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

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


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.
Related Terms: Neurodegenerative Disease

Browse recent Stem Cell Therapeutics Corp. patents - Calgary, CA
Inventors: Samuel Weiss, Christopher Gregg, Allen Davidoff, Joseph E. Tucker
USPTO Applicaton #: #20120270779 - Class: 514 77 (USPTO) - 10/25/12 - Class 514 


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The Patent Description & Claims data below is from USPTO Patent Application 20120270779, Dosing regimens for neural stem cell proliferating agents and differentiating agents for the treatment of neurological disorders.

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CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/687,302, filed Mar. 16, 2007, which 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 stem 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 IU 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 subventricular 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 further 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 ⅙ of the total amount for six days was more effective than delivering ⅓ 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 for 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, intra-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:

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stats Patent Info
Application #
US 20120270779 A1
Publish Date
10/25/2012
Document #
13401323
File Date
02/21/2012
USPTO Class
514/77
Other USPTO Classes
514/97
International Class
/
Drawings
11


Neurodegenerative Disease


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