BRAIN TARGETS FOR NEUROTROPHIC FACTORS TO TREAT

NEURODEGENERATIVE DISEASE

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No.

61/718,141, filed October 24, 2012, which application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] A cause of Parkinson's disease (PD) is believed to be the death of dopaminergic (DAergic) neurons in the substantia nigra zona compacta (SNc) in the ventral midbrain. The cell bodies of the DAergic neurons are located in the SNc, and their axons project to the striatum (caudate -putamen in man). This pathway is called the nigrostriatal projection.

[0003] There is a need for drugs that stimulate sprouting of DAergic nerve terminals in the striatum when administered either to the SNc or to the striatum.

SUMMARY OF THE INVENTION

[0004] Surprisingly, it has been found that the drug MANF (mesencephalic astrocyte - derived neurotrophic factor) significantly reduces neurological deficits when administered in the SNc, This action of MANF differentiates it from GDNF, which does not reduce neurological deficits when administered to the SNc.

Administration of MANF to the SNc and/or striatum by convection enhanced delivery (CED) can increase therapeutic efficacy. Surprisingly, it has been found the lower infusion rates can increase the volume of distribution (Vd) of MANF in CED.

[0005] In some embodiments, the invention provides a method of treating a subject with a condition, comprising contacting the substantia nigra of the subject with an amount of MANF effective to treat the condition.

[0006] In some embodiments, the condition is a neurological disorder. In certain

embodiments, the neurological disorder is Parkinson's disease.

[0007] In certain embodiments, the subject is a mammal. In some embodiments, the

mammal is a human, rat, mouse, monkey, and rabbit. [0008] In some embodiments, the method further comprises contacting the striatum of a subject with an amount of MANF effective to treat said condition.

[0009] In certain embodiments, the method further comprises administering one or more additional therapeutic agents.

[0010] In some embodiments, the subject exhibits an improvement in their clinical scores after treatment. In certain embodiments, the clinical score is a measurement of net ipsilateral rotations or total ipsilateral rotations.

[0011] In certain embodiments, the percentage of viable dopaminergic neurons in a

subject increases; the density of dopaminergic terminals in the putamen of the subject increases; the percentage of viable dopaminergic neurons in the subject and the density of dopaminergic terminals in the putamen of the subject both increase; the viability of cell bodies in the striatum of the subject increases; the percentage of dying dopaminergic neurons in the subject decreases; and/or the percentage of dead dopaminergic neurons in the subject decreases.

[0012] In some embodiments, the amount of MANF administered is at least about 3, 5, 10, 20, 36, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 μg. In certain embodiments, the amount of MANF administered is at least about 1.2, 1.4. 1.6. 1.8, or 2 mg.

[0013] In some embodiments, the amount of MANF administered is less than about 3, 5, 10, 20, 36, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 μg. In certain embodiments, the amount of MANF administered is less than about 1.2, 1.4. 1.6, 1.8, or 2 mg.

[0014] In some embodiments, the amount of MANF administered is about 3, 5, 10, 20, 36, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 μg. In certain embodiments, the amount of MANF administered is about 1.2, 1.4. 1.6. 1.8, or 2 mg.

[0015] In some embodiments, the amount administered is selected to protect and/or

restore the functioning of sick dopaminergic neurons in the striatum; and/or to reduce the death of dopaminergic neurons in the subject.

[0016] Disclosed are methods of treating a neurological disorder in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of MANF to a brain region of the subject by convection enhanced delivery. In some embodiments, the neurological disorder is Parkinson's disease. [0017] In some embodiments, the brain region comprises the striatum, the substantia nigra, or a combination thereof. In some embodiments, the brain region comprises the striatum. In some embodiments, the brain region comprises the substantia nigra. In some

embodiments, the brain region comprises the striatum and the substantia nigra.

[0018] In some embodiments, the effective amount of MANF is from 1 μg to 1000 μg. In some embodiments, the effective amount of MANF is from 10 μg to 500 μg. In some embodiments, the effective amount of MANF is from 50 μg to 250 μg.

[0019] In some embodiments, convection enhanced delivery is performed at a flow rate of from 0.1 μΙ7ηιίη ίο 10 μΙ7ηιίη.

[0020] In some embodiments, convection enhanced delivery is performed acutely.

[0021] In some embodiments, the convection enhanced delivery is repeated two or more times during a course of treatment.

[0022] In some embodiments, convection enhanced delivery is performed chronically.

[0023] In some embodiments, the pharmaceutical composition further comprises a tracer. In some embodiments, diffusion of the MANF in the brain region is monitored in by detection of the tracer. In some embodiments, monitoring comprises magnetic resonance imaging (MRI), computed tomography imaging, or X-ray computed tomography (CT).

[0024] In some embodiments, the pharmaceutical composition further comprises a buffer. In some embodiments, the buffer is a citrate buffer. In some embodiments, the

pharmaceutical composition has a pH of from 5.5 to 6.5.

[0025] In some embodiments, the subject exhibits an improvement in a clinical score after

treatment.

[0026] In some embodiments, the percentage of viable dopaminergic neurons in the subject increases. In some embodiments, the density of dopaminergic terminals in the subject's putamen increases. In some embodiments, both the percentage of viable dopaminergic neurons in the subject and the density of dopaminergic terminals in the subject's putamen increase. In some embodiments, the viability of cell bodies in the striatum increases. In some embodiments, the percentage of dying dopaminergic neurons in the subject decreases. In some embodiments, the percentage of dead dopaminergic neurons in the subject decreases.

[0027] Also disclosed are kits for treating a neurological disorder by convection enhanced

delivery of MANF to a brain region of a subject, the kits comprising: (a) a catheter, that can be stereotactically placed at or near the brain region; (b) a pump, for generating a positive pressure gradient between the catheter and the brain region; (c) a pharmaceutical composition comprising an effective amount of MANF to treat the neurological disorder. [0028] Some embodiments further comprise a delivery sheath to assist in placement of the catheter.

[0029] In some embodiments, the catheter comprises multiple outlet ports.

[0030] In some embodiments, the catheter comprises an outer tubing to provide structural rigidity to the catheter.

[0031] In some embodiments, the pharmaceutical composition comprises from 1 μg to 1000 μg of the MANF. In some embodiments, the pharmaceutical composition comprises from 10 μg to 500 μg of the MANF. In some embodiments, the pharmaceutical composition comprises from 50 μg to 250 μg of the MANF.

[0032] In some embodiments, the pharmaceutical composition further comprises a tracer.

[0033] In some embodiments, the pharmaceutical composition further comprises a buffer. In some embodiments, the buffer is a citrate buffer.

[0034] In some embodiments, the pharmaceutical composition has a pH of from 5.5 to 6.5.

[0035] Also disclosed are methods of treating a neurological disorder in a subject in need

thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of MANF in a 10 mM citrate buffer, pH 6.1 to a brain region of the subject.

INCORPORATION BY REFERENCE

[0036] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that a term incorporated by reference with a term defined herein, this specification shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0038] Figure 1 shows neurorestorative action of MANF and GDNF: MANF 3 and 10 μg, at 4-weeks, and GDNF, 10 μg at 8-weeks after 6-OHDA, caused a significant reduction in neurological deficits in the rodent model of PD, after a partial lesion of the nigrostriatal projection by the injection of 6-OHDA into the striatum unilaterally. Order of Bars: (1) Vehicle; (2) MANF 3 μg; (3) MANF 10 μg; (4) MANF 36 μ¾ (5) GDNF 10 μg.

[0039] Figure 2 shows neurorestorative action of MANF but not GDNF: MANF 10 μg, at 2-weeks, and at 36 μg after 4-weeks, caused a significant reduction in neurological deficits in the rodent model of PD, after a partial lesion of the nigrostriatal projection by the injection of 6-OHDA into the SNc unilaterally. GDNF had no effect at 10 μg, when tested at 2-weeks and 4-weeks after 6-OHDA. Order of Bars: 1-Week; 2-Weeks; 4-Weeks.

[0040] Figure 3 shows the distribution of MANF in the striatum at different flow rates after 0 hours. (A) 0.1 μί/ηιίη (B) 1.25 μί/ηιίη (C) 5 μί/ηώι. All infusions were 10 μg in 2 μΙ_, PBS. Scale bar = 250 μιη.

[0041] Figure 4 shows the distribution of MANF in the striatum at different flow rates after 7 days. GDNF (1 & 3) and no infusion control (2 & 4) were completed at 2.5 μυηιίη into the striatum, as were MANF (5) and no infusion control (6). MANF (7, 9 & 1 1) or PBS (8)/ no infusion (10 & 12) controls were completed at 1.25 μυηιίη into the striatum. Scale bar = 250 μΜ.

[0042] Figure 5 shows calculated volume of distribution of MANF in the striatum at different flow rates after 7 days. MANF infusions were completed at 1.25 μί/ηιίη (n = 2) and 2.5 μί/ηιίη (n=2) into the striatum. GDNF infusions were completed at 2.5 μί/ηιίη (n=2) into the striatum.

[0043] Figure 6 shows (A) an exemplary step-design cannula that can be used in

convection enhanced delivery of therapeutic agents, such as MANF, (B) a delivery sheath that can function as a guide component for the step-design cannula, and (C) an assembly of a step-design cannula wherein the upper portion of delivery sheath is removed.

[0044] Figure 7 illustrates an exemplary neurosurgical device that can be used in

convection enhanced delivery of therapeutic agents, such as MANF, showing (A) a catheter and (B) the catheter inserted into a guide device.

[0045] Figure 8 illustrates 6-OHDA-induced dopaminergic denervation in the striatum.

Brain sections (40 μιη thickness) stained for TH+ and Nissl, mounted on glass slides. (A) Densitometry data were acquired from rostral to caudal at four levels spaced by 320 μιη. A 3x3 grid was applied to define areas for spatial resolution (e.g. , temporal, medial, basal; dorsal, ventral) of densitometry data. Shown is a brain section generated from a vehicle/vehicle animal. (B) 6-OHDA treatment leads to partial denervation in the dorsomedial region. (C) 6-OHDA-induced full denervation of the left striatum.

[0046] Figure 9 illustrates the study design for Example 3. Striatal 6-OHDA

administration at time = 0. Unit of time is weeks. Single administration of growth factors at the indicated time-points. In the neuroprotection protocols the growth factors were administered 6h pre-6-OHDA. Behavioral testing in amphetamine- induced rotations at the indicated time-points. Tissue collections for biochemical and cell biological analyses immediately after the last behavioral test.

[0047] Figure 10 illustrates biochemical and cell biological analyses in Example 3. The 6-OHDA and growth factor administration schedules were as described in Figure 9. MANF and GDNF activities were tested after administration to the striatum or the substantia nigra. Striatal administration analyses included counts of TH ⁺ neurons in the substantia nigra and measurement of dopamine levels in the striatum. Nigral administration analysis included densitometry of TH ⁺ staining in the striatum, levels of dopamine and metabolites in the striatum and determination of the number of dopaminergic neurons in the substantia nigra by stereology.

[0048] Figure 11 shows amphetamine -induced rotational behavior following striatal administration of MANF or GDNF in a neuroprotection protocol. Net ipsilateral rotations (mean ± SEM) during a 2h observation period. Treatment groups as indicated (Vehicle (N=l 1), MANF 3μg (N=l 1), MANF l(^g (N=12), MANF 36μg (N=10), GDNF l0μg (N=l 1)). Time points as indicated (2 weeks, 4 weeks and 8 weeks post 6-OHDA / MANF / GDNF). Statistical analysis with repeated measures two-way ANOVA followed by Fisher's LSD. A) Amphetamine-induced rotations by time -point (week 2, week 4, week 8); (**) P<0.01, (*) P<0.05, (x) P<0.1 (trend) compared to vehicle. B) Amphetamine-induced rotations by treatment group for each time -point. (#) P<0.05 compared to prior time-point.

[0049] Figure 12 shows dopaminergic neurons in the substantia nigra following striatal administration of MANF or GDNF in a neuroprotection protocol. Number of TH ⁺ neurons per field (mean ± SEM). Treatment groups as indicated (Vehicle (N=5), MANF 3μg (N=5), MANF l(^g (N=5), MANF 36μg (N=6), GDNF l(^g (N=6)). 1-wayANOVA followed by Fisher's LSD. (*) P<0.05, (**) PO.01 compared to vehicle; (o) P<0.05 compared to GDNF 10μ§. (A) Ipsilateral TH neurons, (B) Contralateral TH ⁺ neurons, (C) Ratio ipsilateral / contralateral.

[0050] Figure 13 shows striatal dopamine (DA) levels following striatal administration of MANF or GDNF in a neuroprotection protocol. Amounts of dopamine shown as nmol/g wet tissue weight (mean ± SD). Treatment groups as indicated. Statistical analysis by 1-way ANOVA. None of the treatment groups were different from 6- OHD A/vehicle. (A) Ipsilateral, (B) Contralateral.

[0051] Figure 14 shows amphetamine-induced rotational behavior following striatal administration of MANF or GDNF in a neuroregeneration protocol. Net ipsilateral rotations (mean ± SEM). Treatment groups as indicated (Vehicle (N=5), MANF 3μg (N=7), MANF l(^g (N=l 1), MANF 36μg (N=9), GDNF l(^g (N=9)). Time points as indicated (1 week pre, 2 weeks, 4 weeks and 8 weeks post MANF / GDNF administration). Statistical analysis with two-way ANOVA followed by Fisher's LSD. A) Amphetamine -induced rotations by time -point (week -1, week 2, week 4, week 8); None of the treatment groups was statistically different from vehicle treatment at any time point, (x) P<0.1 (trend) versus GDNF 10 μg. B) Amphetamine-induced rotations by treatment group for each time-point. (#) P<0.05, (+) P<0.1 (trend) compared to prior time -point (within treatment group comparisons).

[0052] Figure 15 shows dopaminergic neurons in the substantia nigra following striatal administration of MANF or GDNF in a neuroregeneration protocol. Number of TH ⁺ neurons per field (mean ± SEM). Treatment groups as indicated (Vehicle (N=7), MANF 3μg (N=4), MANF l(^g (N=4), MANF 36μg (N=4), GDNF l(^g (N=4)). 1-wayANOVA followed by Fisher's LSD. (*) P<0.05 compared to vehicle; (o) P<0.05 compared to GDNF 10 μg. (A) Ipsilateral TH ⁺ neurons, (B) Contralateral TH ⁺ neurons, (C) Ratio ipsilateral / contralateral.

[0053] Figure 16 shows striatal dopamine (DA) levels following striatal administration of MANF or GDNF in a neuroregeneration protocol. Amounts of dopamine shown as nmol/g wet tissue weight (mean ± SEM). Treatment groups as indicated (6-OHDA / Vehicle (N=4, ipsi; N=3, contra), 6-OHDA / MANF 3μg (N=3, ipsi; N=4, contra), 6-OHDA / MANF l(^g (N=4, ipsi; N=4, contra), 6-OHDA / MANF 36μg (N=5, ipsi; N=4, contra ), 6-OHDA / GDNF ^g (N=4, ipsi; N=6, contra)). 1-way ANOVA followed by Fisher's LSD. None of the treatment groups were different from 6-OHDA/veh. (A) Ipsilateral, (B) Contralateral.

[0054] Figure 17 shows amphetamine -induced rotational behavior following nigral

administration of MANF or GDNF in a neuroprotection protocol. Net ipsilateral rotations (mean ± SEM). Treatment groups as indicated (Vehicle / vehicle (N=8), 6-OHDA / veh (N=8), 6-OHDA / MANF 3μg (N=10), 6-OHDA / MANF l(^g (N=8), 6-OHDA / MANF 36μg (N=8), 6-OHDA / GDNF l(^g (N=10)). Time points as indicated (2 weeks and 4 weeks post MANF / GDNF administration). Statistical analysis with repeated measures two-way ANOVA followed by

Fisher's LSD. A) Amphetamine-induced rotations by time -point (week 2, week 4); (*) P<0.05, (x) P<0.1 (trend) compared to 6-OHD A/vehicle; (o) P<0.05 compared to GDNF l0μg. B) Amphetamine-induced rotations by treatment group for each time-point. None of the within treatment groups differences were statistically significant.

[0055] Figure 18 shows dopaminergic neurons in the substantia nigra determined by stereo logy following nigral administration of MANF or GDNF in a

neuroprotection protocol. Computed number of nigral TH ⁺ neurons (mean ± SEM) at week 4 post MANF / GDNF administration. Treatment groups as indicated (Vehicle / vehicle (N=5, ipsi, contra), 6-OHDA / vehicle (N=6, ipsi; N=4, contra), 6-OHDA / MANF 3μg (N=6, ipsi; N=4, contra), 6-OHDA / MANF l(^g (N=5, ipsi; N=4, contra), 6-OHDA / MANF 36μg (N=5, ipsi; N=l, contra ), 6-OHDA / GDNF l(^g (N=5, ipsi; N=3, contra)). 1-way ANOVA followed by Fisher's LSD. None of the treatment groups were different from 6-OHD A/vehicle, (x) P<0.05 compared to vehicle/vehicle. (A) Ipsilateral, (B) Contralateral, (C) Ratio ipsilateral / contralateral.

[0056] Figure 19 shows dopaminergic terminals in the global, dorsal and ventral striatum determined by densitometry following nigral administration of MANF or GDNF in a neuroprotection protocol. Density of terminals (mean ± SEM) at week 4 post MANF / GDNF administration. Treatment groups as indicated (Veh/veh (N=5 global, N=6 dorsal, ventral), 6-OHDA/veh (N=6), 6-OHD A/MANF 3μg (N=6), 6- OHDA/MANF l(^g (N=6 global, N=5 dorsal, ventral), 6-OHD A/MANF 36μg (N=5), 6-OHD A/GDNF l(^g (N=6)). 1-way ANOVA followed by Fisher's LSD. (**) P<0.01, (*) P<0.05 compared to 6-OHD A/vehicle; (o) P<0.05 compared to 6- OHDA/GDNF 10 μg. Global striatum: (A) ipsilateral, (B) contralateral, (C) ratio ipsilateral / contralateral; Dorsal striatum: (D) ipsilateral, (E) contralateral, (F) ratio ipsilateral / contralateral; Ventral striatum: (G) ipsilateral, (H) contralateral, (I) ratio ipsilateral / contralateral.

[0057] Figure 20 shows dopaminergic terminals in the temporal, medial and basal

striatum determined by densitometry following nigral administration of MANF or GDNF in a neuroprotection protocol. Density of terminals (mean ± SEM) at week 4 post MANF / GDNF administration. Treatment groups as indicated (Veh/veh (N=6), 6-OHDA/veh (N=6), 6-OHDA/MANF 3μg (N=6), 6-OHDA/MANF l(^g (N=5), 6-OHDA/MANF 36μg (N=5), 6-OHDA/GDNF l(^g (N=6)). 1-way ANOVA followed by Fisher's LSD. (***) PO.001, (*) P<0.05, (x) P<0.1 (trend) compared to 6-OHD A/vehicle; (o) P<0.05 compared to 6-OHDA/GDNF 10 μg. Temporal striatum: (A) ipsilateral, (B) contralateral, (C) ratio ipsilateral / contralateral; Medial striatum: (D) ipsilateral, (E) contralateral, (F) ratio ipsilateral / contralateral; Basal striatum: (G) ipsilateral, (H) contralateral, (I) ratio ipsilateral / contralateral.

[0058] Figure 21 shows striatal dopamine (DA) levels following nigral administration of MANF or GDNF in a neuroprotection protocol. Amounts of dopamine shown as nmol/g wet tissue weight (mean ± SEM) at week 4 post MANF / GDNF administration. Treatment groups as indicated (Vehicle / vehicle (N=5, ipsi, contra), 6-OHDA / Vehicle ( =5 ipsi, contra), 6-OHDA / MANF 3μg (N=5, ipsi, contra), 6-OHDA / MANF l(^g (N=5, ipsi, contra), 6-OHDA / MANF 36μg (N=4, ipsi, contra ), 6-OHDA / GDNF ^g (N=6, ipsi, contra)). 1-way ANOVA followed by Fisher's LSD. (*) P<0.05, (x) P<0.1 (trend) compared to 6-OHDA / vehicle; (o) P<0.05 compared to 6-OHDA/GDNF 10 μg. (A) Ipsilateral, (B) Contralateral, (C) Ratio ipsilateral / contralateral.

[0059] Figure 22 shows striatal DOPAC levels following nigral administration of MANF or GDNF in a neuroprotection protocol. Amounts of DOPAC shown as nmol/g wet tissue weight (mean ± SEM) at week 4 post MANF / GDNF administration. Treatment groups as indicated (Vehicle / vehicle (N=5, ipsi, contra), 6-OHDA / Vehicle ( =5 ipsi, contra), 6-OHDA / MANF 3μg (N=5, ipsi, contra), 6-OHDA / MANF ^g (N=5, ipsi, contra), 6-OHDA / MANF 36μg (N=4, ipsi, contra ), 6- OHDA / GDNF l(^g (N=6, ipsi, contra)). 1-way ANOVA followed by Fisher's LSD. (*) PO.05, (**) P<0.01 and (***) PO.001 compared to 6-OHD A/vehicle; (o) P<0.05, (x) P<0.1 (trend) compared to 6-OHD A/GDNF 10 μ§. (A) Ipsilateral, (B) Contralateral, (C) Ratio ipsilateral / contralateral.

[0060] Figure 23 shows striatal HVA levels following nigral administration of MANF or GDNF in a neuroprotection protocol. Amounts of HVA shown as nmol/g wet tissue weight (mean ± SEM) at week 4 post MANF / GDNF administration.

Treatment groups as indicated (Vehicle / vehicle (N=5, ipsi, contra), 6-OHDA / Vehicle (N=5 ipsi, contra), 6-OHDA / MANF 3μg (N=5, ipsi, contra), 6-OHDA / MANF l(^g (N=5, ipsi, contra), 6-OHDA / MANF 36μg (N=4, ipsi, contra ), 6- OHDA / GDNF l(^g (N=6, ipsi, contra)). 1-way ANOVA followed by Fisher's LSD. (*) PO.05, (**) P<0.01 and (***) PO.001 compared to 6-OHD A/vehicle; (o) P<0.05 , (+) P<0.1 (trend) compared to 6-OHD A/GDNF 10 μg. (A) Ipsilateral, (B) Contralateral, (C) Ratio ipsilateral / contralateral.

[0061] Figure 24 shows amphetamine -induced rotational behavior following nigral

administration of MANF or GDNF in a neuroregeneration protocol. Net ipsilateral rotations (mean ± SEM). Treatment groups as indicated (Vehicle / vehicle (N=l 1), 6-OHDA / veh (N=10), 6-OHDA / MANF 3μg (N=12), 6-OHDA / MANF l(^g (N=10), 6-OHDA / MANF 36μg (N=10), 6-OHDA / GDNF l(^g (N=l 1)). Time points as indicated (1 week pre, 2 weeks and 4 weeks post MANF / GDNF administration). Statistical analysis with repeated measures two-way ANOVA followed by Fisher's LSD. (*) P<0.05 compared to 6-OHD A/vehicle; (x) P<0.1 (trend) compared to 6-OHD A/GDNF 10 μg. A) Amphetamine-induced rotations by time-point (week -1, week 2, week 4); None of the growth factor treatment groups was statistically different from 6-OHD A/vehicle treatment at any time point. No trends of treatment differences to 6-OHD A/vehicle were detected. B) Amphetamine-induced rotations by treatment group for each time -point. (#) P<0.05 compared to prior time -point (within treatment group comparisons).

[0062] Figure 25 shows dopaminergic neurons in the substantia nigra determined by stereo logy following nigral administration of MANF or GDNF in a

neuroregeneration protocol. Computed number of nigral TH ⁺ neurons (mean ± SEM) at week 4 post MANF / GDNF administration. Treatment groups as indicated (Vehicle / vehicle (N=6, ipsi; N=2, contra), 6-OHDA / Vehicle (N=6, ipsi; N=3, contra), 6-OHDA / MANF 3μg (N=6, ipsi; N=3, contra), 6-OHDA / MANF 10μ§ (N=6, ipsi; N=5, contra), 6-OHDA / MANF 36μ§ (N=6, ipsi; N=5, contra ), 6-OHDA / GDNF l(^g (N=6, ipsi; N=4, contra)). 1 -way ANOVA followed by Fisher's LSD. None of the treatment groups were different from 6- OHD A/vehicle, (x) P<0.05 compared to vehicle/vehicle. (A) Ipsilateral, (B) Contralateral, (C) Ratio ipsilateral / contralateral.

[0063] Figure 26 shows dopaminergic terminals in the global, dorsal and ventral striatum determined by densitometry following nigral administration of MANF or GDNF in a neuroregeneration protocol. Density of terminals (mean ± SEM) at week 4 post MANF / GDNF administration. Treatment groups as indicated (Veh/veh (N=6), 6-OHDA/veh (N=6 dorsal, N=5 global, ventral), 6-OHDA/MANF 3μg (N=6), 6-OHDA/MANF l(^g (N=6 global, dorsal, N=5 ventral), 6- OHDA/MANF 36μg (N=6), 6-OHDA/GDNF 10μg (N=6)). 1-way ANOVA followed by Fisher's LSD. (***) PO.001 , (*) P<0.05 compared to 6- OHD A/vehicle; (o) P<0.05, (x) P<0.1 (trend) compared to 6-OHDA/GDNF 10 μg. Global striatum: (A) ipsilateral, (B) contralateral, (C) ratio ipsilateral /

contralateral; Dorsal striatum: (D) ipsilateral, (E) contralateral, (F) ratio ipsilateral / contralateral; Ventral striatum: (G) ipsilateral, (H) contralateral, (I) ratio ipsilateral / contralateral.

[0064] Figure 27 shows Dopaminergic terminals in the temporal, medial and basal

striatum determined by densitometry following nigral administration of MANF or GDNF in a neuroregeneration protocol. Density of terminals (mean ± SEM) at week 4 post MANF / GDNF administration. Treatment groups as indicated (Veh/veh (N=6), 6-OHDA/veh (N=4 basal, contralateral temporal, contralateral medial, all others N=6), 6-OHDA/MANF 3μg (N=6), 6-OHDA/MANF l(^g (N=6), 6-OHDA/MANF 36μg (N=6), 6-OHDA/GDNF l(^g (N=6)). 1 -way ANOVA followed by Fisher's LSD. (***) P<0.001 compared to 6- OHD A/vehicle; (o) P<0.05 compared to 6-OHDA/GDNF 10 μg. Temporal striatum: (A) ipsilateral, (B) contralateral, (C) ratio ipsilateral / contralateral;

Medial striatum: (D) ipsilateral, (E) contralateral, (F) ratio ipsilateral /

contralateral; Basal striatum: (G) ipsilateral, (H) contralateral, (I) ratio ipsilateral / contralateral.

[0065] Figure 28 shows striatal dopamine (DA) levels following nigral administration of MANF or GDNF in a neuroregeneration protocol. Amounts of dopamine shown as nmol/g wet tissue weight (mean ± SEM) at week 4 post MANF / GDNF administration. Treatment groups as indicated (Vehicle / vehicle (N=5, ipsi, contra), 6-OHDA / Vehicle (N=4 ipsi, contra), 6-OHDA / MANF 3μg (N=4, ipsi, contra), 6-OHDA / MANF l(^g (N=5, ipsi; N=3, contra), 6-OHDA / MANF 36μg (N=4, ipsi, contra ), 6-OHDA / GDNF l(^g (N=5, ipsi, contra)). 1-way ANOVA followed by Fisher's LSD. (**) P<0.01, (x) P<0.1 (trend) compared to 6- OHD A/vehicle. (A) Ipsilateral, (B) Contralateral, (C) Ratio ipsilateral / contralateral.

[0066] Figure 29 shows striatal DOPAC levels following nigral administration of MANF or GDNF in a neuroregeneration protocol. Amounts of DOPAC shown as nmol/g wet tissue weight (mean ± SEM) at week 4 post MANF / GDNF administration. Treatment groups as indicated (Vehicle / vehicle (N=5, ipsi, contra), 6-OHDA / Vehicle (N=4 ipsi, contra), 6-OHDA / MANF 3μg (N=4, ipsi, contra), 6-OHDA / MANF l(^g (N=6, ipsi; N=3, contra), 6-OHDA / MANF 36μg (N=4, ipsi, contra ), 6-OHDA / GDNF l(^g (N=5, ipsi, contra)). 1-way ANOVA followed by Fisher's LSD. (**) P<0.01 compared to 6-OHD A/vehicle; (x) P<0.1 (trend) compared to 6-OHD A/GDNF l(^g. (A) Ipsilateral, (B) Contralateral, (C) Ratio ipsilateral / contralateral.

[0067] Figure 30 shows striatal HVA levels following nigral administration of MANF or GDNF in a neuroregeneration protocol. Amounts of HVA shown as nmol/g wet tissue weight (mean ± SEM) at week 4 post MANF / GDNF administration.

Treatment groups as indicated (Vehicle / vehicle (N=5, ipsi, contra), 6-OHDA / Vehicle (N=5 ipsi, contra), 6-OHDA / MANF 3μg (N=4, ipsi, contra), 6-OHDA / MANF l(^g (N=5, ipsi; N=3, contra), 6-OHDA / MANF 36μg (N=4, ipsi, contra ), 6-OHDA / GDNF l(^g (N=5, ipsi, contra)). 1-way ANOVA followed by Fisher's LSD. (**) PO.01 and (***) PO.001 compared to 6-OHD A/vehicle. (A) Ipsilateral, (B) Contralateral, (C) Ratio ipsilateral / contralateral.

[0068] Figure 31 shows distribution of MANF in the striatum at different flow rates on Day 0. 10 μg of MANF or GDNF in 2 μΐ of PBS were delivered by CED. MANF was detected by immunohistochemistry, scale bar = 250 μιη. (A) 0.1 μΐ/min; (B) 1.25 μΐ/min; (C) 5 μΐ/min.

[0069] Figure 32 shows distribution of MANF and GDNF in the striatum at different flow rates after seven days. 10 μg of MANF or GDNF in 2 μΐ of PBS were delivered by CED. MANF and GDNF were detected by immunohistochemistry, scale bar = 250 μηι. (A) GDNF 2.5 μΐ/min; (B) No infusion control; (C) MANF 2.5 μΐ/min; (D) No infusion control; (E) MANF 1.25 μΐ/min; (F) No infusion control.

[0070] Figure 33 shows calculated volumes of distribution in the striatum for MANF and GDNF infusions at different flow rates after 7 days (mean ± SEM). MANF (10 μg in 2 μΐ PBS) striatal infusions were performed at flow rates of 1.25 μΐ/min (N = 2) and 2.5 μΐ/min (N = 2). GDNF (10 μg in 2 μΐ PBS) striatal infusions were performed at a flow rate of 2.5 μΐ/min (N = 2). Statistical analysis by 1-way ANOVA followed by Fisher's LSD. No statistically significant differences in distribution volumes between the treatment groups were found.

DETAILED DESCRIPTION OF THE INVENTION

[0071] The primary cause of Parkinson's disease (PD) is believed to be the death of a significant percentage (70-80%) of dopaminergic (DAergic) neurons in the substantia nigra zona compacta (SNc) in the ventral midbrain. The cell bodies of the DAergic neurons are located in the SNc, and their axons project to the striatum (caudate-putamen in man). This pathway is called the nigrostriatal projection.

[0072] In normal function, the dopaminergic axonal terminals divide repeatedly and contact other neuronal cell bodies and nerve terminals in the striatum. The SNc is one of a system of about eight related brain areas that control normal, slow movements like tying show laces, buttoning a shirt, raising food from plate to mouth and the hand shake greeting. These are some of the movements that are compromised in Parkinsonian patients.

[0073] Results from animal models of PD have shown that if the DAergic cell bodies in the SNc are viable, but the dopaminergic axonal terminals in the striatum are reduced significantly, then function is still compromised. A strategy in treating PD is to find drugs that will both protect the remaining DAergic cell bodies, and stimulate sprouting of the dopaminergic axonal terminals in the putamen.

[0074] The drug GDNF (glial cell line-derived neurotrophic factor) is a standard in the field. However, when GDNF is placed in the SNc, it protects the DAergic cell bodies from death, but does not stimulate sprouting of dopaminergic axonal terminals in the striatum, and therefore does not correct neurological deficits. However, when GDNF is placed in the striatum, it does correct neurological deficits. Sprouting of DAergic terminals in the striatum can be important for the correction of neurological deficits.

[0075] Thus, there is a need for drugs that stimulate sprouting of DAergic nerve

terminals in the striatum when administered either to the SNc or to the striatum. In patients, such a drug could be administered to both targets, the SNc and striatum, and would stimulate the proliferation of DAergic terminals in the striatum and a reduction of neurological deficits.

[0076] The SNc is located deep in the ventral midbrain. Thus, based on results obtained to date (e.g. , with GDNF) the targeting of a drug to the SNc has been perceived as potentially risky, with little potential for benefit. Because of this, during clinical trials, drugs have been administered to the putamen, which is located

subcortically, and more superficially in the brain. Administering drugs to the putamen poses less risk to patients.

[0077] For optimal therapeutic benefit, the ideal drug would be one that is effective when targeted to the putamen and/or the SNc. Such a drug could then be targeted to the putamen and/or the SNc in a treatment, thereby increasing the therapeutic benefit. The additional risk of administering a drug to the SNc (alone or in combination with administration to the putamen) will be justified by the potential reward of a more robust clinical outcome versus then the drug is administered to the putamen alone.

[0078] The experiments disclosed herein surprisingly show that MANF can have a dual action in treatment: (1) to increase in the number of viable DAergic neurons in the substantia nigra over time and (2) to increase in the density of dopaminergic terminals in the putamen over time, e.g., by stimulating neurite outgrowth. Unlike other neurotrophic factors such as GDNF, MANF can be effective when delivered to either the striatum or the substantia nigra.

[0079] The term "or" can be used conjuctively or disjunctively.

[0080] The term "about" means the indicated numerical value ± 10%. All numerical indications in this specification are to be interpreted as being qualified by "about" unless the context clearly indicates otherwise.

[0081] The singular forms "a", "an", and "the" are used herein to include plural

references unless the context clearly indicates otherwise. [0082] Unless otherwise indicated, open terms, for example, "contain," "containing," "include," "including," and the like mean comprising.

[0083] This disclosure relates generally to compositions, methods, and kits for treating a neurological disorder (e.g., by increasing survival of neurons) using

mesencephalic astrocyte-derived neurotrophic factor (MANF). MANF is a neurotrophic factor that selectively protects DA neurons in vitro and corrects the neurological deficits caused by the degeneration of dopaminergic neurons (DA) neurons in the brain in vivo. MANF is expressed at a high level in the ventral mesencephalon, the same region of the brain where the DA neurons that die in PD are located. The actions of MANF indicate that it can be used to treat neurological disorders such as Parkinson's Disease (PD).

[0084] In one aspect, the invention features a method of treating a patient having a

disease or disorder of the nervous system. The method including administering to the patient an effective amount of MANF. The effective amount can be an amount required to promote dopaminergic neuronal survival or neurite outgrowth. The effective amount can be an amount required to reduce one or more symptoms of the disease or disorder. The effective amount can be an amount required to delay the progression of the disease or disorder.

[0085] As demonstrated herein, maintenance, survival or growth of dopaminergic

neurons are enhanced through administration of a MANF. Dopaminergic neurons of the mesencephalon die in patients having Parkinson's disease. The invention thus provides a treatment of Parkinson's disease. In addition, the use of MANF in the treatment of disorders or diseases of the nervous system in which the loss of dopaminergic neurons is present or anticipated is included. The MANF can be substantially purified MANF.

[0086] By "substantially purified" is meant that a polypeptide (e.g., a MANF polypeptide) has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially purified when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. The polypeptide can be at least 75%, at least 90%, or at least 99%), by weight, pure. A substantially purified polypeptide can be obtained, for example, by extraction from a natural source (e.g., a neural cell), by expression of a recombinant nucleic acid encoding the polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0087] A polypeptide is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state. Thus, a polypeptide which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially purified polypeptides include those which naturally occur in eukaryotic organisms but are synthesized in E. coli or other prokaryotes.

[0088] By "polypeptide" or "protein" is meant any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or

phosphorylation.

[0089] By "pharmaceutically acceptable excipient" is meant an excipient, carrier, or diluent that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the polypeptide with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline solution. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington: The Science and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A R., 2000, Lippencott Williams & Wilkins.

[0090] The term "dopaminergic neuronal survival-promoting activity" can indicate that the presence of the compound increases survival of dopaminergic neurons by at least two-fold in a dopaminergic neuronal survival assay relative to survival of dopaminergic neurons in the absence of the compound. The increase in the survival of dopaminergic neurons can be, e.g. , by at least three-fold, at least fourfold, or at least five-fold. The assay can be an in vitro assay or an in vivo assay.

[0091] As shown previously (Commissiong et al, US Application Publication No.

2002/0182198, or Ser. No. 10/102,265), a cell line of mesencephalic origin (termed "VMCL-1 ") secretes a factor that, in turn, promotes differentiation and survival of dopaminergic neurons. This cell line grows robustly in a serum- free medium. Moreover, the CM prepared from these cells contains one or more dopaminergic neuronal survival factors that increase the survival of mesencephalic dopaminergic neurons at least 3-fold, and promotes their development as well.

[0092] Commissiong et al. also demonstrated purification of MANF from the VMCL-1 cell line. The protein was isolated as follows. A 3 L volume of VMCL-1 conditioned medium was prepared, and subjected to five sequential steps of column chromatography. At each purification step, each column fraction was tested for biological activity in the bioassay referred to above. An estimate of the effect of each fraction on dopaminergic neuronal survival was done at 24 hour intervals, over a period of five days, and rated on a scale of 1-10. After the fifth purification step, the biologically active fraction and an adjacent inactive fraction were analyzed by SDS-PAGE. The results of the SDS-PAGE analysis revealed a distinctive protein band in the 20 kDa range in the lane from the active fraction. The "active" band was excised and subjected to tryptic digest, and the molecular mass and sequence of each peptide above background were determined by mass spectrometry analysis. The following two peptide sequences were identified: DVTFSPATIE and QIDLSTVDL. A search of the database identified a match for human arginine-rich protein and its mouse orthologue. The predicted protein encoded by the mouse EST sequence is about 95% identical to the predicted human protein. A search of the rat EST database revealed two sequences, one (dbEST Id: 4408547; EST name: EST348489) having significant homology at the amino acid level to the human and mouse proteins. The full-length rat sequence was not in the GenBank database.

[0093] Furthermore, Commissiong et al. discovered that human ARP is cleaved such that the arginine-rich amino-terminus is separated from the carboxy-terminus to produce human pro-MANF. The cleaved carboxy-terminal fragment contains a signal peptide, resulting in the secretion of human MANF from the cell.

[0094] The term "MANF," unless the context indicates otherwise, is meant to encompass both the secreted and pro forms of the protein, and any biologically functional fragments thereof. MANF can be from any organism, e.g., mammals (e.g., humans, non-human primates, rodents such as mice or rats, dogs, cats, horses, etc.) or insects (e.g., Drosophila species). [0095] Protein Therapy

[0096] MANF, or another therapeutic agent, can be administered to a subject to treat a neurological disorder in the subject. For example, in some embodiments, a subject is treated to enhance maintenance and/or promote growth (e.g., neurite outgrowth) of dopaminergic neurons. The neurological disorder can be Parkinson's disease, or any other neurological disorder associated with the loss of dopaminergic neural function. Various dosage formulations are contemplated for use in the methods disclosed herein.

[0097] A therapeutic approach disclosed herein can involve administration of MANF, either directly to the site of a potential or actual cell loss (for example, by injection) or systemically (for example, by any conventional recombinant protein administration technique).

[0098] The experiments disclosed herein show that MANF can have a dual action in

treatment: (1) to increase in the number of viable DAergic neurons in the substantia nigra over time and (2) to increase in the density of dopaminergic terminals in the putamen over time, for example, by stimulating neurite outgrowth. Unlike other neurotrophic factors such as GDNF, MANF can be effective when delivered to either the striatum or the substantia nigra.

[0099] MANF has been identified in numerous species, including human, rat, mouse, and cow. The source of MANF used (e.g., the species from which it was derived) can depend upon the species being treated. It is contemplated that MANF derived from the same species to be treated will be used in the treatment. It is also contemplated that MANF derived from one species can be used in the treatment of another species. One in the art will recognize that the identification of MANF from other animals can be readily performed using standard methods. Any protein having dopaminergic neuronal survival-promoting activity and encoded by a nucleic acid that hybridizes to the cDNA encoding human ARP is considered part of the invention.

[00100] Modifications

[00101] MANF, or any other protein therapeutic agent, can be modified prior to administration to a subject.

[00102] Modified proteins can possess deletions and/or substitutions of amino

acids; thus, a protein with a deletion, a protein with a substitution, and a protein with a deletion and a substitution are modified proteins. In some embodiments these modified proteins may further include insertions or added amino acids, such as with fusion proteins or proteins with linkers, for example.

[00103] Substitutional or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, for example, to reduce its immunogenicity/antigenicity, reduce any side effects in a subject, or increase its efficacy. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or

phenylalanine; and valine to isoleucine or leucine. An antigenic region of a polypeptide may be substituted for a less antigenic region; the less antigenic region may contain residues that are identical to the corresponding residues in the native protein, yet also contain some conservative substitutions and/or

nonconservative substitutions.

[00104] In addition to a deletion or substitution, a modified protein may possess an insertion of residues, which typically involves the addition of at least one residue in the polypeptide. This may include the insertion of a targeting peptide or polypeptide or simply a single residue. Terminal additions, called fusion proteins, are discussed below.

[00105] The term "biologically functional equivalent" is well understood in the art and is further defined in detail herein. Accordingly, sequences that have between about 70% and about 80%, or between about 81% and about 90%, or even between about 91% and about 99% of amino acids that are identical or functionally equivalent to the amino acids of a native polypeptide are included, provided the biological activity of the protein is maintained. A modified protein may be biologically functionally equivalent to its native counterpart. In various embodiments, a biologically functional equivalent of MANF is administered in a therapeutically effective amount.

[00106] It also will be understood that amino acid and nucleic acid sequences

encoding a therapeutic agent, such as MANF, can include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5 ' or 3' portions of the coding region or may include various internal sequences, e.g., introns, which are known to occur within genes.

[00107] The following discussion is based upon changing of the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, binding sites to substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below. A proteinaceous molecule has "homology" or is considered "homologous" to a second proteinaceous molecule if one of the following "homology criteria" is met: 1) at least 30% of the proteinaceous molecule has sequence identity at the same positions with the second proteinaceous molecule; 2) there is some sequence identity at the same positions with the second proteinaceous molecule and at the nonidentical residues, at least 30% of them are conservative differences, as described herein, with respect to the second proteinaceous molecule; or 3) at least 30%> of the

proteinaceous molecule has sequence identity with the second proteinaceous molecule, but with possible gaps of nonidentical residues between identical residues. As used herein, the term "homologous" may equally apply to a region of a proteinaceous molecule, instead of the entire molecule. If the term "homology" or "homologous" is qualified by a number, for example, "50% homology" or "50% homologous," then the homology criteria, with respect to 1), 2), and 3), is adjusted from "at least 30%" to "at least 50%." Thus it is contemplated that there may homology of at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%), 85%o, 90%), 95%), or more between two proteinaceous molecules or portions of proteinaceous molecules.

[00108] In making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[00109] It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average

hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

[00110] It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[00111] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[00112] Another embodiment for the preparation of modified polypeptides

according to the invention is the use of peptide mimetics. Mimetics are peptide - containing molecules that mimic elements of protein secondary structure. The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen. A peptide mimetic is expected to permit molecular interactions similar to the natural molecule. These principles may be used, in conjunction with the principles outline above, to engineer second generation modified protein molecules having many of the natural properties of a native protein, but with altered and, in some cases, even improved characteristics.

[00113] Fusion proteins

[00114] A specialized kind of insertional variant is the fusion protein comprising

MANF, or another therapeutic agent. This molecule generally has all or a substantial portion of the native molecule, linked at the N- or C-terminus, to all or a portion of a second polypeptide. For example, fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host. Another useful fusion includes the addition of an

immunologically active domain, such as an antibody epitope or other tag, to facilitate targeting or purification of the fusion protein. The use of 6X His and GST (glutathione S transferase) as tags is well known. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. Other useful fusions include linking of functional domains, such as active sites from enzymes such as a hydrolase, glycosylation domains, cellular targeting signals or transmembrane regions.

[00115] Conjugated Proteins

[00116] MANF, or another therapeutic agent, can be conjugated. For example, in various embodiments, provided herein are conjugated polypeptides, such as translated proteins, polypeptides and peptides that are linked to at least one agent to form an conjugate. Of particular use are antibody conjugates, in which the antibody portion targets the agent to a particular site. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is

conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g. , cell growth activity or cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include growth agents, toxins, anti-tumor agents, therapeutic enzymes, radio-labeled nucleotides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or poly-nucleotides. By contrast, a reporter molecule is defined as any moiety that may be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffmity molecules, colored particles or ligands, such as biotin.

[00117] Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label. "Detectable labels" are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and/or further quantified if desired. Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti-cellular agent, and may be termed "immunotoxins."

[00118] Linkers/Coupling Agents

[00119] MANF, or another therapeutic agent, can be joined in a fusion as described above or through a linker or coupling agent as follows. Examples of linker types that can be used to conjugate MANF, or another therapeutic agent, include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide-containing linkers. A linker can be chosen that is, for example, susceptible to cleavage by low pH or susceptible to cleavage by proteases, such as cathepsins (e.g., cathepsins B, C, D). For example, multiple peptides or polypeptides may be joined via a biologically-releasable bond, such as a selectively-cleavable linker or amino acid sequence. For example, peptide linkers that include a cleavage site for an enzyme preferentially located or active within a tumor environment are contemplated. Exemplary forms of such peptide linkers are those that are cleaved by urokinase, plasmin, thrombin, Factor IXa, Factor Xa, or a metallaproteinase, such as collagenase, gelatinase, or stromelysin. Alternatively, peptides or polypeptides may be joined to an adjuvant Amino acids such as selectively-cleavable linkers, synthetic linkers, or other amino acid sequences may be used to separate proteinaceous moieties.

[00120] Protein Purification

[00121] Methods and processes for purifying protein therapeutic agents {e.g. ,

MANF), including endogenous polypeptides and peptides and recombinant polypeptides and peptides are disclosed herein. Generally, these techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using

chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC. In addition, the conditions under which such techniques are executed may be affect characteristics, such as functional activity, of the purified molecules.

[00122] The term "isolated or purified protein or peptide" as used herein, is

intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.

[00123] Generally, "purified" will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%>, about 80%>, about 90%>, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.2%, about 99.4%, about 99.6%, about 99.8%, about 99.9% or more of the proteins in the

composition.

[00124] Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number." The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.

[00125] Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

[00126] There is no general requirement that the protein or peptide always be

provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in

combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column

chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.

[00127] It is known that the migration of a polypeptide can vary, sometimes

significantly, with different conditions of SDS/PAGE (Capaldi et al, 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.

[00128] The use of a peptide tag in combination with the methods and

compositions of the invention is also contemplated. A tag takes advantage of an interaction between two polypeptides. A portion of one of the polypeptides that is involved in the interaction may used as a tag. For instance, the binding region of glutathione S transferase (GST) may be used as a tag such that glutathione beads can be used to enrich for a compound containing the GST tag. An epitope tag, which an amino acid region recognized by an antibody or T cell receptor, may be used. The tag may be encoded by a nucleic acid segment that is operatively linked to a nucleic acid segment encoding a modified protein such that a fusion protein is encoded by the nucleic acid molecule. Other suitable fusion proteins are those with .beta.-galactosidase, ubiquitin, hexahistidine (6X His), or the like.

Furthermore, in some embodiments, MANF or biologically functional fragments thereof are tagged with a fluorescence tag (e.g., GFP, eGFP), which are conventionally known and utilized in detection and monitoring of proteins.

[00129] Combinatorial

[00130] A protein-based therapy disclosed herein can include a combination of

MANF and one or more additional therapeutic agent. Such additional therapeutics include, but are not limited to MANF2 (see, U.S. Patent Application Publication No. 20060084619), CDNF, interferon gamma, nerve growth factor, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), neurogenin, brain derived neurotrophic factor (BDNF), thyroid hormone, bone morphogenic proteins (BMPs), leukemia inhibitory factor (LIF), sonic hedgehog, glial cell line-derived neurotrophic factor (GDNFs), vascular endothelial growth factor (VEGF), interleukins, interferons, stem cell factor (SCF), activins, inhibins, chemokines, retinoic acid and ciliary neurotrophic factor (CNTF) or a combination thereof. Examples of additional therapeutic agents and methods useful with the methods and compositions of the invention are disclosed in U.S. Patent Application Nos: 20080057028; 20070082848, 20070077649

[00131] Pharmaceutical Compositions

[00132] Pharmaceutical compositions of the protein therapeutic agents (e.g.,

MANF) described herein are disclosed herein. Pharmaceutical compositions can comprise MANF, another therapeutic agent, or MANF and one or more additional therapeutic agents, which are administered to a subject to effect prophylactic or therapeutic effect. Such compositions comprise a therapeutically or

prophylactically effective amount of MANF or functional fragments thereof. Examples of such additional one or more therapeutic agents include but are not limited to proteins that are or nucleic acids encoding proteins that are,

"Neurotrophic factors". Neurotrophic factors are growth factors that promote differentiation, maintain a mature phenotype and provide trophic support, promoting growth and survival of neurons. Neurotrophic factors reside in the nervous system or in innervated tissues. The following have been described as neurotrophic factors: basic fibroblast growth factor, (bFGF), acidic fibroblast growth factors (aFGF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophin-3 (NT3), NT4/5, insulin-like growth factor (IGF-1), IGF-II, NT-4, IL-Ι β, TNFa, transforming growth factor β (TGF- β, TGF- β 1), MANF (NTN), persephin (PSP), artemin, and AL-1. Uses of neurotrophic factors are well known to those of skill in the art and can be found, for example, in U.S. Patent Publication No. 2001001 1 126, U.S. Pat. Nos. 6,284,540, 6,280,732, 6,274,624, 6,221 ,676, which are incorporated by reference herein. The use of Par4 is well known to those of skill in the art, as disclosed in U.S. Pat. No. 6, 1 1 1 ,075, which is hereby incorporated by reference. In various embodiments, methods of treating a neurological disorder are contemplated within the scope of the present invention, where MANF or functional fragments thereof are co-administered to a subject with one or more neurogrophic factor.

[00133] In various embodiments of the invention, a pharmaceutical composition comprising MANF or functional fragments thereof, may further contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine), chelating agents [such as ethylenediamine tetraacetic acid (EDTA)]; complexing agents (such as caffeine,

polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or

immunoglobulins); coloring; flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides (preferably sodium or potassium chloride, mannitol sorbitol);

delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants.

(Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company, 1990).

[00134] The pharmaceutical compositions may include one or more inert

excipients, which include water, buffered aqueous solutions, surfactants, volatile liquids, starches, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, acids, bases, salts, emulsions, such as oil/water emulsions, oils such as mineral oil and vegetable oil, wetting agents, chelating agents, antioxidants, sterile solutions, complexing agents, disintegrating agents and the like.

[00135] Excipients can include: water, phosphate buffered saline solutions,

propylene glycol diesters of medium chain fatty acids available under the tradename Miglyol 840 (from Huls America, Inc. Piscataway, N.J.) triglyceride esters of medium chain fatty acids available under the tradename Miglyol 812 (from Huls); perfluorodimethylcyclobutane available under the tradename Vertrel 245 (from E. I. DuPont de Nemours and Co. Inc. Wilmington, Del.);

perfluorocyclobutane available under the tradename octafluorocyclobutane (from PCR Gainsville, Fla.); polyethylene glycol available under the tradename EG 400 (from BASF Parsippany, N.J.); menthol (from Pluess-Stauffer International Stanford, Conn.); propylene glycol monolaurate available under the tradename lauroglycol (from Gattefosse Elmsford, N.Y.), diethylene glycol monoethylether available under the tradename Transcutol (from Gattefosse); polyglycolized glyceride of medium chain fatty adds available under the tradename Labrafac Hydro WL 1219 (from Gattefosse); alcohols, such as ethanol, methanol and isopropanol; eucalyptus oil available (from Pluses-Stauffer International), and mixtures thereof.

[00136] Compounds of the present invention include amino acid derivatives. A preferred surfactant may be the sodium salt form of the compound, which may include the monosodium salt form. Suitable sodium salt surfactants may be selected based on desirable properties, including high speed of polymerization, small resultant particle sizes suitable for delivery, good polymerization yields, stability including freeze-thaw and shelf-life stability, improved surface tension properties, and lubrication properties.

[00137] Surfactants which can be used in pharmaceutical compositions and dosage forms include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. That is, a mixture of hydrophilic surfactants may be employed, a mixture of lipophilic surfactants may be employed, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed.

[00138] Other suitable aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride. Aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p- hydroxybenzoate. [00139] Chelating agents which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, ethylene diaminetetraacetic acid (EDTA), EDTA disodium, calcium disodium edetate, EDTA trisodium, albumin, transferrin, desferoxamine, desferal, desferoxamine mesylate, EDTA tetrasodium and EDTA dipotassium, sodium metasilicate or combinations of any of these.

[00140] Lubricants which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g. , peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, or mixtures thereof.

[00141] Thickening agents which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, isopropyl myristate, isopropyl palmitate, isodecyl neopentanoate, squalene, mineral oil, C ₁₂ - Ci5 benzoate and hydrogenated polyisobutene. Particularly preferred are those agents which would not disrupt other compounds of the final product, such as non- ionic thickening agents. The selection of additional thickening agents is well within the skill of one in the art.

[00142] Other agents may also be added, such as antimicrobial agents, to prevent spoilage upon storage, e.g., to inhibit growth of microbes such as yeasts and molds. Suitable antimicrobial agents are typically selected from the group consisting of the methyl and propyl esters of p-hydroxybenzoic acid (e.g., methyl and propyl paraben), sodium benzoate, sorbic acid, imidurea, purite, peroxides, perborates and combinations thereof.

[00143] Preservatives which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, purite, peroxides, perborates, imidazolidinyl urea, diazolidinyl urea, phenoxyethanol, alkonium chlorides including benzalkonium chlorides, methylparaben, ethylparaben and propylparaben. [00144] Formulations

[00145] Also disclosed are formulations of the one or more therapeutic agents (e.g.,

MANF) disclosed herein. For example, in various embodiments, parenteral formulations (e.g. , for infusion, convection enhanced delivery, acoustic targeted drug delivery) can be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.

[00146] Methods well known in the art for making formulations are to be found in, for example, Remington: The Science and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A R., 2000, Lippencott Williams & Wilkins. Formulations for parenteral administration may, for example, contain as excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes, biocompatible, biodegradable lactide polymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the present factors. Other potentially useful parenteral delivery systems for the factors include ethylene -vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.

[00147] The present factors (e.g., MANF) can be used as the sole active agents, or can be used in combination with other active ingredients, e.g., other growth factors which could facilitate dopaminergic neuronal survival in neurological diseases, or peptidase or protease inhibitors.

[00148] The concentration of the present factors in the formulations of the

invention will vary depending upon a number of issues, including the dosage to be administered, and the route of administration.

[00149] The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of an pharmaceutical composition that contain MANF or additional active ingredient can be determined by the skilled artisan, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g. , human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards, or other regulatory body.

[00150] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is

contemplated.

[00151] The therapeutic agents may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.

[00152] In various embodiments, therapeutic compositions of the invention can be administered intraocularly, intravenously, intradermally, intraarterially, intraperitoneally, intracranially, topically, intramuscularly, intraperitoneally, subcutaneously, intravesicularlly, mucosally, orally, topically, locally, by inhalation (e.g. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), by convection enhanced delivery, by acoustic targeted drug delivery, or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).

[00153] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the methods of preparation can include vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium can be buffered if necessary and the liquid diluent can be rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

[00154] The composition can be stable under the conditions of manufacture and storage, or preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination can be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

[00155] An approach for stabilizing solid protein formulations of the invention is to increase the physical stability of purified, e.g. , lyophilized, protein. This will inhibit aggregation via hydrophobic interactions as well as via covalent pathways that may increase as proteins unfold. Stabilizing formulations in this context often include polymer-based formulations, for example a biodegradable hydrogel formulation/delivery system. As noted above, the critical role of water in protein structure, function, and stability is well known. Typically, proteins are relatively stable in the solid state with bulk water removed. However, solid therapeutic protein formulations may become hydrated upon storage at elevated humidity or during delivery from a sustained release composition or device. The stability of proteins generally drops with increasing hydration. Water can also play a significant role in solid protein aggregation, for example, by increasing protein flexibility resulting in enhanced accessibility of reactive groups, by providing a mobile phase for reactants, and by serving as a reactant in several deleterious processes such as beta-elimination and hydrolysis.

[00156] Protein preparations containing between about 6% to 28% water can be unstable. Below this level, the mobility of bound water and protein internal motions can be low. Above this level, water mobility and protein motions approach those of full hydration. Up to a point, increased susceptibility toward solid-phase aggregation with increasing hydration has been observed in several systems. However, at higher water content, less aggregation is observed because of the dilution effect.

[00157] In particular embodiments, prolonged absorption of an injectable

composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.

[00158] In various embodiments, formulations comprising therapeutic agents of the invention comprise a stabilizing or delivery vehicle. The term "vehicle" in this context refers to a molecule that prevents degradation and/or increases half-life, reduces toxicity, reduces immunogenicity, or increases biological activity of a therapeutic protein. Exemplary vehicles include an Fc domain as well as a linear polymer (e.g., polyethylene glycol (PEG), polylysine, dextran, etc.); a branched- chain polymer (See, for example, U.S. Pat. No. 4,289,872 to Denkenwalter et al., issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tarn, issued Jul. 20, 1993; WO 93/21259 by Frechet et al., published 28 Oct. 1993); a lipid; a cholesterol group (such as a steroid); a carbohydrate or oligosaccharide; or any natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor.

[00159] In one embodiment, this invention provides for at least one peptide to be attached to at least one vehicle (F _ls F ₂ ) through the N-terminus, C-terminus or a side chain of one of the amino acid residues of the peptide(s). Multiple vehicles may also be used; e.g., Fc's at each terminus or an Fc at a terminus and a PEG group at the other terminus or a side chain.

[00160] An Fc domain can be used as a vehicle. The Fc domain may be fused to the N or C termini of the peptides or at both the N and C termini. See, for example WO 97/34631 and WO 96/32478. In such Fc variants, one can remove one or more sites of a native Fc that provide structural features or functional activity not required by the fusion molecules of this invention. One may remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site. The inserted or substituted residues may also be altered amino acids, such as peptidomimetics or D-amino acids. [00161] An alternative vehicle would be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (e.g., a peptidomimetic compound) capable of binding to a receptor or target molecule on a target cell. For example, one could use as a vehicle a polypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also be selected by phage display for binding to the FcRn salvage receptor. Such salvage receptor-binding compounds are also included within the meaning of "vehicle" and are within the scope of this invention. Such vehicles should be selected for increased half-life (e.g. , by avoiding sequences recognized by proteases) and decreased immunogenicity (e.g. , by favoring non-immunogenic sequences, as discovered in antibody

humanization).

[00162] In some embodiments, a vehicle is polyethylene glycol (PEG). The PEG group may be of any convenient molecular weight and may be linear or branched. The average molecular weight of the PEG will preferably range from about 2 kiloDalton ("kDa") to about 100 kDa, more preferably from about 5 kDa to about 50 kDa, most preferably from about 5 kDa to about 10 kDa. In another

embodiment, the average molecular weight of the PEG will be from about 10 kDa to about 20 kDa, or 20 kDa to 30 kDa or 30 kDa to 40 kDa or 40 kDa to 50 kDa. The PEG groups will generally be attached to the compounds of the invention via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the inventive compound (e.g., an aldehyde, amino, or ester group).

[00163] A useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The peptides can be easily prepared with conventional solid phase synthesis as known in the art. The peptides are "preactivated" with an appropriate functional group at a specific site. The precursors are purified and fully characterized prior to reacting with the PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated peptides can be easily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry. [00164] Polysaccharide polymers are another type of water soluble polymer which may be used for protein modification. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by a 1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kDa to about 70 kDa. Dextran is a suitable water-soluble polymer for use in the present invention as a vehicle by itself or in combination with another vehicle (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference. Dextran of about 1 kDa to about 20 kDa is preferred when dextran is used as a vehicle in accordance with the present invention.

[00165] Stability of Formulations and Pharmaceutical Compositions

[00166] It has been found that MANF is surprisingly stable when stored in a

buffered solution. The buffer can be any buffer known in the art. In one embodiment, the buffer is citrate buffer.

[00167] When MANF is stored in a buffer, the concentration of the buffer can be from 0.01 mM to 100 mM. For example, the concentration of the buffer can be 0.01-100 mM, 0.01-50 mM, 0.01-10 mM, 0.01-5 mM, 0.01-1 mM, 0.01-0.1 mM, 0.1-100 mM, 0.1-50 mM, 0.1-10 mM, 0.1-5 mM, 0.1-1 mM, 1-100 mM, 1-50 mM, 1-10 mM, 1-5 mM, 5-100 mM, 5-50 mM, 5-10 mM, 10-100 mM, 10-50 mM, or 50-100 mM. In one embodiment, the concentration of the buffer can be about 10 mM.

[00168] When MANF, or another active agent, is stored in a buffer, the pH of the solution (e.g., the pharmaceutical composition or formulation) can be from 4 to 9. For example, the pH can be 4-9, 4-8, 4-7, 4-6.5, 4-6, 4-5.5, 4-5, 5-9, 5-8, 5-7, 5-

6.5, 5-6, 5-5.5, 5.5-9, 5.5-8, 5.5-7, 5.5-6.5, 5.5-6, 6-9, 6-8, 6-7, 6-6.5, 6.5-9, 6.5-8, 6.5-7, 7-8, 7-9, or 8-9. For example, the pH can be about: 4, 4.1, 4.2, 4.3, 4.4, 4.5,

4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9. In one embodiment, the pH is from 5.5 to 6.5. In another embodiment, the pH is about 6.1. [00169] A buffered solution of MANF can be stable at 4°C for a period of time.

The period of time the MANF solution is stable can be, for example, about: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer. In one embodiment, the buffer is citrate buffer. The citrate buffer can be at a concentration of about 5 mM to 50 mM (e.g., about 10 mM). The citrate buffer can be at a pH of about 5.5 to 6.5 (e.g., about 6.1).

[00170] A buffered solution of MANF can be stable at room temperature (e.g., from 20°C to 25 °C) for a period of time. The period of time the MANF solution is stable can be, for example, about: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer. In one embodiment, the buffer is citrate buffer. The citrate buffer can be at a concentration of about 5 mM to 50 mM (e.g., about 10 mM). The citrate buffer can be at a pH of about 5.5 to 6.5 (e.g., about 6.1).

[00171] A buffered solution of MANF can be stable at -20°C for a period of time.

The period of time the MANF solution is stable can be, for example, about: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer. In one embodiment, the buffer is citrate buffer. The citrate buffer can be at a concentration of about 5 mM to 50 mM (e.g., about 10 mM). The citrate buffer can be at a pH of about 5.5 to 6.5 (e.g., about 6.1).

[00172] Dosage Levels

[00173] The actual dosage amount of a composition (e.g., an effective amount) that is administered to an animal can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration can determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[00174] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.

[00175] In other non-limiting examples, a dose of a pharmaceutical composition or formulation can comprise from about 1 ng/kg/body weight, about 5 ng/kg/body weight, about 10 ng/kg/body weight, about 50 ng/kg/body weight, about 100 ng/kg/body weight, about 200 ng/kg/body weight, about 350 ng/kg/body weight, about 500 ng/kg/body weight, 1 μg/kg/body weight, about 5 μg/kg/body weight, about 10 μg/kg/body weight, about 50 μg/kg/body weight, about 100 μg/kg/body weight, about 200 μg/kg/body weight, about 350 μg/kg/body weight, about 500 μg/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 μg/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

[00176] In further embodiments, the composition may comprise various

antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g. , methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[00177] In additional embodiments of the invention, methods of treatment include administration of therapeutic agents of the invention (e.g., MANF, or

combinations of MANF and one or more additional neurotrophic factors) a certain time period, such as a therapeutically effective time period. In some embodiments, the administration formulations of the invention or a nucleic acid encoding MANF or biologically functional fragments is specifically contemplated. It is

contemplated that the treatment may be administered for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 hours or 1 2, 3, 4, 5, 6, 7 days or 1 , 2, 3, 4, 5 weeks, or 1 , 2, 3, 4, 5, 6, 7, 8, 9 10, 1 1 , 12 months or more. Chronic administration is contemplated for 1 2, 3, 4, 5, 6, 7 days or 1 , 2, 3, 4, 5 weeks, or 1 , 2, 3, 4, 5, 6, 7, 8, 9 10, 1 1 , 12 months or more. Multiple administrations are also contemplated. In some embodiments the administration is given at least twice (repeated at least once).

[00178] A subject may be administered different amounts of therapeutic agents. In some embodiments, the amount of a formulation of the invention (e.g. , protein or nucleic acid) that is administered is between 1 and 1000 ng per kilogram body weight per hour. In other embodiments, the amount is between 1 and 100 ng per kilogram body weight per hour or between 1 and 10 ng per kilogram body weight per hour. It is contemplated that dosages maybe 1 , 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1 100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000 ng per kilogram body weight per hour (ng/kg/hr). It is also contemplated that dosages may also be at least and/or not more than those same amounts.

[00179] The effective amount of MANF in a pharmaceutical composition or

formulation can be from 1 μg to 1000 μg. For example, the effective amount can be 1-1000 μ& 1 -500 μ& 1-250 μ& 1-150 μ& 1-100 μ& 1-75 μ& 1-50 μ& 1 -25 μg, 1-10 μg, 1-5 μg, 5-1000 μg, 5-500 μg, 5-250 μg, 5-150 μg, 5-100 μg, 5-75 μg, 5-50 μg, 5-25 μg, 5-10 μg, 10-1000 μg, 10-500 μg, 10-250 μg, 10-150 μg, 10-100 μg, 10-75 μg, 10-50 μg, 10-25 μg, 25-1000 μg, 25-500 μg, 25-250 μg, 25-150 μg, 25-100 μg, 25-75 μg, 25-50 μg, 50-1000 μg, 50-500 μg, 50-250 μg, 50-150 μg, 50-100 μg, 50-75 μg, 75-1000 μg, 75-500 μg, 75-250 μg, 75-150 μg, 75-100 μg, 100-1000 μg, 100-500 μg, 100-250 μg, 100-150 μg, 150-1000 μg, 150-500 μg, 150-250 μg, 250-1000 μg, 250-500 μ§ or 500-1000 μ§. In some embodiments, the effective amount is from 10 μ to 250 μg. In some embodiments the effective amount of MANF is from 50 μg to 150 μg. In some embodiments the effective amount of MANF is from 10 μ to 500 μg.

[00180] The effective amount of MANF can vary depending upon the species of a subject being treated. For example, the effective amount of MANF for treatment of a rodent, such as a mouse or rat, can be lower than the effective amount for treatment of a human or non-human primate. In one embodiment, the subject is a rodent and the effective amount is from about 1 μg to about 50 μg. In another embodiment, the subject is a human or non-human primate and the effective amount is from about 10 μg to about 500 μg.

[00181] The volume of a pharmaceutical composition or formulation comprising the effective amount of MANF (or other therapeutic agent) that is administered to a subject (e.g., via infusion, convection enhanced deliver, acoustic targeted drug delivery, etc.) can depend upon the species or size of the subject. For example, the volume administered to a rodent can be smaller than the volume administered to a larger mammal such as a human or non-human primate. The effective amount can be in a volume that is from about 1 μΐ _^ to about 2000 μί. For example, the effective amount can be in a volume that is 1 -2000 μί, 1-1500 μί, 1-1000 μί, 1- 750 μΐ,, 1-500 μΐ,, 1-250 μΐ,, 1-100 μΐ,, 1-50 μΐ,, 1-10 μΐ,, 10-2000 μΐ,, 10-1500 μΐ,, 10-1000 μΐ,, 10-750 μΐ,, 10-500 μΐ,, 10-250 μΐ,, 10-100 μΐ,, 10-50 μΐ,, 50- 2000 μΐ,, 50-1500 μΐ,, 50-1000 μΐ,, 50-750 μΐ,, 50-500 μΐ,, 50-250 μΐ,, 50-100 μΐ,, 100-2000 μΐ,, 100-1500 μΐ,, 100-1000 μΐ,, 100-750 μΐ,, 100-500 μΐ,, 100-250 μΐ,, 250-2000 μΐ,, 250-1500 μΐ,, 250-1000 μΐ,, 250-750 μΐ,, 250-500 μΐ,, 500- 2000 μΐ,, 500-1500 μΐ,, 500-1000 μΐ,, 500-750 μΐ,, 750-2000 μΐ,, 750-1500 μΐ,, 750-1000 μΐ,, 1000-2000 μΐ,, 1000-1500 μΐ,, or 1500-2000 μί.

[00182] Routes of Administration

[00183] Penetration of active ingredients into the central nervous system after systemic administration can be limited because of the blood-brain barrier and metabolism of the active ingredient (e.g., by the liver). Improvements in treatment efficacy can be realized by direct administration of active ingredients into the cerebrospinal fluid (CSF) or by infusion directly into brain regions that are target sites for the active ingredient. For example, intrathecal or intraventricular administration can be used to bypass the blood-brain barrier. However, diffusion- dependent methods, which include intrathecal or intraventricular administration, can be limited by nontargeted distribution, nonuniform dispersion, and ineffective volumes of distribution.

[00184] Convection Enhanced Delivery

[00185] Convection Enhanced Delivery (CED) can be a highly technical process that involves stereotactic placement of one or more catheters through cranial burr holes directly into brain tissue. A therapeutic agent can be continuously administered through the catheters by a microinfusion delivery system to create a positive pressure gradient at the catheter tip. As the pressure is maintained, it creates fluid convection or flow to supplement diffusion through the extracellular spaces and enhance the distribution of the therapeutic agent to the targeted area. The goals of CED can be to provide homogenous distribution of a therapeutic agent to a larger volume of brain tissue; provide higher drug concentrations directly to the tissue; and to utilize therapeutic agent in treatment that may not cross the blood brain barrier (BBB).

[00186] Convection enhanced delivery can be chronic delivery, acute delivery, or a combination thereof. For example, the methods of treatment can include chronic delivery of MANF to a brain region, wherein the MANF is delivered at a continuous infusion rate over a time period of days, weeks, months or years. The methods of treatment can include acute delivery of MANF wherein the MANF is delivered in discrete boluses over the course of minutes or hours. The methods of treatment can include a combination of chronic and acute delivery wherein MANF is delivered at a continuous first infusion rate over a time period of days, weeks, months or years interspersed at regular or irregular intervals of limited duration infusions at a second, faster rate.

[00187] CED of a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, can comprise an infusion rate of from about 0.1 μΕ/ηιίη to about 20 μΕ/ηιίη.

[00188] CED of a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, can comprise an infusion rate of greater than about: 0.1 μΕ/ηιίη, 0.5 μΕ/ηιίη, 0.7 μΕ/ηιίη, 1 μΕ/ηιίη, 1.2 μΕ/ηιίη, 1.5 μί/ηιίη, 1.7 μί/ηιίη, 2 μΕ/ηιίη, 2.2 μΕ/ηιίη, 2.5 μί/ηιίη, 2.7 μί/ηιίη, 3 μί/ηιίη, 3.5 μί/ηιίη, 4 μΕ/ηιίη, 5 μί/ηιίη, 7.5 μί/ηιίη, 10 μί/ηιίη, or 15 μί/ηιίη.

[00189] CED of a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, can comprise, or further comprise, an infusion rate of less than about: 25 μί/ηιίη, 20 μί/ηιίη, 15 μί/ηιίη, 12 μΕ/ηιίη, 10 μί/ηιίη, 7.5 μί/ηιίη, or 5 μί/ηιίη.

[00190] CED of a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, can comprise, or further comprise incremental increases in flow rate, referred to as "stepping", during delivery. Stepping can comprise infusion rates of between about 0.1 μΕ/ηιίη and about 20 μί/ηώι. At periodic intervals, stepping CED can comprise one or more increases in infusion rate in steps of about: 0.1 μΕ/ηιίη, 0.2 μΕ/ηιίη, 0.3 μΕ/ηιίη, 0.4 μΕ/ηιίη, 0.5 μΕ/ηιίη, 0.6 μΕ/ηιίη, 0.7 μΕ/ηιίη, 0.8 μΕ/ηιίη, 0.9 μΕ/ηιίη, 1 μΕ/ηιίη, 1.25 μΕ/ηιίη, 1.5 μΕ/ηιίη, 2 μΕ/ηιίη, or more.

[00191] The effective amount of MANF in a pharmaceutical composition or

formulation administered by CED can be from 1 μg to 1000 μg. For example, the effective amount can be 1-1000 μg, 1-500 μg, 1-250 μg, 1-150 μg, 1-100 μg, 1-75 μ _§ , 1-50 μ _§ , 1-25 μ _§ , 1-10 μ _§ , 1-5 μ _§ , 5-1000 μ _§ , 5-500 μ _§ , 5-250 μ _§ , 5-150 μ _§ , 5-100 μg, 5-75 μg, 5-50 μg, 5-25 μg, 5-10 μg, 10-1000 μg, 10-500 μg, 10-250 μg, 10-150 μg, 10-100 μg, 10-75 μg, 10-50 μg, 10-25 μg, 25-1000 μg, 25-500 μg, 25- 250 μ¾ 25-150 μ& 25-100 μ¾ 25-75 μ¾ 25-50 μ& 50-1000 μ¾ 50-500 μ& 50- 250 μ¾ 50-150 μ& 50-100 μ¾ 50-75 μ¾ 75-1000 μ& 75-500 μ¾ 75-250 μ& 75- 150 μg, 75-100 μg, 100-1000 μg, 100-500 μg, 100-250 μg, 100-150 μg, 150-1000 μg, 150-500 μg, 150-250 μg, 250-1000 μg, 250-500 μg or 500-1000 μg. In some embodiments, the effective amount is from 10 μg to 250 μg. In some

embodiments the effective amount of MANF is from 50 μg to 150 μg. In some embodiments the effective amount of MANF is from 10 μg to 500 μg.

[00192] The effective amount of MANF administered by CED can be in a volume o: 1-2000 μΕ, 1-1500 μΕ, 1-1000 μΕ, 1-750 μΕ, 1-500 μΕ, 1-250 μΕ, 1-100 μΕ, 1-50 μΕ, 1-10 μΕ, 10-2000 μΕ, 10-1500 μΕ, 10-1000 μΕ, 10-750 μΕ, 10-500 μΕ, 10-250 μΕ, 10-100 μΕ, 10-50 μΕ, 50-2000 μΕ, 50-1500 μΕ, 50-1000 μΕ, 50-750 μΕ, 50-500 μΕ, 50-250 μΕ, 50-100 μΕ, 100-2000 μΕ, 100-1500 μΕ, 100-1000 μΕ, 100-750 μΕ, 100-500 μΕ, 100-250 μΕ, 250-2000 μΕ, 250-1500 μΕ, 250-1000 μΕ, 250-750 μΐ,, 250-500 μΐ,, 500-2000 μΐ,, 500-1500 μΐ,, 500-1000 μΐ,, 500-750 μΐ,, 750-2000 μΐ,, 750-1500 μΐ,, 750-1000 μΐ,, 1000-2000 μΐ,, 1000-1500 μΐ,, or 1500-2000 μ

[00193] For further teaching on the method of CED, see for example Saito et al,

Exp. Neurol, 196:381-389, 2005; Krauze et al, Exp. Neurol, 196: 104-1 1 1 , 2005; Krauze et al, Brain Res. Brain Res. Protocol, 16:20-26, 2005; U.S. Patent Application Publication No. 2006/0073101 ; and U.S. Pat. No. 5,720,720, each of which is incorporated herein by reference in its entirety. See also Noble et al, Cancer Res. 2006 Mar. 1 ; 66(5):2801 -6; Saito et al, J Neurosci Methods. 2006 Jun. 30; 154(l-2):225-32; Hadaczek et al, Hum Gene Ther. 2006 March;

17(3):291-302; and Hadaczek et al, Mol Ther. 2006 July; 14(l):69-78, each of which is incorporated herein by reference in its entirety.

[00194] Use of tracers in convection enhanced delivery

[00195] An advantage of convection enhanced delivery can be the ability to use imaging technology to allow drug distribution to be seen during infusion.

Gadolinium- and iodine-based imaging compounds can be used as tracers to safely and accurately track active ingredient distribution in real-time using, for example, magnetic resonance imaging or computed tomography imaging. These tracers can show the distribution of both small- and large-molecular- weight compounds with similar convective properties during infusion.

[00196] In methods that comprise delivering a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, by CED, the pharmaceutical composition or formulation can comprise a tracing agent. The tracing agent can enable monitoring the distribution of the tracing agent as it moves through the CNS, and ceasing delivery of the

pharmaceutical composition when the MANF, another therapeutic agent, or a combination thereof is distributed in a predetermined volume within the CNS. The movement of the tracing agent through the solid tissue can be monitored by an imaging technique such as magnetic resonance imaging (MRI), computed tomography imaging, or X-ray computed tomography (CT). The tracing agent can have a mobility in CNS tissue that is substantially similar to the therapeutic agent (e.g., MANF), and delivery can be ceased when the tracing agent is observed to reach a desired region or achieve a desired volume of distribution, or to reach or nearly reach or exceed the borders of the target tissue.

[00197] The desired volume may correspond to a particular region of the brain that is targeted for therapy (e.g., the striatum or substantia nigra). The desired volume of distribution can be "substantially similar" to the volume of distribution observed for a tracing agent that is being monitored to follow the infusion.

"Substantially similar" refers to a difference in volume of less than 20%. More preferably, the difference in volume is less than 15%, more preferably less than 10%, more preferably less than 5%. By monitoring the distribution of the tracing agent, infusion may be ceased when the predetermined volume of distribution is reached.

[00198] The desired volume of distribution can be determined, for example, by using imaging software that is standard in the art, e.g., iFLOW™. See also, for example, Krautze et al., Brain Res. Protocols, 16:20-26, 2005; and Saito et al., Exp. Neurol, 196:3891-389, 2005, each of which is incorporated herein by reference in its entirety.

[00199] The tracer can comprise a paramagnetic ion for use with MRI. Suitable metal ions include those having atomic numbers of 22-29 (inclusive), 42, 44 and 58-70 (inclusive) and have oxidation states of +2 or +3. Examples of such metal ions are chromium (III), manganese (II), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III) and ytterbium (III).

[00200] In embodiments wherein X-ray imaging (such as CT) is used to monitor

CED, the tracer may comprise a radiopaque material. Suitable radiopaque materials include, but are not limited to, iodine compounds, barium compounds, gallium compounds, thallium compounds, and the like. Specific examples of radiopaque materials include barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, iosumetic acid, iotasul, iotetric acid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotriroic acid, ipodate, meglumine, metrizamide, metrizoate, propyliodone, and thallous chloride. [00201] Preoperative Diagnosis

[00202] Treatment methods disclosed herein can comprise or involve preoperative diagnosis.

[00203] Preoperative diagnosis can include neuroimaging, for example, by PET,

SPECT, MRI, X-ray computed tomography, or a combination thereof.

[00204] The neuroimaging can be used for target localization and guided cannula placement. A stereotactic holder can be used in conjunction with neuroimaging to provide for guided cannula placement at or proximal to a target neuronal population.

[00205] Devices for Convection Enhanced Delivery

[00206] It is contemplated that any suitable device can be used in the methods disclosed herein wherein a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, is administered to a brain region by CED.

[00207] A delivery device can comprise a pump that is capable of delivering a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, by CED. The pump can be an osmotic pump or an infusion pump. The device can comprise, or can be used in

conjunction with, a catheter or cannula that facilitates localized delivery to a brain region of a subject (e.g., striatum or substantia nigra). The catheter or cannula can comprise multiple outlet ports. The catheter or cannula can comprise an outer tubing to provide structural rigidity to the catheter or cannula. The catheter or cannula can be a reflux-free step-design cannula.

[00208] One or more catheters or cannuli can inserted into or near one or more brain regions of a subject (e.g., striatum or substantia nigra). Stereotactic maps and positioning devices are available, for example from ASI Instruments, Warren, Mich. Positioning can be conducted by using anatomical maps obtained by CT and/or MRI imaging of the subject's brain to help guide the injection device to the chosen target.

[00209] Reflux-free step-design cannula

[00210] CED can be performed with the use of a CED-compatible reflux-free step- design cannula, such as that disclosed in Krauze et al, J Neurosurg. 2005

November; 103(5):923-9, incorporated herein by reference in its entirety, as well as in U.S. Patent Application Publication No. US 2007/0088295 Al, incorporated herein by reference in its entirety, and U.S. Patent Application Publication No. US 2006/0135945 Al, incorporated herein by reference in its entirety.

[00211] In one embodiment, the step-design cannula is compatible with chronic administration. In another embodiment, the step-design cannula is compatible with acute administration. In another embodiment, the step-design cannula is compatible with a combination of chronic administration interspersed with periods of acute administration at a higher infusion rate or with a higher level of the therapeutic agent.

[00212] Figure 6A is a diagram of an exemplary step-design cannula 10 that can be used in the methods, systems, and kits disclosed herein.

[00213] As shown in Figure 6A, the cannula 10 can comprise a tube 12 having a substantially uniform inner diameter (ID) that defines a central lumen. The outer diameter (OD) of the tube 12, however, is non-uniform and decreases from the proximal end 14 to distal end 16 in order to minimize tissue damage in brain tissue and ensure reflux safety.

[00214] In the embodiment of the step-design cannula 10 shown in Figure 6A, the cannula can comprise a tube made from titanium or other biocompatible material having an ID of, for example, 0.286 mm (29 gauge) and a length of, for example, 234 mm. As illustrated, the OD of the tube decreases from 5 mm at its proximal end 14 to 0.33 mm at its distal end 16 in four steps, thus providing a cannula that has four segments. In this embodiment, the length of the first segment 18 is 40 mm with an OD of 5 mm, the length of the second segment 20 is 124 mm with an OD of 2.1 mm, the length of the third segment 22 is 10 mm with an OD of 0.64 mm, and the length of the fourth segment 24 (needle tip) is 10 mm with an OD of 0.33 mm. The measurements given are exemplary and can be modified by the skilled practitioner according to treatment needs.

[00215] Figure 6B illustrates a delivery sheath 100 that can function as a guide component for the step-design cannula 10, for example, to assist in placement of the infusion cannula. The system can be used in combination with an infusion pump that is either externally or subcutaneously placed. As illustrated, the delivery sheath 100 comprises a tubular member 102 which has, at one end, a coupling 104 that is adapted to be connected to a stereotaxic frame for support and placement. The tubular member 102 can be fabricated from a non-ferromagnetic flexible material such as a biocompatible plastic or a metal such as titanium. The coupling 104 is a conventional coupling that is adapted for connection to a stereotaxic frame at its proximal end 106. The connection between tubular member 102 and coupling 104 can be made using a conventional biocompatible bonding technique such as gluing the components together.

[00216] A central lumen 108 runs through at least a portion of tubular member 102 for receiving an infusion cannula. Accordingly, the inner diameter of central lumen 108 would typically be larger than the largest outer diameter of cannula 14 that will be inserted through the lumen. The distal end 110 of tubular member 102 is open so that the infusion cannula can extend therethrough. A longitudinal passageway or slot 112 communicates between the outer surface 114 of the tubular member and the central lumen for insertion of the infusion cannula into the delivery sheath. A plurality of openings 116 are positioned adjacent the perimeter of passageway 112 though which sutures can be placed.

[00217] Figure 6C illustrates an assembly of a step-design cannula wherein the upper portion of delivery sheath 100 is removed, for example, by cutting the sheath just above where the infusion cannula 200 bends over. A holding bracket 216 and bone screw 218 can be used to affix the assembly in place. A tubing 208 is coupled to the proximal end of the tubular body 202, which can be connected to an infusion pump (not shown). A set screw 220 can be used to lock delivery sheath 100 and cannula 200 in position to prevent relative movement.

[00218] Alternative catheters

[00219] CED can be performed with the use of a neurosurgical apparatus

comprising a CED-compatible catheter, such as that disclosed in WO

2013/127884, incorporated herein by reference in its entirety.

[00220] Figure 7 illustrates a neurosurgical apparatus suitable for CED in the

methods, systems and kits disclosed herein. The neurosurgical apparatus can comprise a guide device and a catheter. The guide device can comprise an elongate tube having a head at its proximal end. In use, the elongate tube can be inserted into the brain towards a target brain region {e.g., striatum or substantia nigra) via a hole formed in the skull. The head can be used to securely attach the guide device to the skull. This insertion may be performed using a stereoguide or surgical robot based technique. An internal channel is provided through the head and bore of the tube. The catheter can then be passed down this channel and into the brain in the vicinity of the selected target.

[00221] As illustrated in Figure 7 A, the catheter 40 comprises a hub 42, a fine tube

44 and a connector tube 46. The hub 42 comprises a body portion 48, a sealing element in the form of a tube having a tapered surface 50 and a pair of protruding wings 52. The wings 52 have apertures 54 formed therein for receiving bone screws.

[00222] As illustrated in Figure 7B, a catheter 40 of the type described is shown when inserted into a guide device 60. The catheter 40 comprises a bore or lumen 62 that runs through the connector tube 46, a hub 42 and a fine tube 44. The internal diameter of the lumen 62 is substantially constant through the catheter 40, although it could be varied if required. The guide device 60 comprises a head 64 and an elongate tube 66. The head 64 comprises external ridges 68 that allow it to be attached to a hole formed in the skull by a press fit action. The guide device 60 comprises a passageway 70 through the head 64 in which the catheter 40 can be located.

[00223] The passageway 70 of the head 64 comprises an internally tapered region

72 that forms a fluid tight seal with the tapered surface 50 of the inserted catheter. This seal prevent fluids from passing along the gap between the fine tube 44 and the elongate tube 66 into the head 64. Fluid leakage and reflux is thus inhibited. However, as shown in figure 7B, this seal does not obstruct the lumen 62 running through the catheter 40.

[00224] Figure 7B shows the arrangement after the catheter has been bent through ninety degrees in the slot formed in the head 64. The wings 52 of the catheter are, when the tapered region 72 of the guide device 60 engages the tapered surface 50 of the catheter 40, arranged to be located very close (e.g. within 0.5mm) to the head 64 of the guide device 60. This provides a visual indication that the tapered surfaces have engaged to form the fluid seal. The wings 52 and head 64 will also engage if further insertion of the catheter is attempted, thereby acting as an insertion limiter or safety stop to prevent buckling or other damage to the catheter.

[00225] This disclosure provides for delivery kits or devices comprising a pump that is capable of effecting delivery of a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, by CED. The kits or devices further comprise a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, such as any of those disclosed herein. The kits or devices further comprises a CED-compatible cannula or catheter. The cannula or catheter can be compatible with chronic or acute administration.

[00226] Kits

[00227] In one aspect, the invention provides kits for the treatment of a CNS

disorders (e.g., Parkinson's Disease). Such kits can comprise one or more pharmaceutical compositions or formulations comprising MANF, another therapeutic agent, or a combination thereof. The kit can further comprise a delivery device useful for CED. The delivery device can comprise a pump. The device can comprise one or more catheters or cannuli, such as those illustrated in Figure 6 and Figure 7. The cannuli can comprise a step-design reflux-free cannula, such as illustrated in Figure 6. Kits can further comprise guide devices for insertion of the cannuli or cather, such as those illustrated in Figure 6 and Figure 7. Kits may additionally comprise connecting parts, tubing, packaging material, instruction pamphlets, and other materials useful for practicing CED of a pharmaceutical composition or formulation comprising MANF, another therapeutic agent, or a combination thereof, to one or more brain regions of a subject having a CNS disorder (e.g., Parkinson's Disease).

[00228] Exemplary embodiments

[00229] In some embodiments, the invention provides a method of treating a

subject with a condition, comprising contacting the substantia nigra of the subject with an amount of MANF effective to treat the condition.

[00230] In some embodiments, the condition is a neurological disorder. In certain embodiments, the neurological disorder is Parkinson's disease.

[00231] In certain embodiments, the subject is a mammal. In some embodiments, the mammal is a human, rat, mouse, monkey, and rabbit.

[00232] In some embodiments, the method further comprises contacting the

striatum of a subject with an amount of MANF effective to treat said condition.

[00233] In certain embodiments, the method further comprises administering one or more additional therapeutic agents. [00234] In some embodiments, the subject exhibits an improvement in their clinical scores after treatment. In certain embodiments, the clinical score is a measurement of net ipsilateral rotations or total ipsilateral rotations.

[00235] In certain embodiments, the percentage of viable dopaminergic neurons in a subject increases; the density of dopaminergic terminals in the putamen of the subject increases; the percentage of viable dopaminergic neurons in the subject and the density of dopaminergic terminals in the putamen of the subject both increase; the viability of cell bodies in the striatum of the subject increases; the percentage of dying dopaminergic neurons in the subject decreases; and/or the percentage of dead dopaminergic neurons in the subject decreases.

[00236] In some embodiments, the amount of MANF administered is at least about

3, 5, 10, 20, 36, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 μg. In certain embodiments, the amount of MANF

administered is at least about 1.2, 1.4. 1.6. 1.8, or 2 mg.

[00237] In some embodiments, the amount of MANF administered is less than about 3, 5, 10, 20, 36, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 μg. In certain embodiments, the amount of MANF administered is less than about 1.2, 1.4. 1.6, 1.8, or 2 mg.

[00238] In some embodiments, the amount of MANF administered is about 3, 5,

10, 20, 36, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000 μg. In certain embodiments, the amount of MANF administered is about 1.2, 1.4. 1.6. 1.8, or 2 mg.

[00239] In some embodiments, the amount administered is selected to protect

and/or restore the functioning of sick dopaminergic neurons in the striatum; and/or to reduce the death of dopaminergic neurons in the subject.

[00240] Disclosed are methods of treating a neurological disorder in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of MANF to a brain region of the subject by convection enhanced delivery. In some embodiments, the neurological disorder is Parkinson's disease.

[00241] In some embodiments, the brain region comprises the striatum, the substantia nigra, or a combination thereof. In some embodiments, the brain region comprises the striatum. In some embodiments, the brain region comprises the substantia nigra. In some

embodiments, the brain region comprises the striatum and the substantia nigra. [00242] In some embodiments, the effective amount of MANF is from 1 μg to 1000 μg. In some embodiments, the effective amount of MANF is from 10 μg to 500 μg. In some embodiments, the effective amount of MANF is from 50 μg to 250 μg.

[00243] In some embodiments, convection enhanced delivery is performed at a flow rate of from 0.1 μΙ7ηήη ίο 10 μΙ7ηιίη.

[00244] In some embodiments, convection enhanced delivery is performed acutely.

[00245] In some embodiments, the convection enhanced delivery is repeated two or more times during a course of treatment.

[00246] In some embodiments, convection enhanced delivery is performed chronically.

[00247] In some embodiments, the pharmaceutical composition further comprises a tracer. In some embodiments, diffusion of the MANF in the brain region is monitored in by detection of the tracer. In some embodiments, monitoring comprises magnetic resonance imaging (MRI), computed tomography imaging, or X-ray computed tomography (CT).

[00248] In some embodiments, the pharmaceutical composition further comprises a buffer. In some embodiments, the buffer is a citrate buffer. In some embodiments, the

pharmaceutical composition has a pH of from 5.5 to 6.5.

[00249] In some embodiments, the subject exhibits an improvement in a clinical score after

treatment.

[00250] In some embodiments, the percentage of viable dopaminergic neurons in the subject increases. In some embodiments, the density of dopaminergic terminals in the subject's putamen increases. In some embodiments, both the percentage of viable dopaminergic neurons in the subject and the density of dopaminergic terminals in the subject's putamen increase. In some embodiments, the viability of cell bodies in the striatum increases.In some embodiments, the percentage of dying dopaminergic neurons in the subject decreases. In some embodiments, the percentage of dead dopaminergic neurons in the subject decreases.

[00251] Also disclosed are kits for treating a neurological disorder by convection enhanced

delivery of MANF to a brain region of a subject, the kits comprising: (a) a catheter, that can be stereotactically placed at or near the brain region; (b) a pump, for generating a positive pressure gradient between the catheter and the brain region; (c) a pharmaceutical composition comprising an effective amount of MANF to treat the neurological disorder.

[00252] Some embodiments further comprise a delivery sheath to assist in placement of the catheter.

[00253] In some embodiments, the catheter comprises multiple outlet ports. [00254] In some embodiments, the catheter comprises an outer tubing to provide structural rigidity to the catheter.

[00255] In some embodiments, the pharmaceutical composition comprises from 1 μg to 1000 μg of the MANF. In some embodiments, the pharmaceutical composition comprises from 10 μg to 500 μg of the MANF. In some embodiments, the pharmaceutical composition comprises from 50 μg to 250 μg of the MANF.

[00256] In some embodiments, the pharmaceutical composition further comprises a tracer.

[00257] In some embodiments, the pharmaceutical composition further comprises a buffer. In some embodiments, the buffer is a citrate buffer.

[00258] In some embodiments, the pharmaceutical composition has a pH of from 5.5 to 6.5.

[00259] Also disclosed are methods of treating a neurological disorder in a subject in need

thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of MANF in a 10 mM citrate buffer, pH 6.1 to a brain region of the subject.

EXAMPLES

[00260] Example 1

[00261] This example compares and contrasts the neurorestorative properties of

MANF and GDNF. In chronic, neurorestorative experiments, MANF or GDNF were administered at various time points after treatment with 6-OHDA, and the effect on neurorestoration was evaluated. The objective of the study was to evaluate the degree to which damaged DAergic neurons can be rescued from death by each drug.

[00262] Methods

[00263] Restoration Experiment: Target, Striatum

[00264] Male Wistar rats, 225 g, were obtained from Harlan Laboratories

(Livermore, CA), housed four per cage, supplied with food and water ad libitum, and kept for at least 24 hours before use. The animal protocol used was approved by thte Institutional Animal Care and Use Committee (IACUC) fo the

Departments of Psychiatry & Biobehavioral Sciences, UCLA, that is AAALAC- certified (Certificate No. 000408). NIH guidelines for the care and use of animals in research were adhered to throughout. Rats were anesthetized with Isoflurane, and mounted in the Kopf stereotaxic apparatus. A 10 Hamilton syringe was used to deliver 12 μg 6-OHDA (free base) (Sigma-Aldrich) in 3 μΐ, 0.05% ascorbic acid solution in phosphate buffered saline (PBS), pH 6.4, is infused into the right striatum using the above stereotaxic coordinates. The 6-OHDA was infused over a period of 3 min, the needle left in place for a further 5 min, withdrawn slowly to Z= -2.5 mm, left in place for a further 5 min, then slowly withdrawn. Two weeks later, the animals were re-anesthetized with Isofluran and mounted in the Kopf stereotaxic apparatus. 10 μg GDNF, or 3.0, 10 or 36 μg MANF dissolved in 10 mM citrate buffer, pH 6.1, were infused to the middle of the right striatum using the following stereotaxic coordinates (relative to bregma) (Paxinos and Watson): AP, +1.0 mm, ML = +3.0 mm, Z = -5.0. MANF or GDNF were delivered over a period of 3 min, the needle left in place for a further 5 min, withdrawn slowly to Z= -2.5 mm, left in place for a further 5 min, then slowly withdrawn. The wound was closed with surgical clips. Control animals were treated with the vehicle used to dissolve 6-OHDA and MANF/GDNF respectively. The MANF, GDNF and 6-OHDA solutions were made fresh, just prior to use. MANF and GDNF were prepared in siliconized, 1.5 mL Eppendorf tubes to prevent the proteins from sticking to the plastic surface. The tube containing the 6- OHDA was wrapped in aluminum foil, and kept on ice during the experiment to minimize degradation.

[00265] Restoration Experiment: Target, Substantia Nigra

[00266] The procedures described above for the restoration experiment with the striatum as target were used, except that drug administration was to the substantia nigra.

[00267] Behavioral Testing

[00268] Once the animals were recovered from the anesthetic, they were housed, 4- animals per cage, in a 12-hr/12-hr light/dark cycle, and allowed free access to water and rat chow. On testing days, the animals were handled gently and introduced into the rotometer with the appropriate harness attachment. For all animals, baseline recordings were made for 60 min. The animals were then treated with amphetamine, 2.5 mg/Kg, ip or vehicle. The acceptance level to be included in the experiment was >4 turns/min, for a 90-min period. Identical behavioral testing was done in all neurorestorative studies.

[00269] Results

[00270] Results showed that MANF significantly reduced neurological deficits in the rodent model of PD when targeted to the striatum or the SNc. When MANF or GDNF were targeted to the striatum, each caused a significant reduction in the neurological deficits in the restorative 6-OHDA model of PD (Figure 1).

However, when MANF and GDNF were targeted to the SNc, MANF but not GDNF causes a significant reduction in the neurological deficits in the 6-OHDA model of PD (Figure 2). The significant reduction in the neurological deficits in the 6-OHDA rodent model of PD when MANF is targeted to the SNc indicates that MANF, unlike GDNF, is able to stimulate the proliferation of DAergic terminals in the striatum when it is delivered to the SNc. This result represents a significant therapeutic breakthrough.

[00271] Discussion

[00272] In this study, dopaminergic neurons responded to a neurorestorative

molecule 3 -weeks after 6-OHDA. This result validates the nigral partial lesion model in which the toxin is delivered to the striatum versus the medial forebrain bundle (MFB). The results of Petrova et al. (2003), {e.g., Fig 6), show that neurons can remain viable after their identifying biomarkers have been downregulated. The larger, relevant issue is the state of the nigral dopaminergic neuronal population in newly diagnosed Parkinsonian patients. This DAergic neuronal population at diagnosis can probably be divided into three groups:

1. Viable: 20-30%,

2. Dead: 70%, and

3. Dying: Unknown, but of potential therapeutic significance.

[00273] The percentage of the dying group is of great interest. Drugs that increase the percentage of dopaminergic neurons over time would justify an invasive neurosurgical procedure in newly diagnosed patients.

[00274] The neurorestorative behavioral data presented in Figures 1 and 2 are

historical, in that the same groups of animals were tested at intervals over time. At 4-weeks, MANF 3 μg, MANF 10 μg and at 8-weeks, GDNF 10 μg all caused a significant reduction in circling in the restoration paradigm (Figure 1). This recovery suggests that the model is physiologically dynamic, with the

dopaminergic neurons being compromised on the lesioned side at 4-weeks, but recovering in the vehicle-treated group at 8-weeks. The data points that stand out at 4-weeks and 8-weeks show the lack of effect of MANF at 36 μg. [00275] A visual inspection of the restoration data at 2-weeks and 4-weeks with the

SNc as target suggests that MANF was effective at 10 μg and 36 μg. Surprisingly, GDNF 10 μg had no effect at 2-weeks or 4-weeks. MANF at a dose of 36 μg had no effect with the striatum as target.

[00276] However, with the SNc as target, MANF had a significant effect at 4- weeks. The positive action of 36 μg MANF at 4-weeks, but not at 2-weeks does suggest that dopaminergic neuronal repair can occur over time. MANF may therefore have a dual action in treatment:

1. an increase in the number of viable DAergic neurons in the SN over time;

2. an increase in the density of dopaminergic terminals in the putamen over time.

[00277] These two actions of MANF are mutually reinforcing, with the potential to produce a synergistic action in a therapeutic.

[00278] Example 2

[00279] Introduction

[00280] Convection-enhanced delivery (CED) has emerged as a novel

neurosurgical technique which has the potential to achieve more effective coverage of the putamen and other brain regions. CED describes a direct method of drug delivery to the brain through very fine microcatheters. By establishing a pressure gradient at the tip of the infusion catheter, CED confers several advantages over conventional drug injection techniques, in particular,

homogeneous drug distribution through large and clinically-relevant brain volumes. This example shows that a single infusion of MANF can be distributed via CED and detected after 7 days.

[00281] Methods

[00282] Convenction enhanced delivery procedures

[00283] All animal work was performed in accordance with the UK Animal

Scientific Procedures Act 1986 and was covered by both Project and Personal licenses that were issued by the Home Office and these were also reviewed and approved by the University of Bristol ethicalcommittee. All efforts were made to minimize animal use and suffering. Adult male Wistar rats (Charles River, Margate, UK, 225 to 275 g) were anaesthetised with intraperitoneal ketamine (Ketaset; 60 mg/kg, Pfizer Animal Health, Sandwich, UK) and medetomidine Dormitor; 0.4 mg/kg, Pfizer), and then placed in a stereotactic frame (Stoelting, Illinois, USA). A midline skin incision was made from glabella to occiput to expose bregma. Bilateral burr holes were drilled using a 2 mm drill. All CED procedures were performed using a custom-made catheter with an outer diameter of 0.22 mm and inner diameter of 0.15 mm, composed of fused silica with a laser cut tip. The cannula was attached to a 1 mL syringe (Hamilton, Bonaduz,

Switzerland) connected to a rate-controlled microinfusion pump (World Precision Instruments Inc., Sarasota, FL, USA) and the tip placed at stereotactic coordinates derived from the Paxinos and Watson stereotactic rat brain atlas (0.5 mm anterior and 3mm lateral to bregma, depth 4.5 mm), in order to target the striatum.

[00284] A total volume of 2 μΕ MANF or GDNF at an infused concentration of 10 μg diluted in PBS or PBS vehicle alone, was delivered into the striatum. CED procedures were performed at an infusion rate of 0.1 μΕ/ηιίη, 1.25 μΕ/ηιίη, 2.5 μΕ/ηιίη, or 5.0 μΕ/ηιίη. On completion of CED, the cannula was left in situ for 10 minutes to minimize reflux, then withdrawn at a rate of 1 mm/ minute. The wound was closed with 4/0 Vicryl, and a dose of intramuscular buprenorphine (Centaur Services, Castle Cary, UK) was administered (30 μg/kg). The anaesthetic was reversed with 0.1 mg/kg i.p. atipamezole hydrochloride (Pfizer) in recovery procedures. Rats were euthanised by anaesthetic overdose with an intraperitoneal injection of 1 mL pentobarbital (Euthatal; Merial Animal Health, Harlow, UK) at pre-defined time -points following CED (0, 3, 24 hours or 7 days). For

immunohistochemical analysis (IHC), animals were transcardially perfused with 4% paraformaldehyde. Brains were removed and placed in 4% paraformaldehyde for 24h, then cryoprotected in 30% sucrose.

[00285] Histology

[00286] Rat brains were cut into 35 μιη thick coronal sections using a Leica

CM1850 cryostat (Leica Microsystems, Wetzlar, Germany) at -20 °C. For fluorescent immunohistochemistry, fixed sections were mounted on gelatine- subbed slides. Once dry, the sections were washed with PBS for 5 minutes x 3. Sections were blocked in PBS plus 0.1% triton-X-100 containing 10% normal donkey serum (Sigma Aldrich, UK) for 1 hour at room temperature (RT). They were then washed with 0.1% triton-X-100 in PBS for 5 minutes. Following washing, sections were incubated in goat anti-GDNF primary antibody (1 :250; R&D Systems, Abingdon, UK) or goat anti-MANF (1 :200; R&D Systems, Abingdon, UK) in order to determine the presence and distribution of infused GDNF/ MANF.

[00287] The next day, primary antibody was removed and sections were washed with 0.1% triton-X-100 in PBS for 5 minutes x 3. Sections were incubated in Donkey Anti-Goat Alexa Fluor® 488 (1 :300, Life Technologies, Paisley, UK) at RT for 2 hours in the dark and then washed with PBS for 5 minutes x 3. Sections were mounted in FluorsaveTM Reagent (Calbiochem®, Merck Millipore, Billerica, MA, USA) before viewing. Images were captured using the Stereo Investigator platform (MicroBrightField Bioscience, Williston, VT, USA) with a Leica DM5500 microscope (Leica Microsystems, Germany) and digital camera (Microbrightfield Bioscience, Williston, VT, USA).

[00288] Volume of Distribution measurements

[00289] Fluorescent imaging was undertaken using a Leica DM5500 microscope

(Leica Microsystems) and digital camera (Leica Microsystems). The volume of distribution of MANF or GDNF recombinant proteins was calculated by tracing contours around the outer margins of the visualised protein using ImageJ software at 2-12 section intervals. Infusions that were associated with obvious reflux of protein into the white matter were excluded from further analysis.

[00290] Results

[00291] Results: 0 hours

[00292] MANF was delivered via CED at flow rates of 0.1 μΐν min, 1.25 μΙ7 min, and 5 μΏ min into the striatum. MANF was successfully detected at 0 hours at all flow rates by IHC (Figure 3).

[00293] Results: 7 days

[00294] Following on from the 0 hour time -point results, the flow rates employed for the 7 day study were 1.25 μί/ηιίη and 2.5 μί/ηιίη. These flow rates were predicted to be low enough for MANF to be detectable after 7 days. MANF was successfully detected by IHC after 7 days post-infusion following CED at both flow rates (1.25 μΕ/ηιίη and 2.5 μΕ/ηώι) (Figure 4). A higher volume of MANF was detectable in those hemispheres infused with a flow rate of 1.25 μΕ/ηιίη (0.0505 mm ³ ± 0.028) compared to 2.5 μΕ/ηώι (0.0185 mm ³ ± 0.008) and GDNF (0.028 mm ³ ± 0.006) (Figure 5). [00295] Conclusions

[00296] After 0 hours, 10 μg of MANF delivered via CED was detectable by IHC at 0.1, 1.25, and 5.0 μΙ ηιίη. After 7 days, 10 μg of MANF delivered via CED was detectable by IHC at 1.25, and 2.5μΕ/ηιίη and detectable levels of MANF were higher in those hemispheres infused with a flow rate of 1.25 μΕ/ηιίη compared to 2.5 μΕ/ηιίη. In addition, 1.25μΙ-Λϊΐϊη administration of MANF lead to a higher volume of distribution compared to GDNF.

[00297] The results of this study have shown successful delivery of MANF via

CED at 0 hours and 7 days, with improved distribution at a lower infusion rate.

[00298] Example 3

[00299] This example investigates MANF in the 6-OHDA rodent model of

Parkinson's Disease , to provide evidence and confirmation of MANF' s activities and experimental support for use of MANF for treatment of PD. Moreover, an additional object was to compare MANF and GDNF activities under the same experimental conditions.

[00300] Methods

[00301] MANF and GDNF protein source and characterization

[00302] The gene of a human MANF variant (R155P) with a C-terminal 6xHis tag and an enterokinase cleavage site was inserted into the kanamycin resistant expression vector pJexpress AW . E. coli BL21(DE3) cells transformed with the resulting expression vector were grown in a 5 liter fermenter in fortified LB medium containing a phosphate buffer and glucose. Cells were grown at 37°C until the glucose was nearly exhausted at which time a glucose feed was started. The glucose concentration was maintained at or below 1 g/L. MANF expression was induced by addition of Isopropyl β-D-l-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and cells were harvested 4 h post induction using a continuous flow centrifuge. Cell paste was stored at -80° C.

[00303] The chromatography system and the columns for MANF purification were sanitized by soaking in 0.5 M NaOH, rinsed with low endotoxin water, and equilibrated in buffers prepared with low endotoxin water. One hundred grams of cells from the fermentor run were resuspended in 1 liter of 20 mM NaH2P04, 0.25 M NaCl, pH 8 with a hand held homogenizer and passed through a micro fluidizer three times at approximately 15,000 psi. The lysate was clarified by centrifugation and filtration. The clarified lysate was applied to a 35 ml IMAC fast flow (FF) column (2.6 cm by 6.3 cm) equilibrated in Buffer NA (20 mM

NaH2P04, 5 mM imidazole, 0.5 M NaCl, pH 8). The column was washed with 2 column volumes (CV) Buffer NA, 2 CV Buffer NA containing 2 M urea and 1 % Triton X-100, 2 CV Buffer NA, and Buffer NA containing 25 mM imidazole. The protein was eluted with Buffer NA containing 200 mM imidazole and the column was purged with Buffer NB (20 mM NaH2P04, 500 mM imidazole, 0.5 M NaCl, pH 8). Fractions were collected in sterile 125 ml capacity PETG bottles. The pooled fractions containing MANF were dialyzed against 2 liters of 20 mM NaH2P04, 50 mM NaCl, 0.1 % Tween 20, pH 7.5 at room temperature. The dialysate was diluted to OD280 ~2 with 20 mM NaH2P04, 50 mM NaCl, 0.1 % Tween 20, pH 7.5, CaC12 was added to 2 mM, and 160 units enterokinase

(EKMax, Invitrogen) was added (to approximately 1 unit/ml). Digestion proceeded at room temperature for 8 hours and terminated by addition of EDTA to 5 mM final concentration. The solution was stored at 4°C overnight. The pH of the EK-digested MANF was adjusted to 6 with HC1 and filtered through a 0.22 μιη cellulose acetate filter (Corning). The entire 80 ml of EK-digested MANF preparation was applied to a sanitized prepacked 5 mL SP HP HiTrap column (GE Healthcare) equilibrated in Buffer SA (10 mM NaH2P04, pH 6) containing 50 mM NaCl, followed by a wash with several CV of Buffer SA containing 50 mM NaCl. Bound proteins were eluted by an initial step to 150 mM NaCl, followed by a continuous gradient to 0.6 M NaCl, and a final step to 1 M NaCl. Fractions containing MANF were combined and stored at 4°C. This pool was combined with the pool from a previous purification run using a similar protocol and was dialyzed against 10 mM Na citrate, 150 mM NaCl, pH 6.0. The dialysate was passed through a Mustang E filter and concentrated using a sanitized Amicon Ultra-15 (10 kDa molecular weight cut off) to an OD280 of 10. The endotoxin level of this protein preparation was less than 10 EU per mg of protein using a Pyrosate® LAL clot assay kit (Cape Cod Associates). The biological activity of MANF was verified in a dopaminergic cell culture assay.

Recombinant human GDNF was obtained from Peprotech (Catalog # 450-

10). The mature sequence of human GDNF with a methionine residue at the N- terminal with no tags was expressed in E.coli. The purity of the recombinant material was reported as greater than 98% by SDS-PAGE and HPLC analyses.

The biological activity of GDNF was tested in a rat C6 cell proliferation assay with an ED50 of <0.1 ng/ml (Peprotech technical information).

[00305] For administration to animals, MANF and GDNF were diluted in filter sterilized 10 mM Na citrate buffer, pH 6.8. The solutions were prepared in siliconized, 1.5 ml Eppendorf tubes just prior to use.

[00306] Animal Housing

[00307] Adult male Wistar rats were housed in groups of four per cage in a

temperature-controlled environment on a 12h: 12h ligh dark cycle. Animals were given free access to food and water. Animals used in the research studies were handled, housed, and sacrificed in accord with the current NIH guidelines regarding the use and care of laboratory animals, and all applicable local, state, and federal regulations and guidelines.

[00308] Administration of 6-OHDA, MANF and GDNF

[00309] Striatal administration of 6-OHDA, MANF and GDNF

[00310] This study consisted of a neuroprotection and neuroregeneration protocol and each protocol included 5 experimental groups (6-OHDA / Vehicle; 6-OHDA / MANF 3 μ& 6-OHDA / MANF 10 μg; 6-OHDA / MANF 36 μ& 6-OHDA / GDNF 10 μg) with 12 animals assigned to each group.

[00311] In preparation of 6-OHDA and growth factor administration, Wistar rats underwent stereotaxic surgery in a Kopf stereotaxic apparatus under isoflurane anesthesia for implantation of a unilateral injection guide cannula above the striatum. Animals were allowed to recover for one week before administration of growth factors or 6-OHDA was initiated.

[00312] In the neuroprotection protocol, MANF (3, 10, 36 μg; 4 μΐ filter sterilized 10 mM

Na citrate, pH 6.8), GDNF (10 μg; 4 μΐ filter sterilized 10 mM Na citrate, pH 6.8) or phosphate -buffered saline (PBS) were injected unilaterally via the previously implanted injection guide cannula with a 10 μΐ Hamilton syringe over a 3 minutes period to the middle of the right or left striatum (Stereotaxic coordinates relative to Bregma: Rostral +1.0 mm, lateral = ±2.7 mm, ventral = 6.0 mm). The needle was left in place for a further 5 min, withdrawn slowly to Z = -2.5 mm, left in place for a further 5 min, and then slowly withdrawn. The wound was closed loosely with surgical clips to regain access later.

Administration of growth factors was performed 6 hours prior to 6-OHDA administration. [00313] For the 6-OHDA administration, animals were re-anesthetized with isoflurane and injected with desipramine (15 mg/kg, i.p.) to protect noradrenergic neurons. 6-OHDA (8 μg free base dissolved in 4 μΐ of filter sterilized PBS / 0.02% ascorbic acid) was injected using the same procedure and to the same sterotaxic location as described for the growth factor administration. Animals were allowed to recover from anesthesia and were then returned to their home cages.

[00314] The neuroregeneration protocol followed the same surgical procedures but differed in two important aspects. (1) The amount of striatally administered 6- OHDA was 20 μg. (2) MANF and GDNF were administered four weeks after the 6-OHDA injection.

[00315] Striatal administration of 6-OHDA, nigral administration of MANF and GDNF

[00316] This study consisted of a neuroprotection and neuroregeneration protocol and each protocol included 6 experimental groups (Vehicle / Vehicle; 6-OHDA / Vehicle; 6-OHDA / MANF 3 μg; 6-OHDA / MANF 10 μ¾ 6-OHDA / MANF 36 μg; 6-OHDA / GDNF 10 μg) with 12 animals assigned to each group. The administration of 6-OHDA, MANF and GDNF in this Phase 2 of the study followed the same general surgical procedures as presented in the previous section. The amount of striatally administered 6-OHDA was 8 μg for the neuroprotection protocol and 20 μg for the neuroregeneration protocol. In the neuroregeneration protocol, MANF (3, 10 or 36 μg, single administration) or GDNF (10 μg, single administration) were administered two weeks after the 6- OHDA injection while in the neuroprotection protocol the growth factors were administered 6 hours prior to 6-OHDA. The growth factors were administered to the substantia nigra using the following coordinates relative to bregma and the skull surface: Caudal 4.9 mm, lateral ±2.0 mm, ventral 8.3 mm.

[00317] Amphetamine-induced rotational behavior

[00318] In order to asses unilateral neuronal damage induced by 6-OHDA injection and effects of treatment by growth factors, D-amphetamine sulphate (2.5 mg/kg free base, i.p.) was administered to animals of the treatment groups in Phases 1 and 2 of this study and circling behavior was monitored over a 2-h period using a video tracking system.

[00319] In the striatal administration neuroprotection protocol, rotational behavior was assessed at 2, 4 and 8 weeks after administration of 6-OHDA / MANF / GDNF. Due to the neuroprotection design of this study there was no mechanism available to identify and exclude animals with minimal 6-OHDA-induced damage.

[00320] In the striatal administration neuroregeneration protocol, the initial

rotational behavior was assessed 3 weeks after 6-OHDA administration (1 week prior to planned growth factor administration). A threshold of 150 unilateral rotations / 2 h after amphetamine treatment was applied to include animals in the study. Based on this data, 13 animals (6 in vehicle group, 3 in MANF 3 μg group, 2 in MANF 36 μg group, 2 in GDNF 10 μg group) were removed from further testing and were not included in the analysis of the results.

[00321] In the nigral administration neuroprotection protocol, rotational behavior was assessed 2 and 4 weeks after administration of 6-OHDA / MANF / GDNF. Due to the neuroprotection design of this study there was no mechanism available to identify and exclude animals with minimal 6-OHDA-induced damage.

[00322] In the nigral administration neuroregeneration protocol, the initial

rotational behavior was assessed 1 week after 6-OHDA administration (1 week prior to planned growth factor administration). A threshold of 150 unilateral rotations / 2 h after amphetamine treatment was applied to include animals in the study. Based on this data 7 animals (2 vehicle; 2 MANF 10 μg; 2 MANF 36 μg; 1 GDNF 10 μg) were excluded from further testing and were not included in the analysis of the results.

[00323] Transcardiac perfusion and tissue collection

[00324] The day following the last behavioral test (striatal: 8 weeks after growth factor administration; nigral: 4 weeks after growth factor administration) half of the animals from each treatment group were euthanized by decapitation, the heads rapidly frozen in liquid nitrogen, stored at -80 °C until the brains were removed and the striata dissected for neurochemical analysis (e.g. , determination of striatal dopamine (DA), 3,4-di-hydroxyphenyl acetic acid (DOPAC) and homovanillic acid (HVA) levels). The other half of the animals from each treatment group were euthanized by deep anesthesia with a lethal dose of pentobarbital (100 mg/kg, i.p.), transcardially perfused with cold phosphate buffered saline and further perfused with 200 ml of cold 4% paraformaldehyde in PBS, pH 7.4 (striatal:

Quantification of TH+ neurons in the substantia nigra; nigral: Quantification of nigral TH+ cells by stereology; Density of striatal dopaminergic (TH+ ) terminals by densitometry). After euthanasia, the brains were removed and cryopreserved in 25 ml 30 % cold sucrose for 24 hours and stored at -80°C until analyzed.

[00325] Quantification of TH ⁺ cells in the substantia nigra after striatal

administration of MANF or GDNF

[00326] A total of 18 sections (40 μιη thickness) were cut through the substantia nigra and mounted on pre-cleaned, Superfrost Plus glass slides (6 sections per slide). The slides were air dried on the lab bench for at least 4 hours prior to staining. The sections were hydrated (lx PBS, 3x 10 min), endogenous peroxidase quenched (0.3% H ₂ 0 ₂ in 50% MeOH, 20 min), washed (PBS, 3x lOmin) and blocked (5% normal horse serum in PBS/ 0.3% Tween for 2 hrs, at room temperature). The primary anti-tyrosine hydroxylase (TH) monoclonal antibody (Sigma, T-2928, 1 :2000, in 5% normal horse serum/lx PBS), was applied over night, at 4 °C (in the refrigerator), followed by washing (3x PBS, 3x 10 min). The secondary Ab, biotinylated anti-Mouse IgG, raised in horse (Vector Lab), 40 μΐ Ab/10 ml 1% horse serum/lX PBS was applied for 2 hrs at room temperature, with the sections protected from light with aluminum foil. After washing (0.1% tween/lx PBS, 4x 10 min), the peroxidase complex (ABC: Vector Lab), made up 45 mins previously according to the instructions of the manufacturer, was applied for 1 hr, at room temperature, followed by washing (4x 0.1% tween in lx PBS). Finally, the freshly prepared diaminobenzadiene (DAB) substrate was applied, and the dark deposit in the substantia nigra developed within 5 to 8 minutes. The sections were rinsed in tap water, and dehydrated through 65%, 80 %, 95% and 100% changes of ethanol. The sections were then cleared in HistoClear, mounted in VectMount (Vector Labs), cover slipped, and dried in the hood for 3 hours, before microscopic analysis. Using a lOx objective, the number of TH ⁺ cells per field in the dorsolateral region of the substantia nigra was determined by counting. This region was selected because the density of neurons is lower in this region and accurate cell counts can be made.

[00327] Embedding and sectioning of rat brains

[00328] Rat brains were treated overnight with 20% glycerol and 2%

dimethylsulfoxide to prevent freeze-artifacts, trimmed to yield the region from substantia nigra through striatum and embedded into six blocks of 12 brains each into a gelatin matrix using MultiBrain® Technology (NeuroScience Associates, Knoxville, TN). After curing, each block was rapidly frozen by immersion in isopentane, chilled to -70°C with crushed dry ice and mounted on an AO 860 sliding microtome. Each MultiBrain® block was cut coronally to generate sections of 40 μιη thickness. All sections were collected sequentially in containers filled with antigen preserve solution (50% PBS pH 7.0, 50% ethylene glycol, 1% polyvinyl pyrrolidone). For the stereological analysis of TH+ cells in the substantia nigra every 8th section was selected and stained. For the striatal densitometry analysis every 8th section (320 μιη spacing) was selected and stained yielding data on a total of four rostral to caudal levels per animal.

[00329] Quantification of TH ⁺ neurons in the substantia nigra by stereology after nigral administration of MANF or GDNF

[00330] TH ⁺ neurons of the substantia nigra were quantified by stereology of

stained sections of the substantia nigra. In brief, four 20 μιη thick sections of the substantia nigra per animal were generated from rostral to caudal spaced by 320 μιη. Sections were stained with a TH-specific antibody and nucleoli were used as the basic counting unit to quantify neurons. Nucleoli were stained using a commercially available, proprietary method to stain the argyrophilic, acidic proteins of the nucleolar organizing region referred to as "AgNORs". Sections stained for TH and AgNOR were incubated in HC1 to enhance permeabilization, bleached to avoid non-specific silver staining, and incubated with a one-quarter strength concentration of the TH antibody to provide optimal contrast with the AgNOR stain while retaining robust pigmentation of TH -positive structures.

[00331] Stereological analysis and quantification of TH+ neurons in the substantia nigra was performed. Every 8th section containing the substantia nigra was selected and the substantia nigra was carefully outlined by using an atlas. TH- AgNORstained cells within these boundaries were quantified using the optical fractionator method and the Stereologer software package (Stereology Resource Center, Chester MD & Tampa- St.Petersburg, FL). A Nikon Eclipse 80i microscope was used which was coupled to a Sony 3CCD color digital video camera and operated an Advanced Scientific Instrumentation MS-2000 motorized stage with input into a Dell Precision 650 server and a high-resolution plasma monitor. The areas of interest were first identified using 4x / 1.3 aperture dry lenses and the stereology was performed at high magnification with 100 ^x / 1.4 aperture oil immersion lenses which allowed for clear visualization of the nucleoli and precise definition of the cell boundaries.

[00332] Quantification of dopaminergic terminals in the striatum after nigral administration of MANF or GDNF

[00333] Dopaminergic (TH+ ) terminals in the striatum were quantified by

densitometry of TH-stained brain sections. For each animal, four 40 μιη thick sections of the ipsilateral and contralateral sides of the substantia nigra / striatum were analyzed from rostral to caudal, spaced by 320 μιη. The sections were then stained free-floating. The dopaminergic terminals were stained using a TH- specific antibody (Pel-Freez Biologicals; 1 : 1600 in cold TBS) and the Vectastain Elite ABC kit (Vector Laboratories). The immunohistochemical staining for TH was followed by a thionine Nissl counterstain and sections were then mounted on gelatinized glass slides. A 3 x 3 grid was placed on each section relative to a landmark (Figure 8) to collect densitometry data from the temporal (positions 1, 2, 3 (left) and 7, 8, 9 (right)), medial (positions 4, 5, 6 (left and right)) and basal (positions 7, 8, 9 (left) and 1, 2, 3 (right)) striatum (Figure 1). Similarly, data for the dorsal (positions 1, 4, 7 (left and right)) and ventral (positions 3, 6, 9 (left and right)) striata were collected. The global striatal data combined all values obtained from the four rostrocaudal levels (4 x 9 = 36 data points) from the ipsilateral or contralateral sides, respectively. The optical densities were measured at the 4 rostrocaudal levels for the temporal, medial and basal striatum with a digital camera and a constant illumination table. To estimate the specific TH staining density, the optical density readings were corrected for the non-specific density as measured on the completely denervated parts of the striatum.

[00334] Determination of striatal levels of dopamine, DOPAC and HVA

[00335] Striatal levels of DA and dopamine metabolites DOPAC and HVA were determined by negative chemical ionization / mass spectrometry. The frozen brains were allowed to thaw on ice, the striata were removed and placed on a pre- cooled piece of aluminum foil and quickly placed back on dry ice. The striatal samples were weighed using a microbalance with a sensitivity of 10 μg, transferred to 1.6 ml Eppendorf tubes, then sonicated on ice in 500 μΐ 0.1 N HC1, and centrifuged at 15,000x g for 10 min. The supernatants were transferred to micro centrifuge tubes and frozen at -80°C. To initiate the analysis, the samples were thawed and centrifuged at 21,000x g for 5 min in an IEC Micromax RF Refrigerated Microfuge (Thermo Fisher Scientivic, Asheville, NC). Two 50 μΐ aliquots were removed from the supernatant and transferred to 0.8 ml amber glass autosampler vials.

[00336] Stock solutions of DA and ² H ₅ -DA, DOPAC and ² H ₅ -DOPAC, and HVA and H5-HVA each at a concentration of 2.0 mg/ml, were diluted by serial dilution and standard curves prepared to match the anticipated concentrations of DA, DOPAC and HVA in the striatum. All calibration samples were prepared and analyzed in duplicate.

[00337] Hslnternal Standards (IS) for each of the three analytes were added to each sample. The samples were dried under a stream of N2 in a 96-well format dryer operated at 60°C and further dried in a vacuum (25 mm Hg) at 70°C for 30 min. For derivatization, pentafluoropropionic anhydride (PFPA) (50 μΐ) and hexafluoroisopropanol (HFIP) (25 μΐ) were added to each dried sample. The samples were capped and incubated at 65°C for 90 min. The caps were removed and the samples were dried under a stream of N ₂ in a 96-well format dryer operated at 60 °C. The samples were reconstituted in 25 μΐ toluene and recapped.

[00338] The reconstituted samples (1 μΐ) were injected on a 15 M RXi®-5MS capillary column (0.25 mm ID, 0.25 μιη film thickness) interfaced to a Thermo TSQ 7000 mass spectrometer. The gas chromatography was performed at a heating rate of 20°C/min from 100 to 200°C with hydrogen (1 ml/min) as the carrier gas. The injector temperature was 250°C. The mass spectrometer was run in single quad mode using chemical ionization with methane reagent gas for negative ions to detect molecular ions and fragments. Co-eluting negative ions were observed at m/z 463 for DOPAC and 468 for the ² H ₅ -DOPAC which correspond to the loss of C2F5CO (Mw=147) from the derivatized precursors. Similarly, negative ions were observed for HVA and H ₅ -HVA at m/z 330 and 334, respectively, and for DA and H ₅ -DA at m/z of 571 and 576, respectively. Product ion (MS/MS) spectra of each analyte were generated at various collision energies using argon (1 mTorr) as the collision gas. Major product ions were observed at m/z 343 and 347 for DOPAC and ² H ₅ -DOPAC, 163 and 166 for HVA and its ² H ₅ -HVA, and 376 and 380 for DA and ² H ₅ -DA. The intensities of the transitions were optimized to 15 eV by varying the collision energy. Samples were analyzed in selected reaction monitoring mode using time segments for the corresponding precursor/product ion transitions for the IS/analyte pairs.

[00339] The concentration of DA in samples was calculated from the equation DA

(nmol/g) = PA _D A/PA _2H 5-DA X (V _H +W _t )/V _A x 1000/W _t , whereas PA _DA = ADC counts for m/z 571, ADC counts for m/z 576, V _H = homogenization volume, VA = volume analyzed, W _t = tissue weight. Concentrations for DOPAC and HVA were calculated using the same method but adapted to their specific molecular ion (m/z): DOPAC m/z 463, ² H ₅ -DOPAC m/z 468; HVA m/z 330, ² H ₅ - HVA m/z 334.

[00340] MANF striatal diffusion by convection enhanced delivery (CED)

[00341] MANF striatal diffusion by convection enhanced delivery (CED) was performed as detailed in Example 2.

[00342] Statistical analyses

[00343] The amphetamine -induced rotations data of the Striatal administration neuroprotection, Nigral administration neuroprotection and Nigral administration neuroregeneration protocols were analyzed by repeated measures two-way ANOVA. The Striatal administration neuroregeneration data was analyzed with simple two-way ANOVA. The TH+ neuron cell counts in the substantia nigra (Striatal administration), the substantia nigra TH+ cell counts determined by stereology (Nigral administration), the density of dopaminergic terminals determined by striatal densitometry (Nigral administration) and striatal dopamine (striatal or nigral administration) and dopamine metabolites levels (Nigral administration) data were analyzed using one-way ANOVA.

[00344] If the ANOVA resulted in a P value less than 0.05, a post-hoc analysis was performed with the Fisher's Least Significant Difference (LSD) test to assess differences between treatment groups (one-way ANOVA, two-way ANOVA) or between different time points within a treatment group (two-way ANOVA). No adjustments for multiple comparisons were made. Significant differences were defined as P<0.05. Trends (P<0.1) of treatment effect differences were detected and are indicated as such in the figures.

[00345] All statistical analyses were performed with Prism Version 6 software. [00346] Results

[00347] Overall study design

[00348] The in vivo activities of MANF and GDNF were evaluated in rats in the 6-

OHDA model of PD. The study consisted of two phases with administration of the growth factors to the striatum (Phase 1) or the substantia nigra (Phase 2), respectively. Both phases included a neuroprotection and a neuroregeneration protocol in which the growth factors were administered shortly before (6h) or weeks after the 6-OHDA administration, respectively (Figure 9).

[00349] This temporal and spatial variation of growth factor administration was paired with a comprehensive test battery of behavioral, biochemical and morphological assessments (Figure 10).

[00350] Striatal administration of growth factors

[00351] The experimental design of for striatal administration of growth factors is described in the previous section (Figures 9 and 10). In brief, 6-OHDA lesions were introduced by intrastriatal administration of the toxin. This consisted of a neuroprotection and a neuroregeneration protocol in which the growth factors {e.g., MANF or GDNF) were administered by a single injection to the striatum either 6h prior to (Neuroprotection) or 4 weeks after (Neuroregeneration) of the 6- OHDA. The effects of growth factor treatment on behavior were investigated in the amphetamine -induced rotations test at weeks 2, 4 and 8 after growth factor administration. These behavioral assessments were complemented by counting dopaminergic neurons {e.g., TH ⁺ neurons) in the substantia nigra and measuring levels of dopamine in the striatum. The former is a measure of surviving dopaminergic cell bodies while the latter provides for a measure of functionality of dopaminergic terminals in the striatum.

[00352] Striatal administration of growth factors experiments included five

treatment groups {e.g., 6-OHDA/vehicle, 6-OHDA/MANF 3μg, 6-OHDA/MANF l(^g, 6-OHDA/MANF 36μg, 6-OHDA/GDNF l(^g) with a planned inclusion of 12 animals per group with a total number of animals of 60.

[00353] Neuroprotection protocol

[00354] The amphetamine -induced rotations test was performed on weeks 2, 4 and

8 after the administration of 6-OHDA and growth factors (Figure 9). Net ipsilateral rotations were recorded over a 2 hours period and data was analyzed by 2-way ANOVA followed by Fisher's test post hoc. A preliminary analysis of the data revealed that four animals displayed ipsilateral rotations with more than 2 standard deviations from the mean of all animals. These animals, one each in the 6-OHDA/veh, 6-OHDA/MANF 3μg, 6-OHDA/MANF 36μ§ and 6-OHDA/GDNF 10μ§, were removed from the further behavioral analysis and the final animal count for this experiment is listed in Figure 11.

[00355] At week 2 after administration of 6-OHDA and growth factors, a

significant reduction in ipsilateral net rotations compared to vehicle treatment was observed for the MANF 3μg and GDNF 10 μg treatment groups (Figure 11A). This normalization of the rotational behavior was sustained at week 4 in the GDNF and MANF 3 μg treatment groups and occurred also in the MANF 10 μg group. The MANF 36 μg group also improved at this time point compared to vehicle but this difference did not reach statistical significance. No significant differences between treatment groups were observed at week 8. This might be due to the fact that the vehicle group displayed substantial recovery at this time point as opposed to weeks 2 and 4 when there was still a significant behavioral impairment observed in the vehicle group. This is further substantiated by the fact that the vehicle group displayed a statistically significant improvement at week 8 compared to week 4 (Figure 1 IB).

[00356] MANF has been tested in an almost identically designed protocol and a significant improvement compared to vehicle was demonstrated at weeks 2 and 4 after the 6-OHDA lesion. The strongest response to treatment was seen with the MANF 10 μg dose level. Moreover, GDNF has been tested in similar protocols and a reduction in net ipsilateral rotations was observed at weeks 2 and 4 and week 6 post-6-OHDA.

[00357] Having observed significant treatment effects with MANF and GDNF in the behavioral assessment, an investigation of the underlying cellular effects may lead to an understanding of how these growth factors mediate their

neuroprotective activity. To this end, the TH ⁺ cells were counted in the substantia nigra at week 8 after administration of 6-OHDA and compared between the different treatment groups (Figure 12). The TH ⁺ cell counts were analyzed for the ipsilateral (Figure 12 A) and contralateral (Figure 12B) sides of the lesion, and ratios between ipsilateral and contralateral sides (Figure 12C) were calculated. The treatment with 6-OHDA led to a reduction of TH cell counts to about 55% on the ipsalateral side compared to the contralateral side, which is in very good agreement with similarly designed studies.

[00358] On the ipsilateral side, a significant increase in TH cells was observed in the MANF 3 μg and 10 μg treatment groups compared to 6-OHD A/vehicle suggesting that growth factor treatment in the striatum protected a significant proportion of dopaminergic neurons (Figure 12A). These effects were not observed on the contralateral side as none of the treatment groups was

significantly different from 6-OHD A/vehicle (Figure 12B). The ratios between ipsilateral and contralateral TH ⁺ counts were significantly increased compared to 6-OHD A/vehicle in the MANF 3 μg and GDNF 10 μg groups. The MANF 3 μg effect was solely dependent on the increased TH ⁺ counts on the ipsilateral side as the contralateral TH ⁺ counts were almost identical between MANF 3 μg and 6- OHD A/vehicle.

[00359] The neuroprotective effect on TH ⁺ cells in the substantia nigra was

demonstrated in a similarly designed study and a similar degree of protection by MANF was observed as in this present study. However, the most active dose was MANF 10 μg while in this present study a significant neuroprotective effect with MANF treatment was observed already at the 3 μg dose level. GDNF

neuroprotective effects on TH ⁺ cells in the substantia nigra after administration to the striatum in a neuroprotection protocol have been demonstrated in several studies and are in good agreement with observations in this present study.

[00360] In order to assess the functionality of dopaminergic neurons and in

particular their axonal projections to the striatum, levels of dopamine in the striatum were measured on the ipsilateral and contralateral sides. It is apparent that 6-OHDA leads to a strong reduction of dopamine levels on the ipsilateral sides compared to the contralateral sides in vehicle treated animals (Figure 13A and B). Treatment with MANF or GDNF did not result in any differences compared to vehicle in striatal dopamine levels on the ipsilateral or contralateral sides.

[00361] The absolute amounts of dopamine detected in the striatum are similar to levels reported in the literature in a similarly designed study. Therefore, the methodology to detect and quantify dopamine employed in this study yielded reliable results similar to the ones of an independently conducted study. The effects of MANF on the integrity of striatal projections were studied in a similarly designed study. MANF at the 10 μg dose level protected about 70% of ipsilateral TH ⁺ fibers but dopamine levels were not measured in that study. Conversely, this present study did not investigate the integrity of TH ⁺ fibers and thus a comparison of MANF effects between these studies is difficult. GDNF administration to the striatum in a neuroprotection protocol has shown mixed results on striatal TH ⁺ fiber density with two studies showing significant protection and another study showing no protection.

[00362] Neuroregeneration protocol

[00363] In this neuroregeneration protocol, MANF or GDNF were administered by a single striatal injection 4 weeks after the unilateral 6-OHDA lesion. This design allowed for an amphetamine -induced rotations test prior to MANF or GDNF administration at week 3 post 6-OHDA to identify and exclude animals that did not display the expected rotational behavior. This procedure led to the exclusion of 6 animals in the 6-OHD A/vehicle, 3 animals in the 6-OHDA/MANF 3 μg, 2 animals in the 6-OHDA/MANF 36 μg and 2 animals in the 6-OHD A/GDNF 10 μg groups.

[00364] The amphetamine -induced rotations test for assessments of treatment

effects was performed on weeks 2, 4 and 8 after the administration of growth factors (Figure 9) {e.g., weeks 6, 8 and 12 after administration of 6-OHDA). Net ipsilateral rotations were recorded over a 2 hours period and data was analyzed by 2-way ANOVA followed by Fisher's test post hoc (Figure 14).

[00365] At week -1 relative to growth factor treatment all groups displayed similar rotational behavior indicating that the treatment groups were well balanced prior to the initiation of growth factor treatment (Figure 14A). At week 2 after growth factor or vehicle treatment, all groups, including the vehicle group, displayed equally improved ipsilateral rotational behavior. At week 4, a further improvement was observed in the vehicle treated group but the MANF 3 μg and 10 μg groups tended to perform better than vehicle even though this difference did not reach statistical significance. Finally, at week 8, a further improvement in the vehicle group and a sustained normalization of the rotational behavior was observed with MANF 3 μg. None of the comparisons between treatment groups were statistically significant. A time-dependent improvement in all treatment groups, including vehicle, was apparent (Figure 14B).

[00366] Striatal administration of MANF and GDNF within a neuroregeneration protocol has been reported previously. Spontaneous recovery in the vehicle treated group was negligible up to 10 weeks post 6-OHDA in two independent studies facilitating the detection of growth factor therapeutic effects. MANF treatment with a 10 μg single dose led to a time-dependent improvement of ipislateral rotations reaching statistical significance versus vehicle in cumulative rotations over a 12 week observation period.

[00367] Similarly, GDNF decreased amphetamine-induced ipsilateral rotations compared to vehicle over a 12 week observation period. The lack of a significant treatment effect by MANF or GDNF in the neuroregeneration protocol of this present study may be due to the comparatively rapid and almost complete spontaneous recovery of the vehicle treated animals.

[00368] In order to assess the potential of MANF and GDNF to restore the TH ⁺ phenotype of neurons in the substantia nigra, the number of nigral TH ⁺ neurons was quantified for all treatment groups for the ipsilateral and contralateral sides (Figure 15 A and B) and ratios of ipsilateral to contralateral TH ⁺ counts were calculated (Figure 15C). The number of TH ⁺ neurons of vehicle treated animals on the ipsilateral side was reduced to about 60% of the number counted on the contralateral side. This extent of nigral cell death is in agreement with the results obtained in the neuroprotection protocol of this study (Figure 12) and with values reported in the literature. None of the MANF treatment groups on the ipsilateral or the contralateral sides was significantly different from the vehicle treated group and thus MANF did not show a regenerative effect on nigral neurons. This result is in agreement with observations in a similarly designed study in which only a modest non- significant effect was shown for MANF 10 μg at 12 weeks post 6- OHDA. GDNF treatment significantly increased the TH ⁺ counts on both the ipsilateral and the contralateral sides compared to 6-OHD A/vehicle (Figure 15A and B). However, when the ratios of ipsilateral and contralateral TH ⁺ counts were compared between GDNF and 6-OHD A/vehicle treatments no statistically significant difference was observed (Figure 15C). TH ⁺ protective effects induced by GDNF in neuroregeneration protocols were reported in similarly designed studies. The effects on TH phenotype restoration by MANF and GDNF are thus similar in this present study and in the literature.

[00369] In order to assess the functionality of dopamineric axonal terminals in the striatum, the levels of dopamine were determined in the ipsilateral (Figure 16A) and contralateral (Figure 16B) striati for each of the treatment groups. In the vehicle group, the ipsilateral dopamine level was strongly reduced compared to the contralateral side, confirming the maintenance of a functional lesion present at the end of the evaluation period (e.g., 12 weeks after 6-OHDA treatment).

Treatment with MANF or GDNF did not lead to any difference in dopamine levels compared to vehicle treated animals on the ipsilateral or the contralateral sides. Hence, none of the growth factor treatment regimens restored dopamine levels at the end of the observation period. This is remarkable in view of the complete functional recovery observed in the rotational behavior with several of the treatment groups (e.g., vehicle, MANF 3 μg, MANF 10 μg, MANF 36 μg).

[00370] Nigral administration of growth factors

[00371] The experimental design for nigral administration of growth factors is described in the previous section (Figures 9 and 10). In brief, 6-OHDA lesions were introduced by intrastriatal administration of the toxin. Neuroprotection and a neuroregeneration protocol in which the therapeutic growth factors (e.g., MANF or GDNF) were administered by a single injection to the substantia nigra either 6h prior to (Neuroprotection) or 2 weeks after (Neuroregeneration) the 6-OHDA injection were performed. The effects of growth factor treatment on behavior were investigated in the amphetamine-induced rotations test at weeks 2 and 4 after growth factor administration. These behavioral assessments were complemented by counting dopaminergic neurons (e.g. , TH ⁺ neurons) in the substantia nigra using computer assisted stereology, quantification of striatal dopaminergic terminals by densitometry and measuring levels of dopamine and its metabolites DOPAC and HVA in the striatum. The stereology provides for a measure of surviving dopaminergic cell bodies in the substantia nigra while the densitometry and dopamine level quantification provides for a measure of functional and structural improvement, respectively, of dopaminergic terminals in the striatum.

[00372] These experiments included six treatment groups (e.g., Vehicle/vehicle, 6- OHD A/vehicle, 6-OHDA/MANF 3μg, 6-OHDA/MANF l(^g, 6-OHDA/MANF 36μg, 6-OHDA/GDNF 10μ§) with a planned inclusion of 12 animals per group with a total number of animals of 60.

[00373] Neuroprotection protocol

[00374] The amphetamine -induced rotations test was performed on weeks 2 and 4 after the administration of 6-OHDA and growth factors (Figure 8). Net ipsilateral rotations were recorded over a 2 hours period and data was analyzed by repeated measures 2-way ANOVA followed by Fisher's test post hoc. Incomplete data collection over the 2 hours period at the week 2 time point caused the exclusion of animals in the vehicle/vehicle (4 animals), 6-OHDA/vehicle (4 animals), 6- OHDA/MANF 3μg (2 animals), 6-OHDA/MANF 10 μg (4 animals), 6- OHDA/MANF 36 μg (4 animals) and GDNF 10 μg (2 animals) groups. The numbers of animals assessed, analyzed and included in the statistical analysis of the rotational behavior data is shown in Figure 17.

[00375] At the week 2 time point, the vehicle/vehicle and 6-OHDA/vehicle values were separated but this difference was not statistically significant (Figure 17A). This was most likely caused by the relatively low number of net ipsilateral rotations observed in the 6-OHDA/vehicle group. Due to the nature of the neuroprotection protocol in which 6-OHDA and the growth factors are

administered at almost the same time it is not possible to know whether these low numbers were due to an ineffective lesion, a rapid recovery, or both. However, at this time point, MANF 10 μg displayed an increase in rotational behavior compared to 6-OHDA/vehicle. While the net ipsilateral rotations in the 6- OHD A/vehicle group decreased from week 2 to week 4, the values for MANF 10 μg and GDNF 10 μg remained elevated. The GDNF 10 μg values were significantly higher compared to 6-OHDA/vehicle while the values for MANF 10 μg displayed a trend towards a difference to 6-OHDA/vehicle. Since MANF has not been administered to the substantia nigra in any of the previous studies there is no literature comparison available. GDNF has been administered to the substantia nigra in several 6-OHDA studies in a neuroprotection design. One did not observe an effect on net ipsilateral rotations 5 months after GDNF treatment. In contrast, another observed an increase in net ipsialateral rotations at week 6 after GDNF was administered to the substantia nigra, similar to the effect observed in this study. Hence, this study and the literature provide evidence that GDNF and MANF increase net ipsilateral rotations in the 6-OHDA model when administered to the substantia nigra in a neuroprotection protocol.

[00376] After nigral administration of MANF or GDNF, the number of TH ⁺

neurons in the substantia nigra was quantified by stereo logy. The numbers of TH ⁺ neurons were determined from nigral sections and a computational model to reconstruct neurons in three-dimensional space for the ispilateral and contralateral sides of animals from all treatment groups (Figure 18A and B). The ipsilateral to contralateral ratios were calculated at the individual animal level (Figure 18C). There was no difference between the vehicle/vehicle and 6-OHDA/vehicle groups on the ipsilateral and the contralateral sides while the ratios were separated slightly but non-significantly. Inspection of the underlying raw data revealed a substantial variability in the computed cell counts with six instances of ipsilateral values substantially higher than the corresponding contralateral cell numbers. Moreover, due to technical difficulties, 13 of the 33 animals with ipsilateral stereology data did not have corresponding contralateral data, leaving the MANF 36 μg group with just one data point.

[00377] There are several studies in which GDNF was administered to the

substantia nigra in a 6-OHDA model and in which survival of nigral TH ⁺ cells was quantified. Generally, GDNF protected TH ⁺ cells to a significant extent, ranging from 60% protection to complete protection. However, TH ⁺ cells seem to be protected but remained in an atrophic state thereby preventing a substantial functional recovery.

[00378] In order to assess whether the dopaminergic axonal terminals of TH+

neurons were affected by 6-OHDA and prior treatment with growth factors, densitometry measurements of the ispilateral and contralateral striata of animals were performed at the end of the observation period (Figure 19). The numbers of dopaminergic (e.g., TH+ ) terminals in the striatum were significantly different between the vehicle/vehicle and 6-OHDA/vehicle groups on the ipsilateral side for the entire striatum (Figure 19A) and for the dorsal (Figure 19D) and ventral (Figure 19G) striata. Hence, as expected, 6-OHDA treatment significantly reduced the number of dopaminergic terminals in the striatum. However, there were subtle differences between the striatal compartments in that the ventral striatum (Figures 19G and I) was more resistant to 6-OHDA than the dorsal striatum (Figures 19D and F). Most importantly, treatment with MANF 36 μ almost completely restored the dopaminergic terminal densities on the ipsilateral side with significant differences to both 6-OHDA/vehicle and GDNF 10 μg treatments in the entire striatum (Figure 19A) and the dorsal (Figure 19D) and ventral (Figure 19G) striata. There was a MANF dose-dependent increase of dopaminergic terminals on the contralateral side (Figure 19B) both in the dorsal (Figure 19E) and ventral (Figure 19H) striata, which however did not reach statistical significance. The calculated ratios between the ipsilateral and contralateral densities yielded a significant difference between MANF 36 μg and GDNF 10 μg (Figures 19C, F and I). An analysis of further striatal spatial subgroups (Figure 20A-I; temporal, medial and basal striatum) revealed essentially the same MANF treatment effects as for the global data (Figure 19 A, B and C). MANF 36 μg protected

dopaminergic terminals significantly compared to 6-OHDA/vehicle and 6-OHDA/ GDNF 10 μg treatment in all three striatal compartments. However, the response to 6-OHDA differed between the temporal, medial and basal striata in that the temporal striatum displayed a marked degree of resistance to 6-OHDA-induced toxicity.

[00379] Densitometry quantifies the amount of TH+ immunoreactivity in the striata but does not yield information on the level of enzymatic activity present in the tissue. Further studies will investigate the functional consequences of MANF - induced increases of striatal TH+ immunoreactivity.

[00380] Nigral administration of GDNF did not increase the density of

dopaminergic terminals in the striatum (Figure 19). Several previous studies have investigated the effect of nigral administration of GDNF on striatal terminals in a 6-OHDA model. No effect on striatal fiber density was observed after GDNF treatment in a neuroprotection protocol and no effects on TH ⁺ terminals by densitometry was observed. Moreover, there was no indication of sprouting in the striatum. This current study is thus in agreement with published accounts of a lack of effect on striatal dopaminergic terminals by nigral administration of GDNF. Conversely, MANF significantly increased the dopaminergic (TH ⁺ ) terminal density when administered to the substantia nigra.

[00381] Given the significant protective effect on striatal dopaminergic terminals by MANF treatment the determination of dopamine levels in the striatum would provide information on the functionality of these terminals. Therefore, levels of dopamine were determined in the ipsilateral (Figure 21 A) and contralateral (Figure 2 IB) striati for each of the treatment groups. There was a significant difference between the vehicle/vehicle and 6-OHDA/vehicle groups on the ipsilateral side and the ipsilateral / contralateral ratios (Figure 21C), but not for the contralateral side. Treatment with MANF 36 μg or any other growth factor treatment, however, did not restore the reduced dopamine levels observed in the 6- OHD A/vehicle group. The MANF 36 μg treatment group displayed a significantly increased dopamine level compared to the GDNF 10 μg treatment in the ipsilateral, contralateral and ratio comparisons.

[00382] Hence, none of the growth factor treatment regimens restored dopamine levels at the end of the observation period. This is remarkable in view of the significant protection of dopaminergic terminals by MANF 36 μg. There appears to be a dissociation between the protection of the TH ⁺ phenotype in the striatal terminals and the dopamine levels in the striatum.

[00383] GDNF administration to the substantia nigra and its effects on dopamine levels in the substantia nigra and the striatum were investigated in a 6-OHDA model. Administration of 6-OHDA to the striatum led to a significant decrease of dopamine levels on the ipsilateral side in both the striatum and the substantia nigra which was almost completely prevented by prior administration of GDNF to the substantia nigra.

[00384] Dopamine is metabolized to DOPAC by MAO and then to HVA by

COMT. An alternative metabolic pathway employs COMT first, yielding 3- methoxytyramine (3-MT) followed by oxidation to HVA by MAO. In this study, striatal levels of DOPAC (Figure 22) and HVA (Figure 23) were determined for each of the six treatment groups for the ipsilateral and the contralateral sides relative to the 6-OHDA lesion.

[00385] DOPAC levels were significantly different between the vehicle/vehicle and 6-OHDA/vehicle groups on the ipsilateral but not on the contralateral side, and for the ipsilateral/contralateral ratio. Neither MANF 3 μg nor MANF 36 μg were different from 6-OHDA/vehicle but MANF 10 μg and GDNF 10 μg had their DOPAC levels significantly reduced. Similar differences were also observed for the ipsilateral/contralateral ratios. The DOPAC levels largely paralleled the effects seen across the treatment groups for the dopamine levels. For both outcomes, the vehicle/vehicle and 6-OHDA/vehicle groups are significantly different, the MANF 10 μg and GDNF 10 μg differ from the 6-OHDA/vehicle group and the MANF 36 μg differs from the GDNF 10 μg group.

[00386] HVA levels were significantly different between the vehicle/vehicle and 6- OHD A/vehicle groups on the ipsilateral (Figure 23 A) but not on the contralateral side (Figure 23B), and for the ipsilateral/contralateral ratio (Figure 23C). MANF 3 μg, MANF 10 μg and GDNF 10 μg had their HVA levels significantly reduced compared to the 6-OHDA/vehicle group. These differences were also observed for the ipsilateral/contralateral ratios but in addition, the MANF 36 μg level was also lower than 6-OHDA/vehicle. Again, the HVA levels largely parallel the effects seen across the treatment groups for the DOPAC and dopamine levels. For all outcomes, the vehicle/vehicle and 6-OHDA/vehicle groups are significantly different, the MANF 10 μg and GDNF 10 μg differ from the 6-OHDA/vehicle group and the MANF 36 μg differs from the GDNF 10 μg group.

[00387] Neuroregeneration protocol

[00388] In this neuroregeneration protocol, MANF or GDNF were administered by a single injection into the substantia nigra 2 weeks after the unilateral 6-OHDA lesion to the striatum. This design allowed for an amphetamine -induced rotations test prior to MANF or GDNF administration at week 1 post 6-OHDA to identify and exclude animals that did not display the expected rotational behavior. This procedure led to the exclusion of 1 animal in the vehicle/vehicle, 2 animals in the 6-OHDA/vehicle, 2 animals in the 6-OHDA/MANF 10 μg, 2 animals in the 6- OHDA/MANF 36 μg and 1 animal in the 6-OHDA/GDNF 10 μg groups.

[00389] The amphetamine -induced rotations test for assessments of treatment effects was performed on weeks 2 and 4 after the administration of growth factors (Figure 9) {e.g., weeks 4 and 6 after administration of 6-OHDA). Net ipsilateral rotations were recorded over a 2 hours period and data was analyzed by repeated measures 2-way ANOVA followed by Fisher's test post hoc (Figure 24).

[00390] The vehicle/vehicle group was significantly different from the 6-

OHD A/vehicle group at all time points (Figure 24 A). Moreover, at the initial assessment of the rotational behavior at week -1 relative to growth factor administration, all treatment groups were similar, indicating that the groups were well balanced prior to the initiation of growth factor treatment. At weeks 2 and 4 after treatment initiation, none of the 6-OHDA groups differed significantly.

Hence, there was no significant effect by MANF or GDNF treatment on ameliorating net ipsilateral rotations compared to vehicle treatment (Figure 24A). In within group comparisons, MANF 10 μg at week 2 was significantly better than at week -1, an effect that was sustained at week 4. Similarly, MANF 36 μg significantly improved between the week 2 and 4 assessments while the GDNF group appeared to get worse in the same period (Figure 24B). However, this time- dependent MANF treatment effect was relatively modest as it could not be separated statistically from the 6-OHDA/vehicle spontaneous recovery. Moreover, compared to the striatal administration data at the same time -point relative to 6- OHDA administration, the spontaneous recovery by 6-OHDA/vehicle was less pronounced.

[00391] There is no literature on MANF administration to the substantia nigra in an

6-OHDA model and it is therefore not possible to compare the results of this current study with independently generated data. However, GDNF was administered to the substantia nigra in a model in which 6-OHDA was injected into the basal forebrain bundle. GDNF was given as single injections of 100 μg or 1000 μg, respectively, at 9 weeks post 6-OHDA. At week 10, the turning behavior was fully restored, an effect that was sustained until the end of the study at week 20.

[00392] As described in the neuroprotection section, the number of TH ⁺ neurons in the substantia nigra was quantified by stereo logy. The numbers of TH ⁺ neurons were determined for the ispilateral and contralateral sides of animals from all treatment groups (Figure 25A and B). The ipsilateral to contralateral ratios were calculated at the individual animal level (Figure 25C). There was no difference between the vehicle/vehicle and 6-OHDA/vehicle groups on the ipsilateral and the contralateral sides while the ratios were separated slightly but non-significantly. While the variation of values observed in the neuroprotection protocol, were less pronounced in the neuroregeneration protocol, there were still three instances of ipsilateral values substantially higher than the corresponding contralateral cell numbers. Moreover, due to technical difficulties, 14 of the 36 animals with ipsilateral stereology data did not have corresponding contralateral data. [00393] The survival of TH neurons in the substantia nigra was assessed after nigral administration of GDNF in a neuroregeneration protocol of the striatal 6- OHDA model. A single treatment with GDNF 10 μg administered 1 week post 6- OHDA, increased the number of ipsilateral TH ⁺ neurons by about 50% compared to vehicle treatment when assessed four weeks post 6-OHDA. In a long-term neuroregeneration study applying 6-OHDA to the basal forebrain bundle, a single dose of GDNF 100 μg and 1000 μg nine weeks post 6-OHDA restored TH enzymatic activity in the substantia nigra but not in the striatum.

[00394] As described in the neuroprotection section, dopaminergic axonal

terminals of TH+ neurons were assessed by TH immunoreactivity densitometry of the ispilateral and contralateral striata of animals in all treatment groups at the end of the observation period. The numbers of dopaminergic (e.g., TH+ ) terminals in the striatum were significantly different between the vehicle/vehicle and 6- OHD A/vehicle groups on the ipsilateral side for the entire striatum (Figure 26A) and the dorsal (Figure 26D) and ventral (Figure 26G) striata. As already seen in the neuroprotection protocol, 6-OHDA treatment significantly reduced the number of dopaminergic terminals in the striatum. The compartmental differentiation of dorsal and ventral striata, respectively, in response to 6-OHDA treatment was similar to the one observed in the neuroprotection protocol, although less pronounced. None of the treatment groups displayed significant differences in the number of dopaminergic terminals on the ipsilateral (Figures 26A, D and G) and the contralateral sides (Figures 26B, E and H). There was a trend towards a difference between MANF 3 μg and MANF 36 μg compared to GDNF 10 μg (Figure 26A). This trend became statistically significant when the ipsilateral / contralateral ratios of MANF 3 μg were compared to GDNF 10 μg (Figure 26C). Nevertheless, none of the growth factors treatments were different from the 6- OHD A/vehicle group. An analysis of further striatal spatial subgroups (Figure 27A-I; temporal, medial and basal striatum) revealed the same absence of MANF treatment effects as observed for the global data. Also, the differential response to 6-OHDA observed in the neuroprotection protocol was less pronounced in the neuroregeneration protocol.

[00395] There are no published reports on MANF administration to the substantia nigra and therefore, these results cannot be compared to independently generated data. However, nigral administration of GDNF was assessed in neuroregenerative protocols of 6-OHDA lesions in the striatum or the medial forebrain bundle. GDNF did not have any effect on TH ⁺ fiber density in the striatum or the striatal TH enzymatic activity. Hence, the results of the present study as they relate to GDNF are in agreement with published information.

[00396] In order to understand the functionality of dopaminergic terminals in the striatum in response to neuroregenerative growth factor therapy the levels of dopamine were determined in the ipsilateral (Figure 28A) and contralateral (Figure 28B) striati for each of the treatment groups, and the ipsilateral / contralateral ratios were calculated at the individual animal level (Figure 28C). There was a significant difference of dopamine levels between the vehicle/vehicle and 6-OHD A/vehicle groups on the ipsilateral side and for the ipsilateral / contralaetral ratios, but not the contralateral side. None of the growth factor treatments was significantly different from 6-OHD A/vehicle group on the ipsilateral side of the lesion. However, MANF 36 μg showed a trend towards normalization of dopamine levels in the striatum. The striatal dopamine levels of GDNF were not different from 6-OHD A/vehicle. This lack of striatal dopamine level restoration was also observed in an independent study of GDNF assessing its neuroregenerative potential in a 6-OHDA medial forebrain bundle lesion.

[00397] As in the neuroprotection protocol, striatal levels of DOPAC (Figure 29) and HVA (Figure 30) were determined for each of the six treatment groups in this neuroregeneration protocol. DOPAC levels were significantly different between the vehicle/vehicle and 6-OHD A/vehicle groups on the ipsilateral (Figure 29A) and for the ipsilateral/contralateral ratio (Figure 29C), but not on the contralateral side (Figure 29B). The MANF 36 μg group showed a trend towards a difference from 6-OHD A/vehicle on the ipsilateral side but none of the other treatment groups was different from 6-OHD A/vehicle. The effect by MANF 36 μg on DOPAC mirrored the results obtained for dopamine.

[00398] HVA levels were significantly different between the vehicle/vehicle and 6- OHD A/vehicle groups on the ipsilateral (Figure 3 OA) and for the

ipsilateral/contralateral ratio (Figure 30C), but not on the contralateral side (Figure 30B). None of growth factor treatment groups was different from the 6- OHD A/vehicle group. [00399] Diffusion of MANF with convection-enhanced delivery

[00400] Convection-enhanced delivery (CED) has emerged as a novel

neurosurgical technique with the potential to achieve more effective coverage of the putamen. CED describes a direct method of drug delivery to the brain through very fine micro catheters. By establishing a pressure gradient at the tip of the infusion catheter, CED confers several advantages over conventional drug injection techniques, in particular, homogeneous drug distribution through large and clinically-relevant brain volumes. The primary objective of this study was to determine whether a single infusion of MANF could be distributed via CED and whether MANF could be detected after 7 days.

[00401] MANF (10 μ§) was delivered via CED at flow rates of 0.1 μΐ/ min, 1.25 μΐ/ min, and 5 μΐ/ min into the striatum. MANF was detected at 0 hours after the infusion at all flow rates by immunohistochemistry (Figure 31).

[00402] Following on from the 0 hour time -point results, the flow rates for MANF delivery by CED for the 7 day study were 1.25 μΐ/ min and 2.5 μΐ/ min. MANF could be detected by immunohistochemistry at 7 days post-infusion following CED at both flow rates (1.25 μΐ/ min and 2.5 μΐ/ min) (Figure 32).

[00403] A higher volume of MANF was detectable in the hemispheres infused with a flow rate of 1.25 μΐ/ min (0.0505 ± 0.028 mm ³ ) compared to 2.5 μΐ/ min (0.0185 ± 0.008 mm ). MANF infusion with this flow rate resulted also in a higher volume of distribution compared to GDNF infusion at 2.5 μΐ/ min (0.028 ± 0.006 mm ) (Figure 33).

[00404] Discussion

[00405] MANF's potent neuroprotective activity was confirmed in this present study both at the behavioral and structural level. A single striatal administration of MANF prevented 6-OHDA-induced behavioral deficits and dopaminergic cell death in the substantia nigra. A comparison of striatal and nigral administration of MANF revealed an intriguing mechanism of distal action of this growth factor. When MANF was administered to the substantia nigra, striatal dopaminergic terminal densities increased and when the striatum was the target of MANF administration an increase of TH ⁺ cells in the substantia nigra was detected.

MANF (and GDNF) administration to the substantia nigra resulted in an increase of ipsilateral rotations and we hypothesize that this effect might be due to activation of contralateral circuits. Therefore, MANF is a potent neuroprotective agent, MANF activity is site specific and MANF may cause effects in nigral neurons of the ipsilateral and contralateral projections to the striatum.

Administration of MANF to the striatum resulted in a robust behavioral affect most notably in the neuroprotection protocol, and to a lesser extent in the neuroregeneration protocol. MANF normalized the 6-OHDA-induced ipsilateral rotations as early as 2 weeks after a single administration and this effect was sustained at 4 weeks post growth factor treatment. The active doses ranged from 3 μg to 10 μg and are in good agreement with the literature. It appears likely that lower doses would be active in this paradigm; the minimally effective dose remains to be determined. These behavioral effects were paralleled by a significant protection of the dopaminergic cell bodies in the substantia nigra. MANF protected dopaminergic neurons in vitro, similar to the effects observed in vivo here. Hence, administration of MANF to the striatum protects the cell bodies of dopaminergic neurons with a concomitant normalization of the rotational behavior. MANF may counteract some of the known intracellular effects of 6- OHDA. 6-OHDA is relatively selective for monoaminergic neurons, resulting from preferential uptake by dopaminergic and noradrenergic transporters. Once inside the neurons 6-OHDA accumulates in the cytosol, generates reactive oxygen species and quinones with the latter leading to the inactivation of biological macromolecules by attack of nucleophilic groups. The mechanism of cell death in mesencephalic neurons involves caspase-dependent pathways. 6-OHDA, but not MPP ⁺ , induced caspase-3 and caspase-9 enzymatic activity in the MN9D dopaminergic neuronal cell line. Application of a caspase inhibitor (zVAD-fmk) prevented 6-OHDA-induced TH ⁺ cell death. The specific neuronal toxicity of 6- OHDA thus involves several mechanisms including the activation of apoptotic pathways and the generation of reactive oxygen species. MANF may counteract the toxicity of 6-OHDA on both levels. MANF decreased caspase-3 activation in serum starved cardiomyocytes. It is thus conceivable that MANF could prevent 6- OHDA-induced caspase activation in dopaminergic neurons and thereby promote the survival of these cells. Moreover, MANF protected cardiomyocytes from reperfusion injury in vivo. It is well known that a key mechanism of reperfusion injury is the generation of reactive oxygen species. MANF could thus counteract the effects of 6-OHDA-generated reactive oxygen species. Finally, MANF might have a more general role in counteracting cellular stress and in particular endoplasmatic stress, as exemplified by its protective activity in thapsigargin- and tunicamycin-induced cell death. These anti-stress effects of MANF may contribute to the observed in vivo effects of MANF in the 6-OHDA model of PD.

[00407] MANF displayed a strong neuroprotective activity that manifested itself in a reduced rotational behavior and a rescue of TH ⁺ neurons in the substantia nigra. Remarkably, this protective effect was not paralleled by a functional

normalization of the striatal dopaminergic terminals as no differences in dopamine levels between the treatment groups were observed.

[00408] The effects of MANF on dopaminergic terminals in the striatum were assessed by densitometry in the current study. Administration of MANF, 36 μg, to the substantia nigra in the neuroprotection protocol resulted in a significant increase of the ipsilateral dopaminergic (TH ⁺ ) terminal density at the end of the observation period compared to 6-OHD A/vehicle treatment. MANF protected dopaminergic terminals almost completely at the highest dose with no difference to vehicle / vehicle animals. It is noteworthy that there was also a non-significant MANF dose-dependent increase of dopaminergic terminal densities on the contralateral side. In contrast, dopamine levels in the striatum were not normalized by treatment with MANF. Therefore, the effect on terminal densities was dissociated from an effect on dopamine levels. In this context, it is important to note that densitometry detects the presence of TH in the dopaminergic terminals. An increase of the densitometry signal thus reflects an increase in the amount of TH protein but not necessarily an increase of active TH. Accordingly, nigral administration of MANF might thus increase or preserve the levels of TH protein in the striatum but may not prevent its inactivation. A further possibility to explain our results is that a non-linear relationship exists between innervating fiber density and dopamine levels, such that nigral administration of MANF in this

experimental situation could increase the former but not the latter. Moreover, MANF could facilitate outgrowth of fibers from sick or surviving neurons. This could be because of a neuroprotective MANF effect on neurons and their projections, and/ or by a sprouting of new fibers into previously occupied striatal terminal fields from surviving neurons or those unaffected by the 6-OH-dopamine lesion.

[00409] The increase of the ipsilateral rotational behavior induced by MANF (or

GDNF) administration to the substantia nigra in the neuroprotection protocol was remarkable but not unprecedented. Previously, a reduction in TH ⁺ fiber density in the striatum after administration of GDNF to the substantia nigra has been observed. In addition, aberrant local sprouting in the ventral thalamus was observed and the combination of these effects may have resulted in more severe motor impairments. Here was observed an increased TH density in the striatum but no data was collected on aberrant sprouting close to the growth factor injection site. An alternative explanation for the increased behavioral abnormalities observed could involve activation of contralateral circuits by striatal

administration of MANF. Neuronal circuits that may be involved in this mechanism are the interhemispheric nigrostriatal and corticostriatal pathways. Importantly, injection of 6-OHDA into the striatum spares these interhemispheric nigrostriatal neurons. It is thus conceivable that nigral administration of MANF could affect the activity of interhemispheric neurons by increasing the

contralateral striatal dopaminergic terminals. Since those terminals are located in an environment conducive to neuronal activity, increased TH protein levels may represent increased active enzymatic activity resulting in increased striatal dopamine levels which could then lead to an increase of ipsilateral rotations. In this context it is interesting to note that both the terminal densities and the dopamine levels on the contralateral side of MANF treated animals were slightly increased.

[00410] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.