11 G proteins. Meanwhile, group II (mGluR2 and mGluR3) and group III (mGluR4 and mGluR6-8) are negatively coupled to adenylyl cyclase/cAMP through G.alpha.i/o G proteins.

The mGluRs are believed to modulate glutamate synaptic transmission via both presynaptic and postsynaptic mechanisms. Inhibition of transmitter release occurs following activation of presynaptic Group II and III mGluRs, most likely through direct G protein-mediated (G.alpha.i/o) inhibition of Ca++ channels, and not through their negative coupling to adenylyl cyclase. In marked contrast, activation of Group I mGluRs enhances glutamate release via a mechanism involving G.alpha.q/11 G protein-mediated PLC/protein kinase C (a product of DAG activity) inhibition of presynaptic K+ channels.

Postsynaptic Group I mGluRs mediate slow depolarization and an increase in cell firing. This effect appears to be due to a depression of K+ currents directly by G.alpha.q/11 rather than as a consequence of their coupling to PLC. Postsynaptic Group I mGluRs may also modulate both AMPA and NMDA iGluR-mediated currents indirectly, probably via PKC-mediated phosphorylation of their respective Ca++ ion channels.

It is known that activation of Group I receptors (and iGluRs) induces seizures and appears to contribute to excitotoxicity and cell death. In contrast, activation of Group II/III mGluRs reduces glutamate release and produces neuroprotective effects.

From the brief discussion above, it is clear that several ligand initiated events can and are affected by both endogenous transmitter and agonist/antagonist receptor interactions. Recent data have shown that G proteins are critical in the signal transduction pathway and when downregulated can affect activity of both the homologous and a heterologous receptor that utilizes the same G protein signaling cascade. As noted above, of all the glutamate receptors, only mGluRs utilize G protein coupling. Moreover, of the three mGluR subgroups, only Group I mGluRs (mGlu1 and mGlu5) use G.alpha.q/11 for signal transduction. Importantly, it is well recognized that G.alpha.q/11 G proteins couple the TRH receptor (TRHr) to PLC for cell signaling. The TRHr is known to be significantly downregulated both by sustained exposure to ligand and following seizures in neurons that co-localize glutamate and TRH as well as their receptors.

Homologous receptor downregulation is essential for G protein downregulation. In this regard, it has been previously demonstrated that sustained exposure (16 hr.) of TRH to the cloned TRHr results in substantial subcellular redistribution and marked dose-dependent downregulation of G.alpha.q/11 G proteins without affecting cellular levels of the .alpha. subunits of Gs, Gi1-3, or Go. Group I mGluRs are the only glutamate receptors that require the G.alpha.q/11 subunit to affect presynaptic glutamate release and postsynaptic ion channel effects (see above), and sustained TRH exposure to its receptor results in relocation and substantial (20-70%) reduction of G.alpha.q/11 G proteins. Therefore, the prolonged exposure of the TRHr to ligand, as from the TRH-polyanhydride microstructure carrier in accordance with the present invention, heterologously modulates (uncouples) G.alpha.q/11 from the Group I mGluR in those cells that express both the TRHr and Group I mGluRs resulting in prolonged Group I desensitization to pre-and postsynaptic glutamate stimulatory effects and potentiation of glutamate-induced Group II/III inhibitory effects.

This mechanism can account, in large part, for TRH effects observed on inhibition of glutamate release and suppression of neuronal Ca++ uptake. This novel mechanism of prolonged desensitization of Group I mGluRs by sustained TRH release in situ could account for the enhanced and prolonged duration of antiepileptogenic and anticonvulsant effects of TRH in the kindling model of temporal lobe epilepsy. This effect would not be limited to seizures, and its related cell damage, but could include modulation of other proposed excitotoxic effects of excessive glutamate release as well, including neurodegeneration associated with neurotrauma, stroke, ischemia and Alzheimer's dementia. Thus, it is clear that heterologous desensitization by TRH could result with other G protein receptors that utilize G.alpha.q/11 coupled signaling cascades. However, receptors that use G.alpha.q/11 coupling are remarkably restricted and include only the M1 acetylcholine receptor and a limited number of neuropeptides and hormones such as neurotensin, vasopressin and bradykinin.

Claim 1 of 14 Claims

What is claimed is:

1. A method for selectively increasing release of a predetermined endogenous compound comprising the steps of:

selecting an in situ locus in the central nervous system which includes agonist receptors and heterologous receptors that are coupled to common G-proteins; and

stereotaxically delivering to the in situ locus at least one biodegradable, non-spherical polymeric microstructure comprising an agonist and a pharmaceutically acceptable carrier, wherein the microstructure has a size and shape sufficient to prevent dispersion from the locus, and wherein the microstructure provides sustained release of the agonist via surface erosion to effectively upregulate G-proteins common to the agonist receptor and the heterologous receptor to potentiate the heterologous receptor in order to increase release of the endogenous compounds released by the heterologous receptor.

 


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