# Patent

## Title
Dosing regimens for neural stem cell proliferating agents and differentiating agents for the treatment of neurological disorders

## Abstract

Effective dosing regimens for neural stem cell proliferating and differentiating agents, kits comprising effective dosing regimens for neural stem cell proliferating and differentiating agents, and uses thereof are provided herein. Such kits and methods can be utilized acutely or chronically to treat a neurodegenerative disease or condition. Furthermore, the compositions and methods can be used continuously or intermittently in various dosing regimens.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to and the benefit of U.S. Provisional Application Ser. No. 60/783,500, filed on Mar. 17, 2006; U.S. Provisional Application Ser. No. 60/789,132, filed on Apr. 5, 2006; and U.S. Provisional Application Ser. No. 60/862,669, filed on Oct. 24, 2006, which are incorporated herein by reference in their entireties.


BACKGROUND
The development of techniques for the isolation and in vitro culture of multipotent neural stem cells (for example, see U.S. Pat. Nos. 5,750,376; 5,980,885; 5,851,832) significantly improved the outlook for the treatment of neurodegenerative diseases and conditions. It was discovered that fetal brains can be used to isolate and culture multipotent neural stem cells in vitro. Moreover, in contrast to the long held belief that adult brain cells are not capable of replicating or regenerating brain cells, it was found that neural stem cells may also be isolated from brains of adult mammals. These stem cells, either from fetal or adult brains, are capable of self-replicating. The progeny cells can proliferate or differentiate into any cell in the neural cell lineage, including neurons, astrocytes and oligodendrocytes. Therefore, these findings not only provide a source of neural cells which can be used in transplantations but also demonstrate the presence of multipotent neural stem cells in adult brain.
Certain agents, neural stem cell proliferating agents, have been found to increase the number of neural stein cells in vitro or in vivo. The mechanisms for such increase may include stimulating proliferation, inhibiting differentiation, and/or preventing death of the neural stem cells. Additional agents, stem cell differentiating agents, have been found to selectively enhance the production of neuronal precursor cells or glial precursor cells in vitro or in vivo. These proliferating and differentiating agents can thus be employed to increase and selectively enhance neurons and glial cells.
SUMMARY
Provided herein are effective dosing regimens for neural stem cell proliferating agents and differentiating agents, kits, and uses thereof. Such compositions of matter and methods can be utilized acutely (e.g., within days after neural injury or onset of neurologic symptoms) or can be used chronically (e.g., for a persisting neural injury or ongoing neurologic symptoms). Furthermore, the compositions and methods can be used continuously or intermittently.
Accordingly, a method for treating or ameliorating a neurodegenerative disease or condition in a mammal is provided. The method comprises administering to the mammal an effective amount of hCG or LH and an effective amount of EPO, wherein the hCG or LH is administered systemically in at least three doses, optionally by use of a kit. The hCG, LH, and/or EPO can be administered either continuously or intermittently. Further, the hCG or LH can be administered in a first treatment period and EPO can be delivered in a second treatment period. For example, hCG or LH can be administered intermittently on days 1, 3, and 5 of a first treatment period, then EPO can be administered continuously on days 1, 2, and 3 of a second treatment period.
Also provided herein is a further method for treating or ameliorating a neurodegenerative disease or condition in a mammal. The method comprises administering to the mammal an effective amount of hCG or LH in a first treatment period followed by an effective amount of EPO in a second treatment period, optionally by use of a kit. The hCG or LH can be delivered intermittently during the first treatment period and the EPO can be delivered continuously during the second treatment period. For example, hCG or LH can be administered intermittently on days 1, 3, and 5 of a first treatment period, then EPO can be administered continuously on days 1, 2, and 3 of a second treatment period.
In the methods and kits, the treatment periods may be, for example, at least three days. The treating methods can be repeated several times or many times with second, third, forth, fifth, etc. treating periods. The treating methods, whether administered once, twice, several, or many times, can take the form of one or more kits.
The details of methods and kits are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the methods and kits will be apparent from the description and drawings, and from the claims.



BRIEF DESCRIPTION OF FIGURES
FIG. 1 shows the effect on functional recovery of a rat subjected to a Middle Cerebral Artery occlusion (MCAo) stroke with increasing dosages of hCG with an intravenous (IV) administration of 1440 ID EPO per day following intramuscular (IM) administration of dosages of hCG.
FIG. 2 shows the differential effect on functional recovery of a rat subjected to a MCAo stroke, compared to untreated controls, of 440 IU of hCG with an IV administration of 1440 IU EPO per day, hCG alone, or EPO alone.
FIG. 3 is a graph indicating % tissue loss (compared to non-stroke hemisphere) in rats subjected to a MCAo stroke, compared to untreated controls, of 440 IU of hCG with an IV administration of 1440 IU EPO per day, hCG alone, or EPO alone.
FIG. 4 shows representative images of tissue loss in rats subjected to a MCAo stroke, compared to untreated controls, of 440 IU of hCG with an IV administration of 1440 IU EPO per day, hCG alone, or EPO alone.
FIG. 5 is a bar graph indicating serum hCG levels as measured after a third IM administration of hCG in Example 2.
FIG. 6 shows the result of six day subcutaneous prolactin infusions in male rats at 10, 15, and 20 times the concentrations used for intracerebroventricular infusions. The total number of bromodeoxyuridine positive (BrdU+) cells in the sub-ventricular zone (SVZ) for 8 sections from each animal is presented. The greatest increase in SVZ proliferation levels was observed with the 15 times dose (170 μg/day for 6 days). (10 times=113 μg/day; 20 times=226 μg/day; Control=rat serum albumin only (RSA)). Significance relative to control: 10×=*p<0.05; 15×=**p<0.01; 20×=p<0.05; n=3 for all conditions; one way analysis of variance (ANOVA) with Tukey posthoc test.
FIG. 7 shows the results of prolactin dosing in male rats using single daily intraperitoneal injections. The total number of BrdU+ cells per section are presented for each dosing regime. (A) A small increase in SVZ proliferation was observed with high 3 day doses. (B) The most robust dosing condition for increasing SVZ proliferation levels used a low, 170 μg/day dose each day over 6 days. Significance is relative to RSA control, n=3; *p<0.05; **p<0.01; one-way ANOVA followed by a Tukey posthoc test.
FIG. 8 shows that single intramuscular injections of hCG on days 1, 3, and 5 post-stroke (stroke=day 0) trigger significantly increased proliferation in the forebrain SVZ. Significant increases in the number of Phospho-Histone H3 positive (pHH3+) cells per ventricle were observed in the 1000 μg dose condition (n=3; *p<0.05; one way ANOVA with Tukey posthoc). Images show the nuclear label. Hoechst and pHH3 expression in the dorsolateral corner of the lateral ventricles in RSA pial strip control rats versus 1000 μg hCG dosed animals, note the increase in total cell number and pHH3 expression in SVZ of 1000 μg dosed animals.
FIG. 9 shows that single intramuscular injections of 1000 μg per day of hCG on days 1, 3, and 5 post-stroke (stroke=day 0) trigger increased neurogenesis in the forebrain SVZ. The number of doublecortin+ neurons was quantified in the dosed animals and was doubled in the 1000 μg dose animals, (n=3; **p<0.01).
FIG. 10 shows the results of single intramuscular injections of hCG given daily for 7 days starting 24 hrs post-stroke (stroke=day 0), (A) The daily 330 μg/injection dosing regime significantly increased the number of proliferating (pHH3+ cells) in the SVZ relative to all other dosing conditions and controls (n=3; *p<0.01; one way ANOVA with Tukey posthoc). (B) Observation of the ischemic lesions in the motor cortex of dosed rats revealed that animals receiving the 330 μg/injection daily dosing regime demonstrated new tissue growth and filling in of the lesion site with a tissue plug.
FIG. 11 shows increased proliferation in the SVZ of 330 μg/injection daily hCG dosed animals as confirmed by counts of BrdU+ cells. The number of BrdU+ cells per ventricle was significantly increased in the 330 μg/injection condition relative to control and 100 μg/injection (p<0.01; n=3; one way ANOVA with Tukey posthoc analysis). These results farther confirmed the increase in proliferation observed with pHH3 staining.



Like reference symbols in the various drawings indicate like elements.
DETAILED DESCRIPTION
Currently there are no neural stem cell proliferating and differentiating agents that have been clinically approved for use in treatment of neurological diseases or conditions. These agents are useful in treating neurological diseases and conditions, thus there is a need for effective dosing regimens using these agents. Effective dosing regimens for neural stem cell proliferating and differentiating agents, kits comprising effective dosing regimens for neural stem cell proliferating agents, and uses thereof are provided herein. Such kits and methods can be utilized acutely (e.g., within days after injury or onset of a neurodegenerative disease or condition) or can be used, chronically (e.g., for a chronic neurodegenerative disease or condition). Furthermore, the compositions and methods can be used continuously or intermittently as further described below.
The methods described herein use neural stem cell proliferating agents for treating or ameliorating a neurodegenerative disease or condition. In these methods, a neural stem cell proliferating agent is administered over the course of a first treatment period. The neural stem cell proliferating agent can be administered continuously or intermittently during the first treatment period. A neural stem cell differentiating agent can further be added during the first treatment period. The examples and description include the use of neural stem cell proliferating agents (e.g., prolactin, hCG, LH, CSF, G-CSF, GM-CSF, VEGF) and differentiating agents (e.g., EPO, BDNF, BMP, PACAP); however, analogs, fragments, or variants of such agents can similarly be used in any of the methods, devices, or kits taught herein. As a specific example, a method is disclosed in which an effective amount of hCG or LH and an effective amount of EPO are administered to a mammal, wherein the hCG or LH is administered systemically in at least three doses.
These methods for using neural stem cell proliferating agents for treating or ameliorating a neurodegenerative disease or condition can further include administering a neural cell differentiating agent in a second treatment period that starts after the end of the first treatment period. The second treatment period can be at least three days. The neural stem cell differentiating agent can be administered continuously or intermittently during the second treatment period. The second treatment period can begin at least one day after the end of the second treatment period. As a specific example, a method is disclosed in which a neural stem cell proliferating agent is administered continuously at least three times systemically over a first treatment period and a neural stem cell differentiating agent is administered over a second treatment period. As a further example, a method is disclosed in which the first treatment period is five days, the neural stem cell proliferating agent is administered intermittently, a second treatment period starts one day after the end of the first treatment period, and the neural stem cell differentiating agent is administered continuously for at least three days. As an additional example, an effective amount of hCG or LH can be administered in a first treatment period followed by an effective amount of EPO in a second treatment period.
As used herein, to deliver or administer a substance continuously to a subject means to deliver or administer the substance at least once per day for a period of consecutive days. For example, the substance may be administered systemically by injection (e.g., IM or subcutaneously) or taken orally daily at least once per day, or administered by infusion in a manner that results in the daily delivery into the tissue or blood stream of the subject. Optionally, the substance is delivered, by infusion or a means other than infusion. As used herein the term systemically does not include intracerebral ventricular infusion. The duration, or treatment period, during which the substance is continuously delivered or administered can last from three days to several years, even for the rest of a subject's life. For example, the duration may be 3-6 days, 3-14 days, 3-21 days, 3-28 days, 1-4 months, 1-6 months, 1-9 months, 1-12 months, 1-2 years, 1-3 years, 1-5 years, 1-10 years, and the like. For further example the treatment period for continuous delivery can be at least three days, at least four days, at least five days, at least six days, at least seven days, or at least fourteen days. Further, the substance can be delivered consecutively on days 1, 2, and 3 of the administration period.
As used herein, to deliver or administer a substance intermittently to a subject means to deliver or administer the substance less than daily, including, for example, once every 2, 3, 4, 5, or 7 days for a period of time. For example, the substance may be delivered or administered every other day of a treatment period, e.g., on days 1, 3, and 5 of a treatment period. The duration, or treatment, period, during which the substance is intermittently delivered or administered can last from three days to several years, even for the rest of a subject's life. For example, the duration may be 3-6 days, 3-14 days, 3-21 days, 3-28 days, 1-4 months, 1-6 months, 1-9 months, 1-12 months, 1-2 years, 1-3 years, 1-5 years, 1-10 years, and the like. For further example the treatment period for intermittent delivery can be at least three days, at least four days, at least five days, at least six days, at least seven days, or at least, fourteen days.
The methods provided herein, for example, can use the proliferating agents prolactin, hCG, LH, CSF, G-CSF, GM-CSF, or VEGF for treatment of a neurodegenerative disease or condition through administration of an effective amount of the proliferating agent to the subject with a neurodegenerative disease or condition. By way of example, the proliferating agents hCG and LH bind the same receptor, and can be used interchangeably in equipotent doses in the specific examples provided herein. As a further example, the proliferating agent hCG can be administered intramuscularly (IM) at a dose of about 120-200 IU/kg/day followed by intravenous (IV) administration of about 570-950 IU/kg/day of EPO. For further example, an hCG can be intramuscularly administered at a dose of 160 IU/kg/day followed by intravenous administration of 765 IU/kg/day of EPO. Intermittent treatment with hCG and LH optionally comprises several days of hCG or LH administration (e.g., on days 1, 3, 5). Such administration of a neural stem cell stimulating agent can be followed by several days of intermittent (e.g., day 7, 9, 11) or continuous (e.g., on days 7, 8, and 9) administration of a differentiating agent such as EPO. Equipotent doses of other neural stem cell, proliferating agents can also be used in similar regimens.
Thus, Example 4 shows a dosing regimen for prolactin (another proliferating agent). Various amounts of prolactin were administered daily for 6 days and the effects on neural stem cell numbers were examined. The results showed that about 150-200 μg/day (including for example 170 μg/day) was the optimal amount in this dosing schedule. This dosing regimen, about 170 μg/day for 6 days, was then varied by shortening the dosing period (170 μg/day for 3 days) or combining a higher daily dose with a shortened period to achieve a similar total dose (about 396 μg/day for 3 days). The results indicated that the continuous delivery of a lower dose over a longer period time is effective.
The methods including continuous delivery or intermittent delivery provided herein can improve neurologic status. Without meaning to be limited, this improvement can be related to an increase in the number of neural stem cells in a mammal. The efficacy of an effective amount of a neural stem cell proliferating agent can be optimized to increasing the number of neural stem cells or the neurologic status in a mammal. The methods comprise administering the neural stem cell proliferating agent to the mammal continuously or intermittently for a period of time, wherein the total dosage of the neural stem cell proliferating agent administered in said period, of time equals the effective amount, and wherein, the agent is administered at least three times over the first treatment period.
The methods described herein can be optimized to increase the efficacy of an effective amount of a neural stem cell proliferating agent in treating or ameliorating a neurodegenerative disease or condition in a mammal. The methods comprise administering the neural stem cell proliferating agent to the mammal continuously or intermittently for a period of time, wherein the total dosage of the neural stem cell proliferating agent administered in said period of time equals the effective amount, and wherein the agent is administered at least three times over the first treatment period.
The neural stem cell proliferating agent can be administered to the mammal within about 14 days (e.g., 0 to about 14 days) of a central nervous system (CNS) injury, onset of symptoms, or diagnosis. As used herein 0 days refers to the time of CNS injury, onset of symptoms, or diagnosis. Optionally, the neural stem cell proliferating agent can be administered within about 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day(s) (e.g., 0 to about 5 days) of a CNS injury, onset of symptoms, or diagnosis. Optionally, the neural stem cell proliferating agent can be administered to the mammal within 24 hours of a CNS injury, onset of symptoms, or diagnosis. Optionally, the neural stem cell proliferating agent can be administered to the mammal within 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hour(s) of a CNS injury, onset of symptoms, or diagnosis.
For intermittent treatment, higher doses of the agent can be used, or continuous treatment can be used. In particular, neural stem cell proliferating agents can be delivered to mammalian subjects at a low dose in a continuous fashion, as opposed to the administration of a high-dose intermittently. The neural stem cell proliferating agent(s) optionally is/are delivered within 24 to 72 hours after neural injury or the onset of neurological symptoms. Optionally, continuous administration of neural stem cell proliferating agents is for 2-5 days. For example, for a given total effective dose of prolactin, or analogs, fragments or variants of prolactin, a dosing regimen comprising daily delivery of 1⁄6 of the total amount for six days was more effective than delivering 1⁄3 of the total amount daily for three days. Equipotent doses of other neural stem cell proliferating agents can be used in similar paradigms.
The methods described herein can also include monitoring levels of the neural stem cell proliferating agent or neural stem cell differentiating agent in a biological fluid of the mammal. The biological fluid monitored can be, for example, cerebral spinal fluid or blood. For example, the level of hCG (or another neural stem cell proliferating agent or neural stem cell differentiating agent) in blood serum can be measured after administration either during or after a treatment period. Equipotent levels of various neural stem cell proliferating agent or neural stem cell differentiating agent can be both determined and monitored in biological fluid.
Also provided herein is a kit far the treatment or amelioration of a neurodegenerative disease or condition in a mammal. The kit comprises at least three dosage units of a neural stem cell proliferating agent for administration during a first treatment period. The total dosage of the neural stem cell proliferating agent administered in that first treatment period can equal an effective amount. The treatment period can be at least three days. The kit can include instructions for use of the kit. The instructions can be for continuous administration or for intermittent administration of the neural stem cell proliferating agent.
The kit can further provide at least three dosage units of a differentiating agent. The differentiating agent can be used over the first treatment period. The total dosage of the differentiating agent administered in the first treatment period can equal an effective amount. The treatment period can be at least three days. The treatment period for the differentiating agent optionally is a second or subsequent treatment period that follows the treatment period or periods with the neural stem cell proliferating agent. The kit can include instructions for use of the differentiating agent. The instructions can be for continuous administration or for intermittent administration of the neural stem cell proliferating agent.
The total dosage of each of the neural stem cell proliferating agent differentiating agent, or other agents in the kit can be provided in one container, a plurality of containers, or any combination thereof. For example, the total dosage for the neural stem cell proliferating agent or agents can be in one container suitable for providing a metered dose or suitable for extraction of a dose, for example, by the person to be treated or by another person, such as a caregiver. Instead of a single container, the neural stem cell proliferating agent or agents can be present in a plurality of containers that provide aliquots for doses suitable for administration daily, weekly, monthly, or the like. A single container or a plurality of containers for the differentiating agent or other agents can similarly be provided in the kit. Combinations may also be included whereby one container of neural stem cell proliferating agent(s) but a plurality of differentiating agent(s) containers or the opposite may be included in the kit. Also, the total dosage of a neural stem proliferating factor for a first treating period may be in a single container or a plurality of containers, the total dosage for a second treating period may be in a single container or a plurality of containers, or any combination thereof.
The neural stem cell proliferating agent and the differentiating agent can optionally be packaged in a kit, such that the total amount, of the neural stem cell proliferating agent and the differentiating agent to be delivered during the treating period(s) is contained in the kit. The kit can optionally contain other components or combinations of other components, including for example a blood sampling device or a component thereof.
The kit can further comprise a device or means for monitoring hematocrit levels in a patient or a suitable device for removing an amount of blood from the patient or both a monitor and a blood sampling device. Blood sampling and monitoring is desirable because hematocrit levels may rise above acceptable levels. Acceptable hematocrit levels can be determined by any standard established in the art.
Optionally, a drug delivery device for administration can be included in a kit containing the neural stem cell proliferating agent(s) and/or the differentiating agent(s).
The kit can be suitable for use in a health care facility such as an inpatient care facility or an emergency care facility. A health care facility includes, for example, a hospital. The kit is also suitable for use after discharge from or without admission in an the inpatient care facility. Packaging in the form of a kit advantageously facilitates early release of patients from a health care facility by permitting patient treatment at a long term care facility or at home, for example, by self-treatment, outpatient treatment, or treatments by a caregiver or health care provider in a home, a long term care facility, or the like. Similarly, packaging in the form of a kit allows immediate treatment of a patient in an acute situation, including an emergency room or by an on-site emergency care provider (e.g., by an emergency medical technician, an athletic trainer, or the like).
In the methods and kits, the period of time may be, for example, at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, fourteen, twenty one, twenty eight days, or any number of days between 3 and 28. Optionally, the methods and kits may comprise administering to the mammal the neural stem cell proliferating agent continuously in a second treating period, wherein the second treating period starts after the end of the period of time by an interval of at least one, two or three days, and wherein the second treating period is at least three days. The second treating period, like the first treating period, may be, for example, at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, fourteen, twenty one, or twenty eight days. The interval between the first treating period and the next treating period may also be, for example, at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, fourteen, twenty one, or twenty eight days. This treating schedule can be repeated several times or many times. The neural stem cell proliferating agent used in the second or subsequent treating period may be the same as or different than the neural stem cell proliferating agent used in the first treating period or used in other treating periods. Furthermore, more than one neural stem cell proliferating agent may be used in a single treating period. Thus, kits useful in the methods may contain one or more neural stem cell proliferating agents for one or more treating periods.
The neural stem cell proliferating agent(s) or other agents (e.g., differentiating agents) can be administered by any method established in the art, such as by intravenous, infra-arterial, intracolonical, intratracheal, intraperitoneal, intranasal, intravascular, intrathecal, intracranial, intramarrow, intrapleural, intradermal, subcutaneous, intramuscular, oral, topical administration, pulmonary administration, or any combination thereof. Optionally, the drug delivery device or component thereof for administration can be included in a kit containing the neural stem cell proliferating agent.
The neural stem cell proliferating agent may be any substance that is capable of increasing the number of mammalian neural stem cells, in vitro or in vivo. As used herein a promoting agent has the same meaning as a proliferating agent. Agents that can increase neural stem cell number include, but are not limited to:

    
    
        1. Follicle-stimulating hormone (FSH), which often acts in concert with LH and induces LH receptor expression, thereby enhancing the effects of LH signaling.
        2. Growth hormone (GH), which can stimulate neural stem cell proliferation.
        3. Insulin growth factors (IGFs), including IGF-1, which are somatomedians that are released from many tissues in response to GH and mediate many of the growth proliferating effects of GH and which stimulate neural stem cell proliferation.
        4. Growth hormone releasing hormone (GHRH), which is secreted from the hypothalamus and induces GH release from the anterior pituitary, resulting in increased levels of circulating GH.
        5. Prolactin (PRL), which is secreted from the anterior pituitary and which is promotes neural stem cell proliferation.
        6. Prolactin releasing peptide (PRP), which triggers the release of prolactin.
        7. Fibroblast growth factor (FGF), a mitogenic agent for neural stem cells.
        8. Estrogen, which promotes the proliferation of neural stem cells, including for example in the hippocampus.
        9. Serotonin, which promotes the proliferation of neural stem cells in the hippocampus.
        10. Epidermal growth factor (EGF), a mitogenic agent for neural stem cells.
        11. Transforming growth factor alpha (TGFα), a mitogenic agent for neural stem cells.
        12. Gonadotropin releasing hormone (GnRH), which triggers the release of LH.
        13. Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) which signal via the gp130 subunit by a signaling pathway that promotes neural stem cell self-renewal, thereby expanding the neural stem cell population of the brain.
        14. Colony stimulating factor (CSF).
        15. Granulocyte colony stimulating factor (G-CSF).
        16. Granulocyte-macrophage colony stimulating factor (GM-CSF).
        17. Vascular, endothelial growth factor (VEGF).
        18. Lutenizing hormone (LH).
        19. Human chorionic gonadotropin (hCG).
    
    


Furthermore, neural cell differentiating agents can be administered to selectively enhance neuron formation or glial cell formation. These differentiating agents can also be delivered according to the dosing regimens and kits. Exemplary differentiating agents include, but are not limited to:

    
    
        1. Erythropoeitin (EPO), which enhances neural stem cell commitment to neuronal cell lineage and is useful for treating mouse and rat models of stroke.
        2. Brain derived neurotrophic factor (BDNF), which is a known survival factor and differentiating agent that promotes the neuronal lineage.
        3. Transforming growth factor beta and bone morphogenetic proteins (BMPs), which are differentiating agents that promote the neuronal lineage and the generation of specific neuronal phenotypes (e.g., sensory interneurons in the spinal cord).
        4. Thyroid hormone (TH, including both the T3 and T4 forms), a differentiating agent that promotes the maturation and generation of oligodendrocytes. See, e.g., Rodriguez-Pena, 1999.
        5. Thyroid stimulating hormone (TSH) and Thyroid releasing hormone (TRH), which promote the release of TH from the anterior pituitary resulting in increased levels of circulating TH. This agent could be used in combination with LH or hCG to promote oligodendrogliogenesis from neural stem cells.
        6. Sonic hedgehog (SHH), a morphogen that patterns the developing CNS during development and, in different concentrations, promotes the generation of specific types of neurons (e.g., motor neurons in the spinal cord) and oligodendrocytes. This agent could be used in combination with LH or hCG to promote neurogenesis and/or oligodendrogliogenesis from, neural stem cells.
        7. Platelet derived growth factor (PDGF), which promotes the generation and differentiating of oligodendrocytes from neural stem cells. This agent could be used in combination with LH or hCG to promote oligodendrogliogenesis from neural stem cells.
        8. Cyclic AMP and agents which enhance the cAMP pathway, such, as pituitary adenylate cyclase activating polypeptide (PACAP) and serotonin, which selectively promote neuron production.
    
    


Any of the methods and kits can comprise a plurality of neural stem cell proliferating agents and/or neural cell differentiating agents. Thus, one or more neural stem cell proliferating agents can be administered together or sequentially and can be administered via separate compositions or in combination within a single composition. Further, one or more neural stem cell proliferating agents and one or more neural stem cell differentiating agents can be administered together or sequentially and can be administered via separate compositions or in combination within a single composition. For example, PRL and LH or hCG can be used in combination to maximize neural stem cell proliferation; PRP can be used in combination with LH or hCG to maximize neural stem cell proliferation; GnRH can be used in combination with or in place of LH or hCG to increase circulating levels of LH and enhance neural stem cell proliferation; and CNTF and LIP can be used in combination with LH or hCG to promote neural stem cell proliferation and increase the size of the neural stem cell population within the CNS. Further for example, prolactin can be used with EPO, LH can be used with EPO, and hCG can be used with EPO. All other combinations, not explicitly set forth, can also be used.
Appropriate dosages for the factors can be determined according to established methods in the art. For example, the dosage for prolactin may range from about 0.510 IU/kg/day to about 100,000 IU/kg/day, such as, for example, about 0.510-100,000; 0.510-75,000; 0.510-50,000; 0.510-25,000; 0.510-10,000; 100-5,000; 100-2,000; 500-2,000; 1,000-2,000; 100-1,000; 200-800 IU/kg/day. The dosage for hCG can range from about 0.5 IU/kg/day to about 3,000,000 IU/kg/day, such as, for example, about 0.5-2,000,000; 0.5-1,000,000; 0.5-500,000; 0.5-250,000; 0.5-100,000; 0.5-50,000; 10-25,000; 10-10,000; 240-216,000; 1,200-2,000; 2,160; or 1,600 IU/kg/day. hCG can also be provided at a dose of 10,000 IU/day. The dosage for LH can range from about 0.5 IU/kg/day to about 500,000 IU/kg/day, such as, for example, about 0.5-300,000; 0.5-200,000; 0.5-100,000; 0.5-50,000; 0.5-25,000; 24-21,600; 1,000; 120-200; 216; or 160 IU/kg/day. LH can also be provided at a dose of 10,000 IU/day. The dosage for EPO can range from about 100 IU/kg/day to about 2000 IU/kg/day, such as, for example, about 100-1500; 100-1000; 160-1000; 570-950; 765; or 1020 IU/kg/day. EPO can also be provided at a dose of 30,000 IU/day. Equipotent doses of other agents can be used. The dosage here refers to the average dose delivered every day or intermittently during the entire treating period. For example, if the neural stem cell proliferating agent or differentiating agent is not delivered everyday, the total amount of the delivered agent during the entire treating period can be divided by the total number of days in the treating period, including intervals, to arrive at the daily dosage.
Specific dosage units (i.e., the amount or a single administration within a series of administrations in a treatment period) can be specified for a neural stem cell proliferating or differentiating agents to be used with the methods disclosed herein. These dosage units can be within the specific dosages and dosage ranges specified herein. Dosage units can be defined with respect to the amount that must be administered to achieve a desired level of a neural stem cell proliferating or differentiating agent in a subject. For example, a dosage unit of a neural stem cell proliferating agent that provides a neural stem cell proliferating or differentiating agent level in blood serum of 0.03 IU/L to 5,000,000 IU/L. Or, as a further example, a dosage unit of a neural stem cell proliferating or differentiating agent that provides a proliferating agent level in cerebral spinal fluid of about 0.003 IU/L to about 5,000 IU/L.
When the neural stem cell proliferating agent and the differentiating agent are administered systemically, a blood brain barrier permeabilizer can be optionally included in the kits or used in the methods to facilitate entry into the brain. Blood brain barrier permeabilizers are known in the art and include, by way of example, bradykinin and the bradykinin agonists described in U.S. Pat. Nos. 5,686,416; 5,506,206 and 5,268,164 (such as NH2-arginine-proline-hydroxyproxproline-glycine-thienylalanine-serine-proline-4-Me-tyrosine.ψ(—CH2NH)-arginine-COOH).
Alternatively, the neural stem cell proliferating agent or the differentiating agent to be delivered can be conjugated to a transferrin receptor antibody as described in U.S. Pat. Nos. 6,329,508; 6,015,555; 5,833,988 or 5,527,527. The neural stem cell proliferating agent and fee differentiating agent can also be delivered as a fusion protein comprising the neural stem cell proliferating or differentiating, agent and a ligand that is reactive wife a brain capillary endothelial cell receptor, such as the transferrin receptor (see, e.g., U.S. Pat. No. 5,977,307).
The pharmaceutical compositions can be prepared by mixing the desired therapeutic agents with an appropriate vehicle suitable for the intended route of administration, optionally for use in an appropriate drag delivery device. In making the pharmaceutical compositions of this invention, the therapeutic agents are usually mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container. When the pharmaceutically acceptable excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the therapeutic agent. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the therapeutic agents, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
Some examples of suitable excipients include artificial cerebral spinal fluid, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The formulations can additionally include; lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the therapeutic agents after administration, to the patient by employing procedures known in the art.
For preparing solid compositions such as tablets, the therapeutic agent is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the therapeutic agents are dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist, disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein. The compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine.
Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from drug delivery devices which deliver the formulation in an appropriate manner. Another formulation employed in the methods of the present invention employs transdermal delivery devices (“patches”). Such transdermal patches may be used to provide continuous or discontinuous infusion of the therapeutic agent of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, for example, U.S. Pat. No. 5,023,252, herein incorporated by reference. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents. Other suitable formulations for use in the present invention can be found in Remington's Science and Practice of Pharmacy, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott Williams & Wilkins, Philadelphia Pa., 2005.
A mammal treated using the methods and kits described herein can be of any age, including a child, juvenile or an adult.
The terms used in this application are defined as follows unless otherwise indicated.
A neural stem cell or neural stem cell is a stem cell in the neural cell lineage. A stem cell is a cell which is capable of reproducing itself. In other words, daughter cells which result from stem cell divisions include stem cells. The neural stem cells are capable of ultimately differentiating into all the cell types in the neural cell lineage, including neurons, astrocytes and oligodendrocytes, astrocytes and oligodendrocytes are collectively called glia or glial cells. Thus, the neural stem cells referred to herein are multipotent neural stem cells.
A neural stem cell proliferating agent is a substance that is capable of increasing the number of neural stem cells, for example, by stimulating proliferation, inhibiting differentiation, and/or preventing death of neural stem cells.
A neurodegenerative disease or condition is a disease or medical condition associated with neuron loss or dysfunction. Examples of neurodegenerative diseases or conditions include neurodegenerative diseases, central nervous system injuries or dysfunctions. Neurodegenerative diseases include, for example, Alzheimer's disease or other dimentia, multiple sclerosis (MS), schizophrenia, macular degeneration, glaucoma, diabetic retinopathy, peripheral neuropathy, Huntington's disease, amyotrophic lateral sclerosis, and Parkinson's disease, CNS injuries include, for example, cerebrovascular events like strokes (e.g., hemorrhagic strokes, focal ischemic strokes or global ischemic strokes), ocular ischemia, and dural sinus thrombosis; traumatic brain or spinal cord injuries (e.g., injuries caused by a brain or spinal cord surgery or physical accidents); concussion; injury caused by drugs, (e.g., chemotherapeutics, recreational drugs, and neuroleptics); coronary artery bypass graft (CABG) surgery; and ischemia at child birth. CNS dysfunctions include, for example, depression, epilepsy, neurosis and psychosis. Examples of neurodegenerative conditions include aging. The number of neural stem cells in the subventricular zone is significantly reduced in aged mice. Accordingly, amelioration of problems associated with aging is achieved by administering neural stem cell proliferating agents and, optionally, neural stem cell differentiating agents according to the methods and kits.
Treating and ameliorating mean the reduction or complete removal of one or more symptoms (including neurologic symptoms or behavioral performance) of a disease or medical condition. Such treatment or amelioration can include the delay or elimination of the onset of one or more symptoms when, administered to a person at risk for the disease or medical condition. Tests for the success of treatment or amelioration are well known in the art.
A polypeptide which shares substantial sequence similarity with a native factor is at least about 30% identical with the native factor at the amino acid level. The polypeptide is preferably at least about 40%, more preferably at least about 60%, yet more preferably at least about 70%, and most preferably at least about 80% identical with the native factor at the amino acid level. Thus, substantial similarity can constitute about 30-99% identity.
The phrase percent identity or % identity of an analog or variant with a native factor refers to the percentage of amino acid sequence in the native factor which are also found in the analog or variant when the two sequences are aligned. Percent identity can be determined by any methods or algorithms established in the art, such as LALIGN or BLAST.
A polypeptide possesses a biological activity of a native factor if it is capable of binding to the receptor for the native factor or being recognized by a polyclonal antibody raised against the native factor. Preferably, the polypeptide is capable of specifically binding to the receptor for the native factor in a receptor binding assay.
A functional agonist of a native factor is a compound that binds to and activates the receptor of the native factor, although it does not necessarily share a substantial, sequence similarity with the native factor.
A lutenizing hormone or LH is a protein comprising a native mammalian (e.g., human) LH or a peptide sequence comparable to a native mammalian LH. As used herein, an LH analog, variant, or fragment (1) comprises a polypeptide that shares substantial sequence similarity with a native mammalian LH, preferably the native human LH; and (2) possesses a biological activity of the native mammalian LH. The native mammalian LH is a gonadotropin secreted by the anterior lobe of the pituitary. LH is a heterodimer consisting of non-covalently bound alpha and beta subunits. The alpha subunit is common among LH, FSH and hCG, and the beta subunit is specific for each hormone. The LH useful in the present methods and kits may have the native alpha subunit, with the beta subunit sharing a substantial sequence similarity with a native mammalian LH. Alternatively, the LH may have the native beta subunit, with the alpha subunit sharing a substantial sequence similarity with a native mammalian LH. The LH analog, variant, or fragment may also have both the alpha and beta subunit sharing a substantial sequence similarity with a native, corresponding subunit. Thus, the term LH analog or variant comprises a deletional, insertional, or substitutional mutant of a native LH subunit. Furthermore, the term LH encompasses the LHs from other species and the naturally occurring variants thereof. In addition, an LH analog may also be a functional agonist of a native mammalian LB receptor.
A human chorionic gonadotropin or hCG is a protein comprising a native mammalian hCG (e.g., human) or a polypeptide sequence comparable to a native mammalian hCG. As used herein, an hCG analog, variant, or fragment (1) comprises a polypeptide that shares substantial sequence similarity with the native hCG; and (2) possesses a biological activity of the native hCG. The native hCG is a heterodimer consisting of non-covalently bound alpha and beta subunits. The alpha subunit is common among LH, FSH and hCG, and the beta subunit is specific for each hormone. However, the beta subunits of hCG and LH share an 85% sequence similarity. The hCG useful in the present methods and kits may have the native alpha subunit, with the beta subunit sharing a substantial sequence similarity with the native hCG. Alternatively, the hCG may have the native beta subunit, with the alpha subunit sharing a substantial sequence similarity with the native hCG. The hCG analog, variant, or fragment may also have both the alpha and beta subunit sharing a substantial sequence similarity with the native, corresponding subunit. Thus, the term hCG analog, variant, or fragment comprises a deletional, insertional, or substitutional mutants of a native hCG subunit. Furthermore, the term hCG encompasses the hCG counterparts from other species and the naturally occurring variants thereof. In addition, an hCG analog may also be a functional agonist of a native mammalian hCG/LH receptor.
A prolactin is a polypeptide comprising a native mammalian prolactin (e.g., human) or a polypeptide sequence comparable to a native mammalian prolactin. As used herein, a prolactin analog, variant, or fragment (1) shares substantial sequence similarity with a native mammalian prolactin, preferably the native human prolactin; and (2) possesses a biological, activity of the native mammalian prolactin. The native human prolactin is a 199 amino acid polypeptide synthesized mainly in the pituitary gland. Thus, the term prolactin encompasses prolactin analogs, variants, or fragments that are the deletional, insertional, or substitutional mutants of the native prolactin. Furthermore, the term prolactin encompasses the prolactins from other species and the naturally occurring variants thereof.
In addition, a prolactin analog, variant, or fragment may also be a functional agonist of a native mammalian prolactin receptor. For example, the functional agonist may be an activating amino acid sequence disclosed in U.S. Pat. No. 6,333,031 for the prolactin receptor; a metal complexed receptor ligand with agonist activities for the prolactin receptor (U.S. Pat. No. 6,413,952); G120RhGH, which is an analog of human growth hormone but acts as a prolactin agonist (Mode et al., 1996); or a ligand for the prolactin receptor as described in U.S. Pat. Nos. 5,506,107 and 5,837,460.
An epidermal growth factor or EGF is a protein comprising a native mammalian EGF or a polypeptide sequence comparable to a native mammalian EGF. As used herein, an EFG analog, variant, or fragment shares a substantial amino acid sequence similarity with a native EGF, as well as at least one biological activity with the native EGF, such as binding to the EGF receptor. Particularly included as an EGF is the native EGF of any species, TGFα, or recombinant modified EGF. Specific examples include, but are not limited, to, the recombinant modified. EGF having a deletion of the two C-terminal amino acids and a neutral amino acid substitution at position 51 (particularly EGF51 gln51; U.S. Patent Application Publication No. 20020098178A1), the EGF mutein (EGF-X16) in which the His residue at position 16 is replaced with a neutral or acidic amino acid (U.S. Pat. No. 6,191,106), the 52-amino acid deletion mutant of EGF which lacks the amino terminal residue of the native EGF (EGF-D), the EGF deletion mutant in which the N-terminal residue as well as the two C-terminal residues (Arg-Leu) are deleted (EGF-B), the EGF-D in which the Met residue at position 21 is oxidized (EGF-C), the EGF-B in which the Met residue at position 21 is oxidized (EGF-A), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, amphiregulin, neuregulin, or a fusion protein comprising any of the above. Other useful EGF analogs, variants, and fragments are described in U.S. Patent Application Publication No. 20020098178A1, and U.S. Pat. Nos. 6,191,106 and 5,547,935.
In addition, an EGF analog, variant, or fragment may also be a functional agonist of a native mammalian EGF receptor. For example, the functional agonist may be an activating amino acid sequence disclosed in U.S. Pat. No. 6,333,031 for the EGF receptor, or an antibody that has agonist activities for the EGF receptor (Fernandez-Pol, 1985 and U.S. Pat. No. 5,723,115).
A pituitary adenylate cyclase activating polypeptide or PACAP is a polypeptide comprising a native mammalian PACAP (e.g., human) or a polypeptide sequence comparable to a native mammalian PACAP. As used herein, a PACAP analog, variant, or fragment is a native PACAP or any PACAP analog, variant, or fragment that shares a substantial amino acid sequence similarity with a native PACAP, as well as at least one biological activity with the native PACAP, such as binding to the PACAP receptor. Useful PACAP analogs, variants, and fragments include, without being limited to, the 38 amino acid and the 27 amino acid variants of PACAP (PACAP38 and PACAP27, respectively), and the analogs and variants disclosed in, e.g., U.S. Pat. Nos. 5,128,242; 5,198,542; 5,208,320; 5,326,860; 5,623,050; 5,801,147 and 6,242,563.
In addition, a PACAP analog, variant, and fragment may also be a functional agonist of a native mammalian PACAP receptor. For example, the functional agonist may be maxadilan, a polypeptide that acts as a specific agonist of the PACAP type-1 receptor (Moro et al., 1997).
An erythropoietin or EPO is a protein comprising a native mammalian EPO (e.g., human) or a polypeptide sequence comparable to a native mammalian EPO. As used herein, an EPO analog, fragment, or variant shares a substantial amino acid sequence similarity with a native EPO, as well as at least one biological activity with the native EPO, such as binding to the EPO receptor. Examples of EPO analogs, variants, and fragments are disclosed, for example, in U.S. Pat. Nos. 6,048,971 and 5,614,184.
In addition, an EPO analog, variant, or fragment may also be a functional agonist of a native mammalian EPO receptor. For example, the functional agonist may be EPO mimetic peptide 1 (EMP1; Johnson et al., 2000); one of the short peptide mimetics of EPO as described in Wrighton et al., 1996 and U.S. Pat. No. 5,773,569; any small molecular EPO mimetic as disclosed in Kaushansky, 2001; an antibody that activates the EPO receptor as described in U.S. Pat. No. 5,885,574, WO 96/40231, WO 97/48729, Fernandez-Pol. 1985 or U.S. Pat. No. 5,723,115; an activating amino acid sequence as disclosed in U.S. Pat. No. 6,333,031 for the EPO receptor; a metal complexed receptor ligand with agonist activities for the EPO receptor (U.S. Pat. No. 6,413,952), or a ligand for the EPO receptor as described in U.S. Pat. Nos. 5,506,107 and 5,837,460.
A LH/hCG-inducing agent is a substance that, when given to an animal, is capable of increasing the amount of LH or hCG in the animal. For example, LH-releasing hormone (LHRH) stimulates the secretion of LH.
A mammalian pheromone can be a protein or a small molecule comprising a native mammalian pheromone (e.g., human) or a polypeptide sequence or analogous small molecule comparable to a native mammalian pheromone. As used herein, a pheromone analog, variant, or fragment is a substance that serves as a signal to another animal of the same species, usually the opposite gender. Preferably, the pheromone is selected from the group consisting of 2-sec-butyl-4,5-dihydrothiazole (SET), 2,3-dehydro-exo-brevicomin (DHB), alpha and beta farnesenes, 6-hydroxy-6-methyl-3-heptanone, 2-heptanone, trans-5-hepten-2-one, trans-4-hepten-2-one, n-pentyl acetate, cis-2-penten-1-yl-acetate, 2,5-dimethylpyrazine, dodecyl propionate, and (Z)-7-dodecen-1-yl acetate (see, e.g., Dulac et al., 2003).
An effective amount is an amount of a therapeutic agent sufficient to achieve the intended purpose. For example, an effective amount of an LH or hCG to increase the number of neural stem cells is an amount sufficient, in vivo or in vitro, as the ease may be, to result in an increase in neural stem cell number. An effective amount of an LH or hCG to treat or ameliorate a neurodegenerative disease or condition is an amount of the LH/hCG sufficient to reduce or remove one or more-symptoms of the neurodegenerative disease or condition. The effective amount of a given therapeutic agent will vary with factors such as the nature of the agent, the route of administration, the size and species of the animal to receive the therapeutic agent, and the purpose of the administration. The effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art.
An equipotent amount of a neural stem cell proliferating agent is the amount of a neural stem cell proliferating agent required to obtain the same or equivalent effect as another neural stem cell proliferating agent. Equipotent amounts can be specified by a relative level or result of an equipotent amount. Thus, an equipotent amount or dose could be the amount or dose of a neural stem cell proliferating agent required to obtain the same level in blood serum or cerebral spinal fluid as another, specific neural stem cell proliferating agent.
A drug delivery device is an object suitable for administration of an effective amount of a neural stem cell proliferating agent or a differentiating agent. A drug delivery device can effect administration of neural stem cell proliferating agent or a differentiating agent by any method established in the art, including, for example, parenteral, intravenous, infra-arterial, intracolonical, intratracheal, intraperitoneal, intranasal, intravascular, intrathecal, intracranial, intramarrow, intrapleural, intradermal, transdermal, subcutaneous, intramuscular, intraperitoneal, oral, rectal, vaginal, topical administration, pulmonary administration, or any combination thereof. Systemic delivery can be accomplished by techniques including, for example, parenteral, intravenous, intra-arterial, intracolonical, intratracheal, intraperitoneal, intranasal, intravascular, intrathecal, intracranial, intramarrow, intrapleural, intradermal, transdermal, subcutaneous, intramuscular, intraperitoneal, oral, rectal, vaginal, topical administration, pulmonary administration, or any combination thereof. A drug delivery device can be for example, an implantable device or a pump (e.g., an osmotic pump), depot (slow release) delivery of formulation, or an injector pen (with or without a needle). Optionally, the drug delivery device is an infusion device or component thereof or, alternatively, is a device for other means than infusion.
The examples below are intended to further illustrate certain embodiments of the invention, and are not intended to limit the scope of the claims.
EXAMPLES
In the examples below, the following abbreviations have the following meanings. Abbreviations not defined have their generally accepted meanings.
° C.=degree Celsius
hr=hour
min=minute
μM=micromolar
mM=millimolar
M=molar ml milliliter
μl=microliter
mg=milligram
μg=microgram
FBS=fetal bovine serum
PBS=phosphate buffered saline
DMEM=Dulbecco's modified Eagle's medium
MEM=modified Eagle's medium
EGF=epidermal growth factor
NSC=neural stem cell
SVZ=sub ventricular zone
PACAP=pituitary adenylate cyclase activating polypeptide
BMP=bone morphogenetic protein
RSA=rat serum albumin
Example 1
Functional Improvement Post-Stroke Using rhCG+EPO
Male rats were injured through temporary occlusion of the right middle cerebral artery (MCA), following which increasing doses of recombinant Human chorionic gonadotropin (rhCG) were administered, followed by 3 days of Erythropoietin (EPO, Epogen 1440 IU/day).
Male Long Evans rats (280-330 g) were fasted overnight but allowed free access to water. Atropine sulfate (0.5 mg/kg, i.p.) was injected 10 min before anesthesia. Anesthesia was induced with 3.5% isoflurane in a mixture of 70% nitrous oxide and 30% oxygen. All rats were orally intubated and mechanically ventilated. During ventilation, the animals were paralyzed with pancuronium bromide (0.6 mg/kg, i.p.).
The MCA was temporarily occluded for 90 minutes as described by Zea Longa et al. (Stroke 20:84 (1989)) and modified (Belayev et al., Stroke 27:1616 (1996)). Following suture placement, the neck incision was closed, and animals were allowed to awaken from anesthesia. At 60 min following MCAo, they were tested on a standardized neurobehavioral battery to confirm the presence of a neurological deficit (Belayev et al, 1996). Rats that did not demonstrate a left upper extremity paresis (total neurological score less than 9; See behavioral tests, below and FIG. 1) were excluded from further study. After 90 min of MCAo, rats were re-anesthetized, temperature probes were re-inserted, and the intraluminal suture were carefully removed.
Behavioral tests were performed in all rats before MCAo and during occlusion (at 60 min) to confirm success of the MCAo. The battery consisted of 2 tests used previously to evaluate various aspects of neurologic function: (1) the postural reflex test, developed by Bederson et al. (Stroke 17:472 (1986)) to examine upper body posture while the animal is suspended by the tail; and (2) the forelimb placing test,—developed by De Ryck et al. (Stroke 20:1383 (1989)) to examine sensorimotor integration in forelimb placing responses to visual, tactile and proprioceptive stimuli. Neurological function was graded on a scale of 0-12 (normal score=0, maximal score=12).
Treatment and experimental groups are as follows:

    
    
        Group 1: n=8; Saline solution (of equal volume to hCG administered IM) administered IM on days 1, 3, and 5, followed by administration of saline IV by ALZET® Pump (Alzet Osmotic Pumps; Cupertino, Calif.) beginning day 7,8,9 after surgery. The first injection was administered 24 hours after stroke surgery.
        Group 2: n=8; Saline solution (of equal volume to hCG administered IM) administered IM on days 1, 3, and 5, after MCA occlusion followed by administration, of EPO (1440 IU/day) IV by ALZET® Pump beginning day 7, 8, 9. The first injection was administered 24 hours after stroke surgery.
        Group 3: n=8; hCG (33 IU/day) delivered by IM on days 1,3, and 5 after MCA occlusion followed by administration of EPO (1440 IU/day) IV by ALZET® Pump beginning day 7, 8, 9. The first injection was administered 24 hours after stroke surgery.
        Group 4: n=8; hCG (100 IU/day) delivered by IM on days 1, 3, and 5 after MCA occlusion followed by administration of EPO (1440 IU/day) IV by ALZET® Pump beginning-day 7, 8, 9. The first injection was administered 24 hours after stroke surgery.
        Group 5: n=8; hCG (300 IU/day) delivered by IM on days 1, 3, and 5 after MCA occlusion followed by administration, of EPO (1440 IU/day) IV by ALZET® Pump beginning day 7, 8, 9. The first injection was administered 24 hours after stroke surgery.
        Group 6: n=8; hCG (1000 IU/day) delivered by IM on days 1, 3, and 5 after MCA occlusion followed by administration of EPO (1440 IU/day) TV by ALZET® Pump beginning day 7, 8, 9. The first injection was administered 24 hours after stroke surgery.
        Group 7: n=8; hCG (3000 IU/day) delivered by IM on days 1, 3, and 5 alter MCA occlusion followed by administration of EPO (1440 IU/day) IV by ALZET® Pump beginning day 7, 8, 9. The first injection was administered 24 hours after stroke-surgery.
        Group 8: n=8; hCG (10,000 IU/day) delivered by IM on days 1, 3, and 5 after MCA occlusion followed by administration of EPO (1440 IU/day) IV by ALZET® Pump beginning day 7, 8, 9. The first injection was administered 24 hours after stroke surgery.
        Group 9: n=8; hCG (30,000 IU/day) delivered by IM on days 1, 3, and 5 after MCA occlusion followed by administration of EPO (1440 IU/day) IV by ALZET® Pump beginning day 7, 8, 9. The first injection was administered 24 hours after stroke surgery.
    
    


As can be seen in FIG. 1, functional improvement for those animals receiving hCG followed by EPO was better than those receiving EPO alone.
Conversion to human administration follows an allometric scaling factor of 8 to convert from mg/kg administered to rats to mg/m2 for human administration. Following the guidelines established for this conversion (Guidance for Industry: Estimating the Maximum Safe Starting Does in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers, U.S. Department of Health and Human Services, FDA Center for Drug Evaluation and Research, July 2005), the observed optimal dose for rats of 300 IU/day converts to:

HD=[AD*AKm]/HKm 

Wherein,
HD=Human dose,
AD=Animal dose
AKm=Animal km factor
HKm=Human km factor
Therefore, 300 IU/day (which is equivalent to 30 μg of hCG per day, i.e., 1 μg hCG=10 IU hCG in this study) for an average rat of weight 305 g, is equivalent to 98.4 μg/kg dose in the animal. With a rat km factor of 8 and a human km factor of 37, the optimal dose of hCG for human administration is therefore calculated as

HD=98.4 μg/kg*8/37=21.28 μg/kg or 212.8 IU/kg day

The human dose of EPO can be calculated, with an activity in the present example of the 1440 IU/day dose of EPO for an average rat of weight 305 g, is equivalent to 4,721.3 IU/kg dose in the animal. With a rat km factor of 8 and a human km factor of 37, the optimal dose of EPO for human administration is therefore calculated as

HD=4,721.3 IU/kg*8/37=1020.82 IU/kg

Example 2
Functional Improvement Post-Stroke Using hCG+EPO
A second group of male rats were injured through temporary occlusion of the right middle cerebral artery (MCA) as described in Example 1, following which doses of human derived Human chorionic gonadotropin (hCG) were administered, followed by 3 days of Erythropoietin (EPO, Epogen 1440 IU/day) as described in Example 1.
Male Long Evans rats (280-330 g) were fasted overnight but allowed free access to water. Atropine sulfate (0.5 mg/kg, i.p.) was injected 10 min before anesthesia. Anesthesia was induced with 3.5% isoflurane in a mixture of 70% nitrous oxide and 30% oxygen. All rats were orally intubated and mechanically ventilated. During ventilation, the animals were paralyzed with pancuronium bromide (0.6 mg/kg, i.p.).
Behavioral tests were performed in all rats before MCAo and during occlusion (at 60 min) to confirm, success of the MCAo as described in Example 1.
Treatment and experimental groups were as follows:

    
    
        Group 1: n=10; Saline solution (of equal volume to hCG administered IM) was administered IM on days 1, 3, and 5, followed by administration of Saline IV by ALZET® Pump beginning day 7, 8, 9 after surgery.
        Group 2: n=10; hCG (440 IU/day) was delivered by IM on days 1, 3, and 5 after MCA occlusion followed by administration of EPO (1440 IU/day) IV by ALZET® Pump beginning day 7, 8, 9.
        Group 3: n=10; hCG (440 IU/day) was delivered by IM on days 1, 3, and 5 after MCA occlusion followed by administration of Saline at an equivalent rate of volume delivered using IV by ALZET® Pump beginning day 7, 8, 9,
        Group 4: n=10; Saline was delivered by IM on days 1, 3, and 5 after MCA occlusion followed by administration of EPO (1440 IU/day) IV by ALZET® Pump beginning day 7, 8, 9.
        Group 5: n=5. No MCAo, no treatment; animals were trained and tested in four behavioral tasks at week −1, 1, 2, 3, 4, and 6.
    
    


FIG. 2 shows the difference in neurological function, as graded on a scale of 0-12, between the test groups at the same time point post-stroke. As can be seen, there is a marked functional improvement arising from administration of hCG followed by EPO in the manner described herein. Further, FIG. 3 shows a graph indicating the % tissue loss (as compared to non-stroke hemisphere) for these test groups, and FIG. 4 shows images representative of the tissue loss for each group.
Additionally, serum hCG levels were measured after the third IM administration of hCG. As shown in FIG. 5, circulating hCG levels were significant in dosed animals.
Example 3
Using hCG+ EPO in the Treatment of Acute Stroke in Humans
A study has begun in human stroke patients that will involve providing a patient with 3 once-daily IM doses of hCG (at 10,000 IU/dose) on days 1, 3, and 5 of study participation, followed by a one day wash, out period (day 6), followed by three once-daily I.V. doses of erythropoietin (at 30,000 IU/dose) on days 7, 8, and 9 of study participation. The first IM hCG dose is targeted to be delivered between 24 and 48 hours after a moderate-severe stroke event. Patients will be examined at several points during treatment, as well as 6 weeks and 3 months after stroke onset. Baseline assessments will include clinical/safety, neurological, hematological, and vascular status, as well as a brain MRI. Assessments of clinical/safety, neurological, hematological, and vascular status will be repeated at 1 day, 15 days, and 80 days after completing the treatment. A brain MRI will be repeated 80 days after completing the treatment (which will be approximately 90 days after stroke onset) for comparison purposes.
Example 4
Administration of Prolactin
Male rats (250-300 g) were used in two prolactin dosing experiments. Prolactin was given by subcutaneous mini-osmotic pump infusions (ALZET® minipumps)—one injection daily. Stock prolactin was diluted in bicarbonate buffer and the stock was further diluted in 1 mg/ml Rat Serum Albumin (RSA) in saline for injections. The rats did not receive ischemic injuries. On the 6th day the animals received 6 BrdU injections (Sigma-Aldrich) (60 mg/kg, i.p.) over 10 hrs and were sacrificed 30 min following the final BrdU injection. The brains were cryosectioned and BrdU+ cells were quantified in the SVZ using 8 sections per animal. The results are presented as total number of BrdU+ cells in the SVZ or as an average per section as indicated in the figure legend.
Experiment #1:
Rats were dosed for 6 days and received subcutaneous infusions of RSA (control) or rat prolactin (National Hormone and Peptide Program, Torrance, Calif.) at the following doses (3 rats in each group):
*10×=99 ul/pump (2 mg/0.25 ml PRL)—113 μg/day
**15×=148.5 ul/pump (2 mg/0.25 ml PRL)—170 μg/day
***20×=198 ul/pump (2 mg/0.25 ml PRL)—226 μg/day
wherein
*10×=10 times the dose given for intracerebroventricular infusions (approx 11 μg/day).
**15×=15 times the dose given for intracerebroventricular infusions.
***20×=20 times the dose given for intracerebroventricular infusions.
Results:
As shown in FIG. 6, 170 μg/day resulted in the largest increase in proliferation (number of BrdU+ cells) within the forebrain SVZ.
Experiment #2:
Rats were dosed for 3 or 6 days and received daily single intraperitoneal injections of RSA or rat prolactin (National Hormone and Peptide Program, Torrance, Calif.) at the following doses (3 rats in each group):
170 μg/day for 3 days
396 μg/day for 3 days
170 μg/day for 6 days
Results:
As shown in FIG. 7, 170 μg/day delivered for 6 days resulted in the largest increase in proliferation (number of BrdU+ cells) within the forebrain SVZ.
Example 5
Administration of hCG
The purpose of this study is to determine the dose of hCG that maximizes cell proliferation in the forebrain germinal zone and tissue regeneration of adult male rats that have received a pial-strip devasculaizing ischemic injury to the motor cortex.
Methods
Animals and Surgery
250-300 g male rats received a pial-strip devascularization ischemic injury to the motor cortex as previously described (Gonzalez and Kolb. A comparison of different models of stroke on behaviour and brain morphology. Eur J Neurosci. 2003. 18(7): 1950-1962). With the animals under sodium pentobarbital anesthesia (60 mg/kg), a rectangular hole was drilled into the frontal and parietal bones running from +4 to −2 mm anterior/posterior to the bregma and running laterally from 1.5 to 4.5 mm from midline. The dura was removed and a sterile saline-soaked cotton swab was used to wipe the pia and attached blood vessels from the cortical surface.
Dosing
Beginning one day post-stroke (24 hrs later), animals received a single intramuscular (i.m.) injection of hCG (National Peptide and Hormone Program, Torrance, Calif.)). Doses were given as described in Table 1 and were delivered in either three injections over 5 days (dosed on days 1, 3, and 5) or as daily injections over one week and injections were given at 9:00 am each day. Control rats received injections of rat serum albumin in saline (RSA; Sigma, 1 mg/ml). On the day of the final dose animals received 6 BrdU injections over 10 hrs, beginning 30 min after the hCG injection. BrdU (Sigma-Aldrich) was given at a dose of 60 mg/kg, i.p. Animals were transcardially perfused with 4% paraformaldehyde. Brains were dissected, cryoprotected in sucrose and cryosectioned. Brains were cryosectioned at 14 microns in two series of 8 slides each with 8 sections per slide. Immunostaining was performed using rabbit anti-phosphohistone H3 (anti-pHH3; 1:100; Upstate Biotechnologies), Rat anti-BrdU (1:100; Seralab), goat, anti-doublecortin (DCX; 1:100; Santa Cruz Biotechnologies). The number of phosphohistone H3 (pHH3—a marker of mitotically-active cells), BrdU, and doublecortin (DCX—a marker of immature neurons) positive cells in the forebrain subventricular zone (SVZ) around the lateral ventricle of each animal was quantified in 8 sections and presented as the average number of positive cells per lateral ventricle.
Statistics
Values are means+standard error of the mean (SEM). Significance was determined using a one-way ANOVA followed by a Tukey HSD posthoc test (*p<0.05; **p<0.01). Three animals were included in each group.
Results
The present study examines the ability of intramuscular injections of hCG to promote the proliferation of neural stem cells and progenitor cells residing in the adult forebrain subventricular zone (SVZ) following stroke. Animals underwent pial strip devascularization surgery to induce a focal ischemic injury in the motor cortex and treatments began 24 hrs later. In a high bolus dose strategy, animals received 3 doses of hCG over five days on days 1 (24 hrs post-stroke), 3 and 5 as summarized in Table 1. Animals were sacrificed on day 5 for analysis of the levels of proliferation in the forebrain SVZ. As shown in Table 2 and FIG. 8, this regime was effective in increasing proliferation compared to stroked animals receiving RSA control injections. At a dose of 1000 μg, proliferation was increased by almost 2.5 fold and, as shown in FIG. 9, the number of newly generated doublecortin positive (DCX+) neurons in the SVZ of these animals was similarly significantly increased.
In another study, animals received daily dosing with hCG as summarized in Table 1 for 7 days, beginning 24 hrs post-stroke, and the animals were given BrdU on day 7 for 10 hrs and then sacrificed. As shown in FIG. 10A, the number of dividing cells in the SVZ, as indicated by pHH3 immunoreactivity, was significantly increased in the 330 μg/injection group relative to all other groups. This increase was confirmed by quantifying the number of BrdU+ cells in the SVZ of these animals relative to RSA controls (FIG. 11). There was a trend level increase in the 100 μg treatment group relative to pial strip RSA controls (FIGS. 10A and 11). Note that the untreated animals in FIG. 10 received no injections and no pial strip stroke. As an internal control, a group received the same total dose as the 330 μg/injection group (see Table 1), but the hCG was given in 3 injections of 770 μg/injection on days 1, 3 and 5 and the animals were sacrificed on day 5. Based on this study, a low, regular dose of hCG given at 330 μg/injection daily was most effective for increasing proliferation in the forebrain SVZ following ischemic damage in the brain.
To determine whether any of the dosing regimes might result in the growth of new cortical tissue we analyzed the lesion site in cortex of hCG treated animals. Tissue regrowth was particularly evident in the low, regular daily dosing regime the 330 μg/injection dosed group of animals (FIG. 10B).
Any patents or publications mentioned in the specification are indicative of the level of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
The present invention is not limited in scope by the embodiments disclosed in the examples which are intended as illustrations of a few aspects of the invention and any embodiments which are functionally equivalent are within the scope of this invention. Various modifications of the methods and kits in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims. Further, while only certain representative combinations of the compositions disclosed herein are specifically discussed in the embodiments above, other combinations of the compositions will become apparent to those skilled in the art and also are intended to fall within the scope of the appended claims. Thus a combination of steps or compositions may be explicitly mentioned herein; however, other combinations of steps or compositions are included, even though not explicitly stated







TABLE 1







hCG Dosing Strategy. Rats received either three intramuscular (I.M.)


injections of hCG over 5 days or daily injections or 7 days beginning


24 hrs post-stroke. Control rats received injections of RSA only.











Dose/injection


Total Dose
Dose/injection
(micrograms


(IUs hCG)
(IUs hCG)
(μg) hCG)





FIGS. 8 and 9




Dosed on days 1, 3, and 5


RSA (no stroke)


RSA


 330
110
11


 990
330
33


 9900
3300
330


30000
10000
1000


FIGS. 10 and 11


Dosed daily for 7 days


Untreated (no stroke and no injections)


RSA


 7000
1000
100


23100
3300
330


46200
6600
660


70000
10000
1000


Dosed on days 1, 3, and 5


23100
7700
770
















TABLE 2





Actual values ± SEM presented as the average number of positive cells per


lateral ventricle for quantification of pHH3+, BrdU+ and DCX+ cells in


animals dosed with hCG 24 hrs following pial strip devascularizing stroke


relative to controls.

















Dosing Condition (μg/injection)
pHH3+ Cells
BrdU+ Cells








Daily Dosing for 1 Week
Number of Positive Cells per Ventricle












Untreated No Stroke
8.7 ± 2 
—


RSA
 9.3 ± 0.3
374 ± 15 


 10
19.3 ± 5  
459 ± 138


330
 27 ± 3**
874 ± 91*


660
12.3 ± 2  
—


1000 
17 ± 2
—


770 (dosed on days 1, 3 and 5)
16 ± 1
—











5 Day Dosing with Injections on Days 1, 3 and 5
DCX+ Cells












RSA
8.7 ± 1 
280 ± 15 


11
 8 ± 2
—


33
  8 ± 0.1
—


330 
13 ± 1
—


1000 
 19 ± 1*
533 ± 42*

## Claims

1. A method of treating or ameliorating a neurodegenerative disease or condition in a mammal comprising administering to the mammal an effective amount of human chorionic gonadotropin (hCG) during a first treatment period lasting at least three days and administering to the mammal an effective amount of erythropoietin (EPO) during a second treatment period lasting at least three days, wherein the neurodegenerative disease or condition is a stroke, and wherein the second treatment period starts after the end of the first treatment period.

2. The method of claim 1, wherein the hCG is administered systemically.