English:
[0001] This application claims priority to and the benefit of U.S. Provisional Application Serial No. 60/783,500, filed on March 17, 2006; U.S. Provisional Application Serial No. 60/789,132, filed on April 5, 2006; and U.S. Provisional Application Serial No. 60/862,669, filed on October 24, 2006, which are incorporated herein by reference in their entireties.
[0002] 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.
[0005] 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.
[0006] 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.
[0009] Figure 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
[0010] Figure 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.
[0011] Figure 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.
[0012] Figure 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.
[0013] Figure 5 is a bar graph indicating serum hCG levels as measured after a third IM administration of hCG in Example 2.
[0015] Figure 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.
[0016] Figure 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 RSApial 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.
[0017] Figure 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).
[0018] Figure 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.
[0019] Figure 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0030] 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.
[0043] 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:
[0044] 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 LIF 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, hCG can be used with EPO. All other combinations, not explicitly set forth, can also be used.
[0045] 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 every day, 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.
[0048] 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 the 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 with a brain capillary endothelial cell receptor, such as the transferrin receptor (see, e.g., U.S. Pat. No. 5,977,307).
[0050] 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.
[0054] 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.
[0056] The terms used in this application are defined as follows unless otherwise indicated.
[0069] 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. 20020098178Al), the EGF mutein (EGF-Xi 6 ) 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. 20020098178Al, and U.S. Pat. Nos. 6,191,106 and 5,547,935.
[0070] 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).
[0072] 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).
[0074] 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 (EMPl ; 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. 5,723,1 15; 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.
[0075] 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.
[0077] 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 case 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.
[0080] The examples below are intended to further illustrate certain embodiments of the invention, and are not intended to limit the scope of the claims.
[0081] In the examples below, the following abbreviations have the following meanings. Abbreviations not defined have their generally accepted meanings.
[0082] 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).
[0084] 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 Figure 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.
[0085] 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.
[0087] As can be seen in Figure 1, functional improvement for those animals receiving hCG followed by EPO was better than those receiving EPO alone.
[0089] 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 k m factor of 8 and a human k m factor of 37, the optimal dose of hCG for human administration is therefore calculated as
[0090] 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 305g, is equivalent to 4,721.3 IU/kg dose in the animal. With a rat k m factor of 8 and a human k m factor of 37, the optimal dose of EPO for human administration is therefore calculated as
[0091] 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.
[0093] 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.
[0095] Figure 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, Figure 3 shows a graph indicating the % tissue loss (as compared to non-stroke hemisphere) for these test groups, and Figure 4 shows images representative of the tissue loss for each group.
[0096] Additionally, serum hCG levels were measured after the third IM administration of hCG. As shown in Figure 5, circulating hCG levels were significant in dosed animals.
[0097] 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.
[0099] Rats were dosed for 6 days and received subcutaneous infusions of RSA (control) or rat prolactin (National Hormone and Peptide Program, Torrance, CA) at the following doses (3 rats in each group):
[00100] As shown in Figure 6, 170 μg/day resulted in the largest increase in proliferation (number of BrdU+ cells) within the forebrain SVZ.
[00102] As shown in Figure 7, 170 μg/day delivered for 6 days resulted in the largest increase in proliferation (number of BrdU+ cells) within the forebrain SVZ.
[00103] 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 devascularizing ischemic injury to the motor cortex.
[00105] Beginning one day post-stroke (24hrs later), animals received a single intramuscular (i.m.) injection of hCG (National Peptide and Hormone Program, Torrance, CA)). 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, lmg/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.
[00106] 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.
[00107] 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 Figure 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 Figure 9, the number of newly generated doublecortin positive (DCX+) neurons in the SVZ of these animals was similarly significantly increased.
[00108] In another study, animals received daily dosing with hCG as summarized in Table 1 for 7 days, beginning 24hrs post-stroke, and the animals were given BrdU on day 7 for lOhrs and then sacrificed. As shown in Figure 1OA, 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 (Figure 11). There was a trend level increase in the 100 μg treatment group relative to pial strip RSA controls (Figures 1OA and 11). Note that the untreated animals in Figure 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.
[00109] 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 (Figure 10B).
[00111] 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 \o 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.

中文:
[0001]本申请要求2006年3月17日提交的美国临时申请No.60/783,500,2006年4月5日提交的美国临时申请No.60/789,132,和2006年10月24日提交的美国临时申请No.60/862,669的优先权和权益,其全文在此引入作为参考。
[0002]分离和体外培养多潜能神经干细胞的技术的发展(例如,见美国专利No.5,750,376;5,980,885;5,851,832)显著改进了神经变性疾病和病症的治疗前景。已经发现胎儿脑可用于分离和体外培养多潜能神经干细胞。而且,与长期以来认为胎儿脑细胞是不能复制或再生的脑细胞的观点相反,已经发现神经干细胞也可从成体哺乳动物脑中分离。这些干细胞,不管是来自胎儿还是成体脑中,是能够自我复制的。子代细胞可增殖或分化为神经细胞系中的任何细胞,包括神经元,星形胶质细胞和少突神经胶质细胞。因此,这些发现不仅提供了可用于移植的神经细胞的来源,还证实了成体脑中多潜能神经干细胞的存在。
[0005]相应的,提供了治疗或改善哺乳动物中的神经变性疾病或病症的方法。本方法包含给予哺乳动物有效量的hCG或LH和有效量的EPO,其中hCG或LH以至少三个剂量全身给药,任选的通过利用试剂盒。hCG,LH和/或EPO可连续或间歇地给药。而且,hCG或LH可在第一治疗期内给药,EPO可在第二治疗期内给药。例如,hCG或LH可在第一治疗期的第1、3和5天间歇给药,然后EPO可在第二治疗期的第1、2和3天连续给药。
[0006]此处还提供了治疗或改善哺乳动物的神经变性疾病或病症的其它方法。本方法包括在第一治疗期给予哺乳动物有效量的hCG或LH,紧接着在第二治疗期给予有效量的EPO,任选的通过利用试剂盒。hCG或LH可在第一治疗期间歇给药并且EPO可在第二治疗期连续给药。例如,hCG或LH可在第一治疗期的第1、3和5天间歇给药,然后EPO可在第二治疗期的第1、2和3天连续给药。
[0009]图1显示了进行了下述治疗的遭受了大脑中动脉闭塞(MCAo)中风的大鼠功能恢复的效果,用逐渐增加剂量的hCG,在肌肉内(IM)给予hCG剂量之后,每天静脉内(IV)给予1440IU的EPO。
[0010]图2显示了进行了下述治疗的遭受了MCAo中风的大鼠与未治疗对照相比功能恢复的区别效果,每天用440IU的hCG并IV给予1440IU的EPO、单独用hCG、或单独用EPO。
[0011]图3是显示进行了下述治疗的遭受了MCAo中风的大鼠与未治疗对照相比的组织损失%(与非中风半球相比)的图,每天用440IU的hCG并IV给予1140IU的EPO、单独用hCG、或单独用EPO。
[0012]图4显示了进行了下述治疗的遭受了MCAo中风的大鼠与未治疗对照相比的组织损失的示意图,每天用440IU的hCG并IV给予1140IU的EPO、单独用hCG、或单独用EPO。
[0013]图5是显示实施例2中在第三次IM给予hCG之后测量的血清hCG水平的柱形图。
[0015]图7显示了对雄性大鼠利用每天单次腹膜内注射的催乳素给药的结果。展现了每个给药方案的每个切片的BrdU+细胞总数量。(A)对于高的3天剂量观察到SVZ增殖的小的增加。(B)对于增加SVZ增殖水平的最强给药条件利用低的,每天170μg/天的剂量进行6天。显著性是相对于RSA对照的。n=3;*p**p<0.01;接着用Tukeyposthoc测试进行单因素ANOVA。
[0016]图8显示了在中风后(中风=第0天)第1、3和5天单次肌肉内注射hCG引起前脑SVZ显著增加的增殖。在1000μg剂量条件(n=3;*p<0.05;用Tukey posthoc进行单因素ANOVA)下观察到每脑室的磷酸-组蛋白H3阳性(pHH3+)细胞数量的显著增长。图象显示了RSA软膜条对照大鼠对比给药1000μghCG的动物的侧脑室背外侧角中的核标记Hoechst和pHH3的表达。注意对于给药1000μg的动物SVZ中总细胞数量和pHH3的表达增加。
[0017]图9显示了在中风后(中风=第0天)第1、3和5天每天单次肌肉内注射1000μg的hCG引发了前脑SVZ中神经发生的增加。确定给药的动物中的doublecortin+神经元的数量,在1000μg给药的动物中该数量翻倍。
[0018]图10显示了中风后(中风=第0天)24小时开始每天单次肌肉内注射hCG进行7天的结果。(A)每天330μg/注射的给药方案相对于所有其它给药条件和对照(n=3;*p<0.01;用Tukey posthoc进行单因素ANOVA)显著增加了SVZ中增殖的数量(pHH3+细胞)。(B)观察给药的大鼠的运动皮层中的缺血性病变,表明每天接受330μg/注射的给药方案的动物显示出新的组织生长并且用组织填料(tissue plug)填充病变位点。
[0019]图11显示了通过计数BrdU+细胞确认的每天以330μg/注射的hCG给药的动物SVZ中增加的增殖。在330μg/注射的条件下相对于对照和100μg/注射(p<0.01;n=3;用Tukey posthoc分析的单因素ANOVA)每脑室的BrdU+细胞数量显著增加了。这些结果进一步确认了用pHH3染色观察到的增殖的增加。
[0024]如此处所用,连续对受试者递送(deliver)或给予(administer)物质意味着将该物质在连续数天的时期内至少一天一次的递送或给予。例如,可通过注射(例如,IM或皮下)或口服至少每天一次全身给药,或通过以导致每天递送到受试者的组织或血流中的方式输注给药。任选的,该物质通过输注或输注以外的方式递送。如此处所用的,“全身”一词不包括大脑内脑室输注。该物质持续递送或给予的持续期或治疗期可持续三天到几年,甚至在受试者剩余的生命中持续。例如,持续期可以是3-6天,3-14天,3-21天,3-28天,1-4个月,1-6个月,1-9个月,1-12个月,1-2年,1-3年,1-5年,1-10年等。对于进一步的实施例,持续递送的治疗期可以是至少三天,至少四天,至少五天,至少六天,至少七天,或至少十四天。而且,该物质可在给药期第1,2和3天连续递送。
[0025]如此处所用的,间歇的对受试者递送或给予物质意味着以少于每天地递送或给予该物质,例如,在一段时期内每2,3,4,5或7天一次。例如,该物质可在一定治疗期内每隔一天递送或给予,例如,在治疗期第1,3和5天。该物质间歇递送或给予的持续期或治疗期可持续三天到几年,甚至在受试者剩余生命中持续。例如,持续期可以是3-6天,3-14天,3-21天,3-28天,1-4个月,1-6个月,1-9个月,1-12个月,1-2年,1-3年,1-5年,1-10年等。对于进一步的实施例,间歇递送的治疗期可以是至少三天,至少四天,至少五天,至少六天,至少七天,或至少十四天。
[0026]此处提供的方法,例如,可通过给予具有神经变性疾病或病症的受试者有效量的增殖试剂,利用增殖试剂催乳素、hCG、LH、CSF、G-CSF、GM-CSF或VEGF治疗神经变性疾病或病症。作为例子,增殖试剂hCG和LH结合相同的受体,并可互换地在此处提供的特定实施例中以等效剂量利用。作为进一步的实施例,增殖试剂hCG可以大约120-200IU/kg/天的剂量肌肉内给药,然后静脉内给予大约570-950IU/kg/天的EPO。对于进一步的实施例,可以160IU/kg/天的剂量肌肉内给予hCG,然后静脉内给予765IU/kg/天的EPO。用hCG和LH间歇治疗任选地包含数天的hCG或LH给药(例如,在第1,3,5天)。所述神经干细胞试剂的给药可紧接着数天的间歇(例如,第7,9,11天)或连续(例如,第7,8,9天)给予分化试剂例如EPO。等效剂量的其它神经干细胞增殖试剂也可被用于类似的方案中。
[0030]神经干细胞增殖试剂可在大约14天内(例如,0到大约14天)给予中枢神经系统(CNS)损伤、症状发作、或诊断的哺乳动物。如此处所用的,0天指CNS损伤、症状发作、或诊断的时间。任选的,神经干细胞增殖试剂可在CNS损伤、症状发作、或诊断的大约13,12,11,10,9,8,7,6,5,4,3,2,或1天内(例如,0到大约5天)给予。任选的,神经干细胞增殖试剂可在CNS损伤、症状发作、或诊断的24小时内给予哺乳动物。任选的,神经干细胞增殖试剂可在CNS损伤、症状发作、或诊断的12,11,10,9,8,7,6,5,4,3,2,1小时内给予哺乳动物。
[0043]而且,可给予神经细胞分化试剂以选择性增强神经元形成或神经胶质细胞形成。这些分化试剂也可根据给药方案和试剂盒递送。示例性的分化试剂包括但不限于:
[0044]任何方法和试剂盒可包含多种神经干细胞增殖试剂和/或神经细胞分化试剂。因而,一种或多种神经干细胞增殖试剂可共同或相继给药,并且可通过分别的组合物或组合在单个组合物中的形式给药。而且,一种或多种神经干细胞增殖试剂和一种或多种神经干细胞分化试剂可共同或相继给药,并且可通过分别的组合物或组合在单个组合物中的形式给药。例如,PRL和LH或hCG可组合使用以使神经干细胞增殖最大化;PRP可与LH或hCG组合使用以使神经干细胞增殖最大化;GnRH可与LH或hCG组合使用或替代其使用以增加LH的循环水平并且增强神经干细胞增殖;并且CNTF和LIF可与LH或hCG组合使用以促进神经干细胞增殖和增加CNS中神经干细胞群的大小。进一步例如,催乳素可与EPO一起使用,LH可与EPO一起使用,hCG可与EPO一起使用。没有明确阐述的所有其它组合也可使用。
[0045]这些因子的适当的剂量可根据本领域已经建立的方法确定。例如,催乳素的剂量范围为可以是从大约0.510IU/kg/天到大约100,000IU/kg/天,例如,大约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;200-800IU/kg/天。hCG的剂量范围可以是从大约0.5IU/kg/天到大约3,000,000IU/kg/天,例如,大约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;或1,600IU/kg/天。hCG也可以10,000IU/天的剂量提供。LH的剂量范围可以是从大约0.5IU/kg/天到大约500,000IU/kg/天,例如,大约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;或160IU/kg/天。LH也可以10,000IU/天的剂量提供。EPO的剂量范围可以是从大约100IU/kg/天到大约2000IU/kg/天,例如,大约100-1500;100-1000;160-1000;570-950;765;或1020IU/kg/天。EPO也可以30,000IU/天的剂量提供。可利用等效剂量的其它试剂。此处的剂量指在整个治疗期中每天或间歇地递送的平均剂量。例如,如果神经干细胞增殖或分化试剂不是每天递送,在整个治疗期中的递送试剂总量可除以包括间隔的治疗期的总天数,以获得每天的剂量。
[0048]作为选择地,如美国专利No.6,329,508;6,015,555;5,833,988或5,527,527所述,待递送的神经干细胞增殖试剂或分化试剂可与转铁蛋白受体抗体配合。神经干细胞增殖试剂和分化试剂还可作为融合蛋白给药,所述融合蛋白包含神经干细胞增殖或分化试剂和与脑毛细管内皮细胞受体具有反应性的配体,例如转铁蛋白受体(见例如美国专利No.5,977,307)。
[0050]适当的赋形剂的一些例子包括人工脑脊髓液,乳糖,葡萄糖,蔗糖,山梨醇,甘露醇,淀粉,阿拉伯树胶,磷酸钙,藻酸盐,黄芪胶,明胶,硅酸钙,微晶纤维素,聚乙烯吡咯烷酮,纤维素,无菌水,糖浆,和甲基纤维素。制剂中可另外包括:润滑剂例如滑石,硬脂酸镁,和矿物油;湿润剂;乳化和悬浮剂;防腐剂例如甲基苯甲酸盐和丙羟基苯甲酸盐;甜味剂;和调味剂。本发明的组合物可配制为在利用本领域已知方法给予患者之后提供治疗剂的快速,持续或延迟释放。
[0054]溶液、悬浮液、或粉末组合物可从以适当方式递送制剂的药物递送装置优选口服或经鼻给药,用于本发明的方法的另一种制剂利用透皮药物递送装置(“贴片”)。所述透皮贴片可用于提供以控制量的本发明治疗剂的连续或不连续的输注。用于给药的透皮贴片的结构和用途是本领域公知的。见例如,美国专利No.5,023,252,此处全文引入作为参考。所述贴片可以为连续的,脉动的,或根据给药需要而构造。其它适于用于本发明的制剂可在Remington′s Science and Practice ofPharmacy,21st Edition,ed.University of the Sciences in Philadelphia,Lippincott,Williams & Wilkins,Philadelphia Pa.,2005中找到。
[0056]本申请中使用的词语如下定义,除非有相反的提示。
[0069]表皮生长因子或EGF是包含天然哺乳动物EGF或类似于哺乳动物EGF的多肽序列的蛋白。如此处所用,EGF类似物、变体、或片段与天然EGF具有基本氨基酸序列类似性,并具有天然EGF的至少一种生物学活性,例如与EGF受体的结合。特别包括的作为EGF的是任何物种的天然EGF、TGFα、或重组修饰的EGF。特定的例子包括但不限于,具有两个C末端氨基酸的缺失和位点51的中性氨基酸取代的重组修饰的EGF(特别是EGF51 gln51;美国专利申请公开No.20020098178A1),位点16的His残基被中性或酸性氨基酸取代的EGF突变蛋白(EGF-X16)(美国专利No.6,191,106),缺乏天然EGF的氨基末端残基的EGF的52-氨基酸缺失突变体(EGF-D),N-末端残基以及两个C-末端残基(Arg--Leu)被删去的EGF缺失突变体(EGF-B),位点21的Met残基被氧化的EGF-D(EGF-C),位点21的Met残基被氧化的EGF-B(EGF-A),肝素结合性EGF样生长因子(HB-EGF),β-细胞素,双调蛋白,神经调节蛋白,或包括上述任何蛋白的融合蛋白。其它有用的EGF类似物,变体和片段描述于美国专利申请公开No.20020098178A1,和美国专利No.6,191,106和5,547,935。
[0070]另外,EGF类似物、变体、或片段也可以是天然哺乳动物EGF受体的功能性激动剂。例如,功能性激动剂可以是公开于美国专利No.6,333,031中的EGF受体的活化氨基酸序列,或具有EGF受体激动剂活性的抗体(Fernandez-Pol,1985和美国专利No.5,723,115)。
[0072]另外,PACAP类似物、变体、和片段也可以是天然哺乳动物PACAP受体的功能性激动剂。例如,功能性激动剂可以是maxadilan,一种作为PACAP 1型受体的特异性激动剂的多肽(Moro et al.,1997)。
[0074]另外,EPO类似物、变体、或片段也可以是天然哺乳动物EPO受体的功能性激动剂。例如,功能性激动剂可以是EPO模拟肽1(EMP1;Johnson et al.,2000);描述于Wrighton et al.,1996和美国专利No.5,773,569中的一种EPO的短肽模拟物;公开于Kaushansky,2001中的任何小分子EPO模拟物;描述于美国专利No.5,885,574、WO 96/40231、WO 97/48729,Fernandez-Pol,1985或美国专利No.5,723,115中的活化EPO受体的抗体;公开于美国专利No.6,333,031中的EPO受体的活化氨基酸序列;具有EPO受体的激动剂活性的金属复合受体配体(美国专利No.6,413,952),或描述于美国专利No.5,506,107和5,837,460中的EPO受体配体。
[0075]LH/hCG-诱导试剂是当给予动物时能够增加动物中的LH或hCG量的物质。例如,LH-释放激素(LHRH)刺激LH的分泌。
[0077]有效量是足以获得期望目的的治疗剂的量。例如,增加神经干细胞数量的LH或hCG的有效量是如该情况可能成为的足以在体内或体外导致神经干细胞数量增加的量。LH或hCG治疗或改善神经变性疾病或病症的有效量是足以降低或去除神经变性疾病或病症的一种或多种症状的LH/hCG的量。给定的治疗剂的有效量将随着例如试剂性质,给药途径,接受治疗剂的动物大小和物种,以及给药目的的因素而改变。本领域熟练技术人员可根据本领域已经建立的方法根据经验确定每个个体病例中的有效量。
[0080]下面的实施例意图进一步阐述本发明的特定实施方式,而不意图限制权利要求的范围。
[0081]在下面的实施例中,下述缩写具有下述意思。没有定义的缩写具有一般所接受的含义。
[0082]通过临时闭塞右侧大脑中动脉(MCA)而损伤雄性大鼠,然后给予逐步增加剂量的重组人绒毛膜促性腺激素(rhCG),紧接着给予3天的红细胞生成素(EPO,Epogen 1440IU/天)。
[0084]如Zea Longa et al.(Stroke 20:84(1989))所述和(Belayev et al.,Stroke 27:1616(1996))的修改将MCA临时闭塞90分钟。缝合处置之后,封闭颈部切口,允许动物从麻醉中苏醒。MCAo之后60分钟,在标准神经行为成套测试中测试,以确认神经缺陷的存在(Belayev et al,1996)。没有表现出左上肢轻瘫的大鼠(总神经得分小于9;见行为测试,下面和图1)排除在进一步研究之外。MCAo 90分钟之后,大鼠被重新麻醉,重新插入温度探针,并且小心的去除腔内缝线。
[0085]MCAo之前和闭塞过程中(在60分钟时)在所有大鼠中进行行为测试以确认MCAo的成功。成套测试由先前利用的2个测试组成以评价神经功能的不同方面:(1)姿势反射测试,为Bederson et al.(Stroke17:472(1986))所研发,以检查当动物通过尾巴被悬挂时的上身姿势;和(2)前肢放置测试(forelimb placing test),为De Ryck et al.(Stroke20:1383(1989))所研发,以检查对视觉、触觉和本体感受刺激的前肢放置反应中的感觉运动综合。神经功能被分级为0-12等级(正常得分=0,最大得分=12)。
[0087]如从图1中所见,这些接受了hCG然后接受了EPO的动物的功能性恢复好于仅接受了EPO的动物。
[0089]因此,对于体重为305g的普通大鼠300IU/天(在本研究中其相当于每天30μg hCG,也就是:1μg hCG=10IU hCG)相当于动物中的98.4μg/kg剂量。大鼠km因子为8,人km因子为37,对于人给药的hCG最佳剂量因而计算为:
[0090]EPO的人剂量可被计算,根据本实施例中体重为305g的普通大鼠的1440IU/天的EPO剂量的活性相当于动物的4721.3IU/kg的剂量。用大鼠km因子为8,人km因子为37,对于人给药的EPO最佳剂量因而计算为:
[0091]第二组雄性大鼠通过如实施例1所述临时闭塞右侧大脑中动脉(MCA)而被损伤,然后给予人源的人绒毛膜促性腺激素(hCG),紧接着如实施例1所述给予3天的红细胞生成素(EPO,Epogen 1440IU/天)。
[0093]如实施例1所述在MCAo之前和闭塞过程中(在60分钟)对所有大鼠进行行为测试,以确认MCAo的成功。
[0095]图2显示了中风后同一时间点上测试组之间神经功能上的差异,分为0-12级。如所见的,以在此处所述的方案给予hCG然后给予EPO产生显著的功能改进。而且,图3显示了这些测试组的组织损失%(与非中风大脑半球相比)的图表,图4显示了每个组的组织损失的代表图。
[0096]另外,在第三次IM给予hCG之后测量血清hCG水平。如图5所示,循环的hCG水平在给药动物中是显著的。
[0097]对于人中风患者开始了一项研究,涉及到在研究参与的第1,3和5天提供给患者3个每日一次的hCG(10000IU/天)IM剂量,接着一天的洗出期(第6天),然后再研究参与的第7,8和9天三个一天一次的红细胞生成素(30000IU/天)IV剂量。首次IM hCG剂量在中重度的中风事件之后24到48小时之间递送。治疗过程中在几个点以及在中风发作之后6周和3个月检查患者。基线评价包括临床/安全、神经学、血液学、和血管状态,以及脑MRI。临床/安全、神经学、血液学、和血管状态的评价将在完成治疗后第1天,15天和80天重复。为对比目的,脑MRI将在完成治疗(中风发作之后大约90天)之后80天重复。
[0099]大鼠给药6天,并且接受皮下输注以下剂量的RSA(对照)或大鼠催乳素(National Hormone and Peptide Program,Torrance,CA)(每组三只大鼠):
[00100]如图6所示,170μg/天导致了在前脑SVZ之内的增殖(BrdU+细胞数量)的最大增加。
[00102]如图7所示,170μg/天递送6天导致了在前脑SVZ之中增殖(BrdU+细胞数量)的最大增加。
[00103]本研究的目的是为了确定使成体雄性大鼠的前脑胚带的细胞增殖和组织再生最大化的hCG剂量,所述大鼠已经受到运动皮层的软膜条(pial strip)血行阻断缺血性损伤。
[00105]中风后一天开始(24小时后),动物接受单独肌肉内(i.m.)注射hCG(National Peptide and Hormone Program,Torrance,CA)),剂量如表1所述给出,并且以5天三次注射(在第1,3和5天给药)或一周内每天注射,注射在每天9:00am给予。对照大鼠接受盐水中的大鼠血清白蛋白(RSA;Sigma,1mg/ml)注射。在最后给药的当天,hCG注射之后30分钟开始,动物10小时内接受6次BrdU注射。BrdU(Sigma-Aldrich)以60mg/kg剂量,i.p.给予。动物用4%多聚甲醛透心输注。切割大脑,在蔗糖中冷冻保藏并冷冻切割。大脑以14微米冷冻切割为两系列的8个切条,每个切条具有8个切片。利用兔抗-磷酸组蛋白H3(phosphohistone H3)(抗pHH3;1:100;Upstate Biotechnologies)、大鼠抗-BrdU(1:100;Seralab)、山羊抗-doublecortin(DCX;1:100;Santa CruzBiotechnologies)进行免疫染色。每个动物的侧脑室周围的前脑室下区(SVZ)中的磷酸组蛋白H3(phosphohistone H3)(pHH3-一种有丝分裂-活性细胞标记物)、BrdU、和doublecortin(DCX-不成熟神经元的标记物)阳性细胞的数量在8个切片中量化,并且表示为每个侧脑室的阳性细胞平均数量。
[00106]数值是平均值+平均值标准误差(SEM)。显著性使用单因素方差分析然后进行Tukey HSD posthoc测试(*p**p<0.01)而确定。每组中包括三只动物。
[00107]本研究检查了肌肉内注射hCG促进中风后成体前脑室下区中的神经干细胞和祖细胞的增殖的能力。动物进行了软膜条血行阻断外科手术以诱导运动皮层的局部缺血性损伤,治疗在24小时后开始。在弹丸浓注的高剂量策略中,如表1中概括的,动物在5天内的第1(中风后24小时)、3和5天接受3剂hCG。动物在第5天被处死分析前脑SVZ的增殖水平。如表2和图8所示,本方案与接受RSA对照注射的中风动物相比在增加增殖上是有效的。在1000μg的剂量时,增殖增加了几乎2.5倍,并且,如图9所示,这些动物的SVZ中新产生的doublecortin阳性(DCX+)神经元的数量类似地显著增加。
[00108]在另一研究中,动物如表1所概括的每天接受hCG给药7天,开始于中风后24小时,在第7天给予动物BrdU10小时,然后处死。如图10A所示,通过pHH3免疫反应性显示的SVZ中分裂细胞数量在330μg/注射的组中相对于所有其它组显著增加了。这种增加通过相对于RSA对照量化这些动物的SVZ中的BrdU+细胞数量而确认(图11)。相对于软膜条RSA对照在100μg治疗组中有增加的趋势(图10A和11)。注意图10中的未治疗动物没有接受注射和没有软膜条中风。作为内部对照,一个组接受与330μg/注射的组相同的总剂量(见表1),但在第1,3和5天以770μg/注射3次注射给予hCG,并且动物在第5天处死。基于此研究,以每天330μg/注射给予的低的,常规剂量的hCG对于大脑缺血性损伤之后的前脑SVZ中的增殖的增加是最有效的。
[00109]为确定是否任何给药方案都可能导致新的皮层组织的生长,我们分析了hCG治疗动物的皮层的损害位点。在以330μg/注射的低的常规剂量的给药方案给药的动物组中组织再生长是特别明显的(图10B)。
[00111]本发明不限于实施例中公开的实施方式的范围,实施例只是意图作为本发明的某些方面的例证,任何功能等同的实施方式在本发明的范围内。在此展示和描述之外的本方法和试剂盒的多种修饰对于本领域熟练技术人员来说是明显的,并且落在所附权利要求的范围内。而且,尽管只有在此公开的组合物的某些代表性组合在上述实施方式中进行了特别讨论,组合物的其它组合对于本领域熟练技术人员来说是明显的,并且也落在所附权利要求的范围内。因而步骤或组合物的组合可在此明确提及;然而,步骤或组合物的其它组合尽管没有明确阐述也包括在内。